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Papers by David Fenske

Research paper thumbnail of Deuterium and phosphorus-31 nuclear magnetic resonance investigations of human plasma low- and high- density lipoproteins

Research paper thumbnail of Ionophore-mediated liposome loading

Research paper thumbnail of Regioselectively and stereoselectively deuteriated sialyl glycerolipids for dynamic studies by 2 H-NMR spectroscopy

Research paper thumbnail of Entrapment of small molecules and nucleic acid-based drugs in liposomes

Methods in enzymology, 2005

In the past two decades there have been major advances in the development of liposomal drug deliv... more In the past two decades there have been major advances in the development of liposomal drug delivery systems suitable for applications ranging from cancer chemotherapy to gene therapy. In general, an optimized system consists of liposomes with a diameter of approximately 100 nm that possess a long circulation lifetime (half-life >5 h). Such liposomes will circulate sufficiently long to take advantage of a phenomenon known as disease site targeting, wherein liposomes accumulate at sites of disease, such as tumors, as a result of the leaky vasculature and reduced blood flow exhibited by the diseased tissue. The extended circulation lifetime is achieved by the use of saturated lipids and cholesterol or by the presence of PEG-containing lipids. This chapter will focus on the methodology required for the generation of two very different classes of liposomal carrier systems: those containing conventional small molecular weight (usually anticancer) drugs and those containing larger gene...

Research paper thumbnail of Regio- and stereo-selectively deuteriated sialyl glycerolipids for dynamic studies by 2H NMR spectroscopy

Journal of the Chemical Society, Chemical Communications, 1990

ABSTRACT

Research paper thumbnail of Glycosphingolipids: sup 2 H NMR study of the influence of carbohydrate headgroup structure on ceramide acyl chain behavior in glycolipid-phospholipid bilayers

Biochemistry, 1991

Galactosyl- and glucosylceramide, globoside, and dihydrolactosylceramide, bearing (2,2-²Hâ)stear... more Galactosyl- and glucosylceramide, globoside, and dihydrolactosylceramide, bearing (2,2-²Hâ)stearic acid, have been studied at a concentration of 10 mol % in bilayers of dimyristoylphosphatidylcholine by ²H NMR. The quadrupolar splittings Îv{sub Q} of the C2 deuterons were measured at several temperatures in the range of 30-60°C. Spin-lattice relaxation times T⁠of C2 deuterons were determined in the same temperature range for

Research paper thumbnail of Structural and motional properties of vesicles as revealed by nuclear magnetic resonance

Chemistry and Physics of Lipids, 1993

Research paper thumbnail of Insoluble complex formation between low density lipoprotein and heparin. A 31P-NMR study

Chemistry and Physics of Lipids, 1990

31P-NMR has been used to probe the motions of the phosphate moiety of phospholipid head-groups in... more 31P-NMR has been used to probe the motions of the phosphate moiety of phospholipid head-groups in samples of human low density lipoprotein (LDL) in which particle tumbling has been greatly reduced by increasing the viscosity of the medium, by forming an LDL gel by ultracentrifugation, or by precipitation with heparin. The 31P-NMR spectra of LDL gel give broad "powder-like" lineshapes, with the sign and magnitude of the anisotropy characteristic of the bilayer mesophase, which narrow as the temperature is raised from 5 to 45 degrees C. This narrowing occurs over the same temperature range as the core cholesteryl ester liquid-crystalline to liquid phase transition, suggesting interactions between the surface and core. The 31P lineshapes of LDL-heparin insoluble complexes are also "powder-like", but are broader than native LDL at all temperatures studied. The spectra were simulated assuming an axially-symmetric shielding tensor motionally narrowed by Brownian isotropic diffusion [Burnell et al. (1980) Biochim. Biophys. Acta 603, 63-69], allowing determination of the lateral diffusion coefficients, DT, and the chemical shift anisotropy, delta sigma, of the monolayer phospholipids. Relative to LDL gel, the temperature-dependence of DT was reduced in the LDL-heparin insoluble complexes, and delta sigma was increased from 50 to 60 ppm. The results suggest that insoluble complex formation slows phospholipid lateral diffusion in the LDL monolayer and alters the orientation and/or order of the head-group.

Research paper thumbnail of Soluble complex formation between low-density lipoprotein and glycosaminoglycans. A 2H and 31P-NMR, and quasi-elastic light scattering study

Chemistry and Physics of Lipids, 1988

Soluble complex formation between LDL and heparin (HEP) and chondroitin sulfate (CS) has been stu... more Soluble complex formation between LDL and heparin (HEP) and chondroitin sulfate (CS) has been studied by 2H- and 31P-NMR and light scattering. The 2H-NMR linewidths of [2H]HEP and [2H]C4S increase substantially upon binding to LDL, with the [2H]HEP linewidths broader at low glycosaminoglycan (GAG)/low density lipoprotein (LDL) ratios. Preliminary analysis of the bound C2H3 group correlation times suggests that the observed linewidths are determined by the complex size, and that both [2H]GAGs have similar motions when bound to LDL. The 31P-NMR data demonstrate that large LDL-HEP complexes (diameter approx. 50 nm) are formed only over a narrow range of HEP concentrations, whereas the size of LDL-CS complexes increases continuously over the range of CS concentrations studied, reaching values of 32-35 nm for both C4S and C6S. At the lower protein concentrations studied by light scattering (less than or equal to 1 mg/ml), the same trends are observed, although the mean diameters are less than those estimated by 31P-NMR. Soluble complex formation was unaffected by the presence of 2 mM Ca2+. Dilution studies demonstrate that complex size varies with protein concentration. The binding of GAGs to LDL was also examined by HEP-CS competition studies. HEP has the higher affinity while no differences in binding could be detected between C4S and C6S.

Research paper thumbnail of Phosphorus-31 two-dimensional solid-state exchange NMR. Application to model membrane and biological systems

Biophysical Journal, 1991

Two-dimensional solid-state 31P NMR has been used to investigate the orientational exchange of ph... more Two-dimensional solid-state 31P NMR has been used to investigate the orientational exchange of phospholipids in gel and liquid-crystalline aqueous multilamellar dispersions and oriented multibilayers, and in biological membranes. In liquid-crystalline L. multilamellar dispersions, orientational exchange originates from the lateral diffusion of phospholipid molecules over the curved surface of the liposomes and is manifest by an increase in off-diagonal intensity, which correlates the 90 and 00 orientations of the membrane normal with respect to the magnetic field when the system is fully exchanged. Spectral simulations of the time evolution of exchange allowed determination of the correlation times Td for lateral diffusion. For DMPC and DPPC at comparable reduced temperatures, Td values of 44 and 8 ms were obtained, respectively. The nature and rate of exchange observed for POPE at 300C is similar to that of DMPC at the same temperature. The measured correlation times are consistent with diffusion rates obtained by FRAP for liposomes with radii in the 1 pum range. In the gel phase of DPPC (30°C), little orientational exchange is observed at mixing times up to 200 ms, demonstrating that the lateral diffusion is very slow. The correlation time for orientational exchange obtained from spectral simulations was-900 ms; thus, exchange in the gel state is at least two orders of magnitude slower than in the liquid-crystalline state. In the P,. (ripple) phase, at temperatures between 34 and 390C, significant exchange is observed for mixing times between 50 and 200 ms. Exchange is also observed in oriented samples of DPPC in the PW. phase for mixing times of 50 ms, but not for oriented liquid-crystalline samples for mixing times up to 100 ms. The exchange observed in the ripple phase could originate from rapid lateral diffusion of 'fast" diffusing phospholipid within defect structures, and/or from "slow" lateral diffusion of ordered phospholipid over the ripples. 2D experiments were also performed on pig erythrocyte ghosts and on intact pig spinal cord. Significant orientational exchange was observed with the erythrocyte ghosts at a mixing time of 200 ms, but almost no exchange was observed with the spinal cord at the same mixing time. Spectral simulations suggest Td values of-400 ms and 1.3 s for the erythrocyte ghosts and spinal cord at 30°C. The results demonstrate that exchange in the biological membranes is significantly slower than in the model membrane systems, which suggests that the cell surfaces are relatively "smooth," i.e., any local surface perturbations are either present in small number or have little effect on the mean orientation of the phospholipids with respect to the membrane normal.

Research paper thumbnail of Effect of calcium on the dynamic behavior of sialylglycerolipids and phospholipids in mixed model membranes. A deuterium and phosphorus-31 NMR study

Biochemistry, 1991

DTSL, a sialic acid bearing glyceroglycolipid, has been deuteriated at the C3 position of the sia... more DTSL, a sialic acid bearing glyceroglycolipid, has been deuteriated at the C3 position of the sialic acid headgroup and at the C3 position of the glycerol backbone. The glycolipid was studied as a neat dispersion and in multilamellar dispersions of DMPC (at a concentration of 5-10 mol 7% relative to phospholipid), using 2H and 31P NMR. The quadrupolar splittings, Au of the headgroup deuterons were found to differ in the neat and mixed dispersion, suggesting different leadgroup orientations in the two systems. In DTSL-DMPC liposomes, two quadrupolar splittings were observed, indicating that the axial and equatorial deuterons make different angles with respect to the axis of motional averaging. The splittings originating from the equatorial and axial deuterons were found to increase and decrease with increasing temperature, respectively, indicating a temperature-dependent change in average headgroup orientation. Longitudinal relaxation times, TIT, were found to be short (3-6 ms). The field dependence of TIZ suggests that more than one motion governs relaxation. At 30.7 MHz a T I Z minimum was observed at approximately 40 "C. At 46.1 MHz the TIZ values were longer and increased with temperature, demonstrating that the dominant rigid-body motions of the headgroup at this field are in the rapid motional regime (>lo8 s-l). DTSL labeled at the glycerol C3 position was studied in DMPC multilamellar dispersions. Whereas two quadrupolar splittings have been observed for other glycolipids labeled at this position, only a single Au was observed. This shows that the orientation of the C2-C3 segment of DTSL relative to the bilayer norma? differs from that of other glycolipids. T I Z values were short (3-7 ms) and increased with temperature, demonstrating that motion is in the rapid motional regime. Quadrupolar splittings and TIZ values were also obtained for the headgroup-labeled DTSL in the presence of 5 and 50 mM Ca2+. As the Ca2+ concentration was increased, the ratio of outer to inner quadrupolar splittings increased, suggesting a small change in headgroup orientation. From the longitudinal relaxation times the rate of the dominant headgroup motion(s) appeared to decrease. The DTSL-DMPC liposomes were also studied by 31P N M R and by two-dimensional solid-state 31P NMR, the latter technique giving information on the orientational exchange of phospholipid molecules. DTSL appeared to alter the headgroup orientation of DMPC and also to increase the rate of orientational exchange. The latter most likely reflects an increase in the rate of lateral diffusion of the phospholipid. Ca2+ was found to reverse both of these effects partially.

Research paper thumbnail of Lipid-lipid interactions in reconstituted high-density lipoproteins by deuterium nuclear magnetic resonance

Biochemistry, 1988

Lipid-lipid interactions between the core and monolayer have been studied by using reconstituted ... more Lipid-lipid interactions between the core and monolayer have been studied by using reconstituted high-density lipoproteins (rHDLs) composed of apoHDL, with either dipalmitoylphosphatidylcholine (DPPC) or egg phosphatidylcholine (egg PC) as the monolayer and either cholesteryl oleate (CO) or triolein (TO) as the core. The effect of the monolayer on the core was observed by deuterium nuclear magnetic resonance (2H NMR) studies of rHDLs containing the core component cholesteryl [ 18,18,18-*H3]oleate ([2H3]CO) or t i [ 16,16-2H2]oleoylglycerol (['Ha]To) surrounded by a monolayer of either DPPC or egg PC as a function of temperature. The reverse effect, that of the core on the monolayer, was examined by both 2H and 31P N M R studies of rHDLs containing [5,5-2H2]PC in the presence of CO or TO as a function of temperature. The 2H N M R line widths of [2H3]C0 and ['H6]T0 were considerably broader and showed a greater temperature dependence in rHDLs containing DPPC than in those containing egg PC. Similarly, the C-2H order parameters of [2H2]PC were higher and showed a greater temperature dependence in rHDLs containing C O than in those containing TO. In contrast, the 31P N M R line widths were identical for both [2H2]-PC/CO/apoHDL3 and [2H2]PC/TO/apoHDL3 at 25 and 6 "C, showing only a slight temperature de-'This work was supported by the British Columbia Heart Foundation.

Research paper thumbnail of Structure and motion of phospholipids in human plasma lipoproteins. A phosphorus-31 NMR study

Biochemistry, 1990

The structure and motion of phospholipids in human plasma lipoproteins have been studied by using... more The structure and motion of phospholipids in human plasma lipoproteins have been studied by using 31P N M R. Lateral diffusion coefficients, DT, obtained from the viscosity dependence of the 31P N M R line widths, were obtained for very low density lipoprotein (VLDL), low-density lipoprotein (LDL), highdensity lipoproteins (HDL2, HDL3), and egg PC/TO microemulsions at 25 OC, for VLDL at 40 "C, and for LDL at 45 OC. At 25 "C, the rate of lateral diffusion in LDL (DT = 1.4 X cm2/s) is an order of magnitude slower than in the HDLs (DT = 2 X cm2/s). At 45 "C, DT for LDL increases to 1.1 X IO-* cm2/s. In contrast, DT for VLDL increases only slightly going from 25 to 40 O C. The large increase in diffusion rate observed in LDL occurs over the same temperature range as the smectic to disordered phase transition of the core cholesteryl esters, and provides evidence for direct interactions between the monolayer and core. In order to prove the orientation and/or order of the phospholipid head-group, estimates of the residual chemical shift anistropy, Ac, have been obtained for all the lipoproteins and the microemulsions from the viscosity and field dependence of the 31P N M R line widths. For VLDL and LDL, the anisotropy is 47-50 ppm at 25 OC, in agreement with data from phospholipid bilayers. For the HDLs, however, significantly larger values of 69-75 ppm (HDL2) and >120 ppm (HDL3) were obtained. These results suggest differences in the orientation and/or ordering of the head-group in the HDLs. The dynamic behavior of the phosphate moiety in LDL and HDL3 has been obtained from the temperature dependence of the 31P spin-lattice relaxation rates. Values of the correlation time for phosphate group reorientation and the activation energy for the motion are nearly identical in LDL and HDL3 and are similar to values obtained for phospholipid bilayers. This argues against long-lived protein-lipid interactions being the source of either the slow diffusion in LDL or the altered head-group orientation in the HDLs.

Research paper thumbnail of Glycosphingolipids: deuterium NMR study of the influence of carbohydrate headgroup structure on ceramide acyl chain behavior in glycolipid-phospholipid bilayers

Biochemistry, 1991

Galactosyl-and glucosylceramide, globoside, and dihydrolactosylceramide, bearing [ 2,2-2H2] stear... more Galactosyl-and glucosylceramide, globoside, and dihydrolactosylceramide, bearing [ 2,2-2H2] stearic acid, have been studied at a concentration of 10 mol 76 in bilayers of dimyristoylphosphatidylcholine by 2H NMR. The quadrupolar splittings AvQ of the C2 deuterons were measured at several temperatures in the range of 30-60 OC. Spin-lattice relaxation times T I of C 2 deuterons were determined in the same temperature range for all lipids but globoside. T1 values at 30 and 50 OC were unexpectedly short (6-8 ms), indicating reduced mobility of the ceramide acyl chains compared to that of the host phospholipid. At all temperatures, both AvQ and T1 were essentially identical for the monoglycosylated species, GalCer and GlcCer, indicating that the order and dynamics of the upper portion of the fatty acyl chain are insensitive to this small change in the headgroup structure. In the case of globoside, where the glycolipid headgroup is equivalent to that of GlcCer extended by three sugar residues, values for the quadrupolar splittings associated with the acyl chain C2-position were very close to those obtained for Gal-and GlcCer. In contrast, the AuQ values obtained for the diglycosyl species, LacCer, were significantly different at all temperatures. This different behavior of LacCer relative to that of the other glycolipids most likely originates from an orientational change of the acyl chain at the CZposition due to the absence of a 4 5 double bond in dihydrosphingosine. T , values for the GlcCer and GalCer systems increased with temperature, indicating that the motions responsible for relaxation were in the short correlation time regime. T1 for deuterons a t the acyl chain C2-position of LacCer was observed to decrease with increasing temperature, indicating that the motion(s) dominating relaxation are in the long correlation time regime. Thus the mobility of the acyl chain at the 2-position is reduced in the LacCer with respect to GlcCer and GalCer.

Research paper thumbnail of Effect of unsaturated phosphatidylethanolamine on the chain order profile of bilayers at the onset of the hexagonal phase transition. A deuterium NMR study

Biochemistry, 1990

The quadrupolar splitting profiles of methylene groups along the acyl chains of perdeuteriated di... more The quadrupolar splitting profiles of methylene groups along the acyl chains of perdeuteriated dimyristoylphosphatidylcholine (DMPC-dS4) in mixtures with dioleoylphosphatidylethanolamine (DOPE) were studied by 2H N M R. The quadrupolar splittings, obtained for lipid mixtures in the bilayer state, were measured as functions of temperature and PE:PC ratio and were used to obtain the approximate gauche probabilities a t a given chain position, pB. Ratios (R) ofpB for C13, C12, and C1 1 relative to that of the plateau region were used to characterize the effect of increasing P E on the gauche content of PC chains. At all temperatures studied (including the bilayer to hexagonal phase transition region), for each ratio R (e.g., RC13,P), the relative gauche content of the D M P C chains was similar over the range of 2 5 4 5 % PE. DOPE is viewed in simple terms as having a "conical" shape; if this geometry applies to the acyl chain region of the molecule, a greater lateral pressure would be expected toward the center of the bilayer as the P E content is increased, resulting in a decreased gauche content, relative to the plateau, of those methylene groups of PC. The failure to observe the predicted increase in lateral pressure has ramifications for the cone-shape molecular model. The overall "cone shape" of P E is seen to arise from the smaller size of the head-group relative to the acyl chains; however, the acyl chain region itself is not rigidly cone-shaped and is better represented by a flexible ''balloon''. These results were supported by small-angle X-ray diffraction, which showed a decreasing trend in the area per molecule with increasing P E content.

Research paper thumbnail of Surface diffusion in human serum lipoproteins

Biochemical and Biophysical Research Communications, 1987

From the viscosity dependence of the 31P RMR signals, the diffusion coefficients DT of phospholip... more From the viscosity dependence of the 31P RMR signals, the diffusion coefficients DT of phospholipid molecules in the surface monolayer of HDL, LDL and VLDL have been determined. DT for HDL, and HDL, are found to be 2.3~10-~ cm>/5 and 1.8~10-~ cm'/s, respectively. These values are similar to values reported for diffusion of phosphoiipid molecules in phospholipid bilayers above the gel to liquid crystalline phase transition temperature. Viscosity dependence of [16,16,16-ZH,]phosphatidylcholine incorporated into HDL, yielded a value similar to that determined by 31P (D 3 = 1.9x1O-8 cm2/s). Slower diffusion coefficients were measured for LDL, an VLDL. VLDL had a value DT = 9.1x10-' cm2/s. The diffusion coefficient for LDL, was 1.4~10-~ cml/s. Thus, diffusion of phospholipids in LDL, is a full order of magnitude slower at 25OC than diffusion of phospholipids in the HDLs. B

Research paper thumbnail of Deuterium magnetic resonance study of the interaction between chondroitin sulfate and human plasma low density lipoproteins

Biochemical and Biophysical Research Communications, 1984

Research paper thumbnail of Glycosphingolipid interdigitation in phospholipid bilayers examined by deuterium NMR and EPR

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990

Glycosphingolipid fatty acids commonly have up to eight methylene carbons more than do their surr... more Glycosphingolipid fatty acids commonly have up to eight methylene carbons more than do their surrounding phospholipid-attached counterparts. The resultant 'extra' segment may very well modulate glycosphingolipid function as receptor and structural element. As part of an investigation of this phenomenon, galactosylceramide was prepared with a deuterated 18-carbon fatty acid chain. Deuterium-labelled galactosylceramide was assembled at 10 tool% into unsonlcated phosphatidylcholine bilayers having all 14-carbon or all 18-carbon saturated fatty acid chains (DMPC and DSPC, respectively). The systems were studied by ZH-NMR spectroscopy above and below the phase transition temperatures, Tin, of the host matrices. At comparable reduced temperatures in fluid membranes the degree of motional order exhibited by the glycolipid fatty acid was significantly higher in the phospholipid host matrix that was four carbons shorter. The fatty acid chain segment least affected by the change from long to short chain host matrix was the terminal (deutero)methyl group (an increase of 8% in quadrupolar splitting for the terminal methyl vs. 16% for deuterons at C tT and 23-28% for the remainder of the chain). Order parameter profiles for galactosylceramide were qualitatively very similar in the two host membranes, arguing against any major conformational difference between the arrangement of the 18-carbon glycolipid fatty acid in the 18-carbon vs. 14-carbon host matrices. Similarly a nitroxide spin probe covalently attached to carbon-12 of the galactosylceramide fatty acid gave clear indication of greater order in the fluid 14-carbon fatty acid phospholipid bilayer. These results are consistent with 'tethering' of the extra length of fatty acid via interdigitation into the opposing monolayer. There was no spectroscopic evidence of any intrinsic difference in glycolipid behaviour in the two fluid host matrices. 2H-NMR specWa of galactosylceramide at comparable reduced temperatures below T m of the phospholipid bilayer were very different for 14-carbon vs. 18-carbon host matrices. The glycolipid fatty acid showed evidence of relatively reduced mobility in the shorter chain matrix.

Research paper thumbnail of Effects of fatty acid alpha-hydroxylation on glycosphingolipid properties in phosphatidylcholine bilayers

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1992

The role of glycosphingolipid fatty ~-~d a-hyd.,'oxylation as a moduli:*er of glycolipJd organiza... more The role of glycosphingolipid fatty ~-~d a-hyd.,'oxylation as a moduli:*er of glycolipJd organization and dynamics was considered by 2H-NMR in bilayer tr, embranes. For these experiments, galactosylceramides were prepared in which the notural fatty, acid mixture was ~placed with perdeuterated 18-earhen hydroxylated or non-hydroxylated stzaric acid. The L-stereoisomer of N-(a-O.q-stearoyl-d~4)gnlactosylceramide and its naterally-accureing t~a-Otl analogue, were isolated for independent study. Bilayers were formed using 10 tool% gnlactosylceramide in n shorter chain phospholi#d, dim.vristoyiphosphatidylcholine, in an attempt to reproduce several features of glyculipid-phospholipid interactions typical of c¢il membranes. Spectra of deuterated gnlactosylceramide in gel pha~ phospholipid membranes indicated Chat a-hydr~xyla*..~.-. !ed to greater motional freedom and/or conformational di~rder, .~.lh no measurable difference hetwcen n-and L-a-OH fatty a:,M derivatives, in fluid phosphatidylcholine bilayer~ the effects were modest Glycolipid fatty acid hydroxylation led to broadening of the range of order pnramett.,s assueiated with ra~zhy|cne groups near the membrane surface (frequently referred to as the 'plateau re#n#)-this ergo: being .'ue~ mad,-ed for the naturally.occurring (n) slt,reoisomer. The dctWee of overall molectdar order sensed by the glycolipid fatty acid chain in a fluid host matrix was minil:.mlly affecled by ¢~bydroxylation; although the plateau region of the n isomer was slightly i~ore ordered than that ef the t. isomer and the non-bydroxylated species. These results suggest that a significant aspect of the a-hydrexy grou.9 effect on glycosphingolipid 6ehavlour in bilayer membranes with levy glyeolipid content was interference with glycolipid packing amongst host phospholipids in the upper portion of the acyl cimins. For the u ster,'elsomer, there was some evidence titat the hydroxy group led to strengthening of interlipid interr .'tion near the membrane surface.

Research paper thumbnail of Medical Applications of Lipid Nanoparticles

From Basic Research to Application, 2014

Research paper thumbnail of Deuterium and phosphorus-31 nuclear magnetic resonance investigations of human plasma low- and high- density lipoproteins

Research paper thumbnail of Ionophore-mediated liposome loading

Research paper thumbnail of Regioselectively and stereoselectively deuteriated sialyl glycerolipids for dynamic studies by 2 H-NMR spectroscopy

Research paper thumbnail of Entrapment of small molecules and nucleic acid-based drugs in liposomes

Methods in enzymology, 2005

In the past two decades there have been major advances in the development of liposomal drug deliv... more In the past two decades there have been major advances in the development of liposomal drug delivery systems suitable for applications ranging from cancer chemotherapy to gene therapy. In general, an optimized system consists of liposomes with a diameter of approximately 100 nm that possess a long circulation lifetime (half-life >5 h). Such liposomes will circulate sufficiently long to take advantage of a phenomenon known as disease site targeting, wherein liposomes accumulate at sites of disease, such as tumors, as a result of the leaky vasculature and reduced blood flow exhibited by the diseased tissue. The extended circulation lifetime is achieved by the use of saturated lipids and cholesterol or by the presence of PEG-containing lipids. This chapter will focus on the methodology required for the generation of two very different classes of liposomal carrier systems: those containing conventional small molecular weight (usually anticancer) drugs and those containing larger gene...

Research paper thumbnail of Regio- and stereo-selectively deuteriated sialyl glycerolipids for dynamic studies by 2H NMR spectroscopy

Journal of the Chemical Society, Chemical Communications, 1990

ABSTRACT

Research paper thumbnail of Glycosphingolipids: sup 2 H NMR study of the influence of carbohydrate headgroup structure on ceramide acyl chain behavior in glycolipid-phospholipid bilayers

Biochemistry, 1991

Galactosyl- and glucosylceramide, globoside, and dihydrolactosylceramide, bearing (2,2-²Hâ)stear... more Galactosyl- and glucosylceramide, globoside, and dihydrolactosylceramide, bearing (2,2-²Hâ)stearic acid, have been studied at a concentration of 10 mol % in bilayers of dimyristoylphosphatidylcholine by ²H NMR. The quadrupolar splittings Îv{sub Q} of the C2 deuterons were measured at several temperatures in the range of 30-60°C. Spin-lattice relaxation times T⁠of C2 deuterons were determined in the same temperature range for

Research paper thumbnail of Structural and motional properties of vesicles as revealed by nuclear magnetic resonance

Chemistry and Physics of Lipids, 1993

Research paper thumbnail of Insoluble complex formation between low density lipoprotein and heparin. A 31P-NMR study

Chemistry and Physics of Lipids, 1990

31P-NMR has been used to probe the motions of the phosphate moiety of phospholipid head-groups in... more 31P-NMR has been used to probe the motions of the phosphate moiety of phospholipid head-groups in samples of human low density lipoprotein (LDL) in which particle tumbling has been greatly reduced by increasing the viscosity of the medium, by forming an LDL gel by ultracentrifugation, or by precipitation with heparin. The 31P-NMR spectra of LDL gel give broad "powder-like" lineshapes, with the sign and magnitude of the anisotropy characteristic of the bilayer mesophase, which narrow as the temperature is raised from 5 to 45 degrees C. This narrowing occurs over the same temperature range as the core cholesteryl ester liquid-crystalline to liquid phase transition, suggesting interactions between the surface and core. The 31P lineshapes of LDL-heparin insoluble complexes are also "powder-like", but are broader than native LDL at all temperatures studied. The spectra were simulated assuming an axially-symmetric shielding tensor motionally narrowed by Brownian isotropic diffusion [Burnell et al. (1980) Biochim. Biophys. Acta 603, 63-69], allowing determination of the lateral diffusion coefficients, DT, and the chemical shift anisotropy, delta sigma, of the monolayer phospholipids. Relative to LDL gel, the temperature-dependence of DT was reduced in the LDL-heparin insoluble complexes, and delta sigma was increased from 50 to 60 ppm. The results suggest that insoluble complex formation slows phospholipid lateral diffusion in the LDL monolayer and alters the orientation and/or order of the head-group.

Research paper thumbnail of Soluble complex formation between low-density lipoprotein and glycosaminoglycans. A 2H and 31P-NMR, and quasi-elastic light scattering study

Chemistry and Physics of Lipids, 1988

Soluble complex formation between LDL and heparin (HEP) and chondroitin sulfate (CS) has been stu... more Soluble complex formation between LDL and heparin (HEP) and chondroitin sulfate (CS) has been studied by 2H- and 31P-NMR and light scattering. The 2H-NMR linewidths of [2H]HEP and [2H]C4S increase substantially upon binding to LDL, with the [2H]HEP linewidths broader at low glycosaminoglycan (GAG)/low density lipoprotein (LDL) ratios. Preliminary analysis of the bound C2H3 group correlation times suggests that the observed linewidths are determined by the complex size, and that both [2H]GAGs have similar motions when bound to LDL. The 31P-NMR data demonstrate that large LDL-HEP complexes (diameter approx. 50 nm) are formed only over a narrow range of HEP concentrations, whereas the size of LDL-CS complexes increases continuously over the range of CS concentrations studied, reaching values of 32-35 nm for both C4S and C6S. At the lower protein concentrations studied by light scattering (less than or equal to 1 mg/ml), the same trends are observed, although the mean diameters are less than those estimated by 31P-NMR. Soluble complex formation was unaffected by the presence of 2 mM Ca2+. Dilution studies demonstrate that complex size varies with protein concentration. The binding of GAGs to LDL was also examined by HEP-CS competition studies. HEP has the higher affinity while no differences in binding could be detected between C4S and C6S.

Research paper thumbnail of Phosphorus-31 two-dimensional solid-state exchange NMR. Application to model membrane and biological systems

Biophysical Journal, 1991

Two-dimensional solid-state 31P NMR has been used to investigate the orientational exchange of ph... more Two-dimensional solid-state 31P NMR has been used to investigate the orientational exchange of phospholipids in gel and liquid-crystalline aqueous multilamellar dispersions and oriented multibilayers, and in biological membranes. In liquid-crystalline L. multilamellar dispersions, orientational exchange originates from the lateral diffusion of phospholipid molecules over the curved surface of the liposomes and is manifest by an increase in off-diagonal intensity, which correlates the 90 and 00 orientations of the membrane normal with respect to the magnetic field when the system is fully exchanged. Spectral simulations of the time evolution of exchange allowed determination of the correlation times Td for lateral diffusion. For DMPC and DPPC at comparable reduced temperatures, Td values of 44 and 8 ms were obtained, respectively. The nature and rate of exchange observed for POPE at 300C is similar to that of DMPC at the same temperature. The measured correlation times are consistent with diffusion rates obtained by FRAP for liposomes with radii in the 1 pum range. In the gel phase of DPPC (30°C), little orientational exchange is observed at mixing times up to 200 ms, demonstrating that the lateral diffusion is very slow. The correlation time for orientational exchange obtained from spectral simulations was-900 ms; thus, exchange in the gel state is at least two orders of magnitude slower than in the liquid-crystalline state. In the P,. (ripple) phase, at temperatures between 34 and 390C, significant exchange is observed for mixing times between 50 and 200 ms. Exchange is also observed in oriented samples of DPPC in the PW. phase for mixing times of 50 ms, but not for oriented liquid-crystalline samples for mixing times up to 100 ms. The exchange observed in the ripple phase could originate from rapid lateral diffusion of 'fast" diffusing phospholipid within defect structures, and/or from "slow" lateral diffusion of ordered phospholipid over the ripples. 2D experiments were also performed on pig erythrocyte ghosts and on intact pig spinal cord. Significant orientational exchange was observed with the erythrocyte ghosts at a mixing time of 200 ms, but almost no exchange was observed with the spinal cord at the same mixing time. Spectral simulations suggest Td values of-400 ms and 1.3 s for the erythrocyte ghosts and spinal cord at 30°C. The results demonstrate that exchange in the biological membranes is significantly slower than in the model membrane systems, which suggests that the cell surfaces are relatively "smooth," i.e., any local surface perturbations are either present in small number or have little effect on the mean orientation of the phospholipids with respect to the membrane normal.

Research paper thumbnail of Effect of calcium on the dynamic behavior of sialylglycerolipids and phospholipids in mixed model membranes. A deuterium and phosphorus-31 NMR study

Biochemistry, 1991

DTSL, a sialic acid bearing glyceroglycolipid, has been deuteriated at the C3 position of the sia... more DTSL, a sialic acid bearing glyceroglycolipid, has been deuteriated at the C3 position of the sialic acid headgroup and at the C3 position of the glycerol backbone. The glycolipid was studied as a neat dispersion and in multilamellar dispersions of DMPC (at a concentration of 5-10 mol 7% relative to phospholipid), using 2H and 31P NMR. The quadrupolar splittings, Au of the headgroup deuterons were found to differ in the neat and mixed dispersion, suggesting different leadgroup orientations in the two systems. In DTSL-DMPC liposomes, two quadrupolar splittings were observed, indicating that the axial and equatorial deuterons make different angles with respect to the axis of motional averaging. The splittings originating from the equatorial and axial deuterons were found to increase and decrease with increasing temperature, respectively, indicating a temperature-dependent change in average headgroup orientation. Longitudinal relaxation times, TIT, were found to be short (3-6 ms). The field dependence of TIZ suggests that more than one motion governs relaxation. At 30.7 MHz a T I Z minimum was observed at approximately 40 "C. At 46.1 MHz the TIZ values were longer and increased with temperature, demonstrating that the dominant rigid-body motions of the headgroup at this field are in the rapid motional regime (>lo8 s-l). DTSL labeled at the glycerol C3 position was studied in DMPC multilamellar dispersions. Whereas two quadrupolar splittings have been observed for other glycolipids labeled at this position, only a single Au was observed. This shows that the orientation of the C2-C3 segment of DTSL relative to the bilayer norma? differs from that of other glycolipids. T I Z values were short (3-7 ms) and increased with temperature, demonstrating that motion is in the rapid motional regime. Quadrupolar splittings and TIZ values were also obtained for the headgroup-labeled DTSL in the presence of 5 and 50 mM Ca2+. As the Ca2+ concentration was increased, the ratio of outer to inner quadrupolar splittings increased, suggesting a small change in headgroup orientation. From the longitudinal relaxation times the rate of the dominant headgroup motion(s) appeared to decrease. The DTSL-DMPC liposomes were also studied by 31P N M R and by two-dimensional solid-state 31P NMR, the latter technique giving information on the orientational exchange of phospholipid molecules. DTSL appeared to alter the headgroup orientation of DMPC and also to increase the rate of orientational exchange. The latter most likely reflects an increase in the rate of lateral diffusion of the phospholipid. Ca2+ was found to reverse both of these effects partially.

Research paper thumbnail of Lipid-lipid interactions in reconstituted high-density lipoproteins by deuterium nuclear magnetic resonance

Biochemistry, 1988

Lipid-lipid interactions between the core and monolayer have been studied by using reconstituted ... more Lipid-lipid interactions between the core and monolayer have been studied by using reconstituted high-density lipoproteins (rHDLs) composed of apoHDL, with either dipalmitoylphosphatidylcholine (DPPC) or egg phosphatidylcholine (egg PC) as the monolayer and either cholesteryl oleate (CO) or triolein (TO) as the core. The effect of the monolayer on the core was observed by deuterium nuclear magnetic resonance (2H NMR) studies of rHDLs containing the core component cholesteryl [ 18,18,18-*H3]oleate ([2H3]CO) or t i [ 16,16-2H2]oleoylglycerol (['Ha]To) surrounded by a monolayer of either DPPC or egg PC as a function of temperature. The reverse effect, that of the core on the monolayer, was examined by both 2H and 31P N M R studies of rHDLs containing [5,5-2H2]PC in the presence of CO or TO as a function of temperature. The 2H N M R line widths of [2H3]C0 and ['H6]T0 were considerably broader and showed a greater temperature dependence in rHDLs containing DPPC than in those containing egg PC. Similarly, the C-2H order parameters of [2H2]PC were higher and showed a greater temperature dependence in rHDLs containing C O than in those containing TO. In contrast, the 31P N M R line widths were identical for both [2H2]-PC/CO/apoHDL3 and [2H2]PC/TO/apoHDL3 at 25 and 6 "C, showing only a slight temperature de-'This work was supported by the British Columbia Heart Foundation.

Research paper thumbnail of Structure and motion of phospholipids in human plasma lipoproteins. A phosphorus-31 NMR study

Biochemistry, 1990

The structure and motion of phospholipids in human plasma lipoproteins have been studied by using... more The structure and motion of phospholipids in human plasma lipoproteins have been studied by using 31P N M R. Lateral diffusion coefficients, DT, obtained from the viscosity dependence of the 31P N M R line widths, were obtained for very low density lipoprotein (VLDL), low-density lipoprotein (LDL), highdensity lipoproteins (HDL2, HDL3), and egg PC/TO microemulsions at 25 OC, for VLDL at 40 "C, and for LDL at 45 OC. At 25 "C, the rate of lateral diffusion in LDL (DT = 1.4 X cm2/s) is an order of magnitude slower than in the HDLs (DT = 2 X cm2/s). At 45 "C, DT for LDL increases to 1.1 X IO-* cm2/s. In contrast, DT for VLDL increases only slightly going from 25 to 40 O C. The large increase in diffusion rate observed in LDL occurs over the same temperature range as the smectic to disordered phase transition of the core cholesteryl esters, and provides evidence for direct interactions between the monolayer and core. In order to prove the orientation and/or order of the phospholipid head-group, estimates of the residual chemical shift anistropy, Ac, have been obtained for all the lipoproteins and the microemulsions from the viscosity and field dependence of the 31P N M R line widths. For VLDL and LDL, the anisotropy is 47-50 ppm at 25 OC, in agreement with data from phospholipid bilayers. For the HDLs, however, significantly larger values of 69-75 ppm (HDL2) and >120 ppm (HDL3) were obtained. These results suggest differences in the orientation and/or ordering of the head-group in the HDLs. The dynamic behavior of the phosphate moiety in LDL and HDL3 has been obtained from the temperature dependence of the 31P spin-lattice relaxation rates. Values of the correlation time for phosphate group reorientation and the activation energy for the motion are nearly identical in LDL and HDL3 and are similar to values obtained for phospholipid bilayers. This argues against long-lived protein-lipid interactions being the source of either the slow diffusion in LDL or the altered head-group orientation in the HDLs.

Research paper thumbnail of Glycosphingolipids: deuterium NMR study of the influence of carbohydrate headgroup structure on ceramide acyl chain behavior in glycolipid-phospholipid bilayers

Biochemistry, 1991

Galactosyl-and glucosylceramide, globoside, and dihydrolactosylceramide, bearing [ 2,2-2H2] stear... more Galactosyl-and glucosylceramide, globoside, and dihydrolactosylceramide, bearing [ 2,2-2H2] stearic acid, have been studied at a concentration of 10 mol 76 in bilayers of dimyristoylphosphatidylcholine by 2H NMR. The quadrupolar splittings AvQ of the C2 deuterons were measured at several temperatures in the range of 30-60 OC. Spin-lattice relaxation times T I of C 2 deuterons were determined in the same temperature range for all lipids but globoside. T1 values at 30 and 50 OC were unexpectedly short (6-8 ms), indicating reduced mobility of the ceramide acyl chains compared to that of the host phospholipid. At all temperatures, both AvQ and T1 were essentially identical for the monoglycosylated species, GalCer and GlcCer, indicating that the order and dynamics of the upper portion of the fatty acyl chain are insensitive to this small change in the headgroup structure. In the case of globoside, where the glycolipid headgroup is equivalent to that of GlcCer extended by three sugar residues, values for the quadrupolar splittings associated with the acyl chain C2-position were very close to those obtained for Gal-and GlcCer. In contrast, the AuQ values obtained for the diglycosyl species, LacCer, were significantly different at all temperatures. This different behavior of LacCer relative to that of the other glycolipids most likely originates from an orientational change of the acyl chain at the CZposition due to the absence of a 4 5 double bond in dihydrosphingosine. T , values for the GlcCer and GalCer systems increased with temperature, indicating that the motions responsible for relaxation were in the short correlation time regime. T1 for deuterons a t the acyl chain C2-position of LacCer was observed to decrease with increasing temperature, indicating that the motion(s) dominating relaxation are in the long correlation time regime. Thus the mobility of the acyl chain at the 2-position is reduced in the LacCer with respect to GlcCer and GalCer.

Research paper thumbnail of Effect of unsaturated phosphatidylethanolamine on the chain order profile of bilayers at the onset of the hexagonal phase transition. A deuterium NMR study

Biochemistry, 1990

The quadrupolar splitting profiles of methylene groups along the acyl chains of perdeuteriated di... more The quadrupolar splitting profiles of methylene groups along the acyl chains of perdeuteriated dimyristoylphosphatidylcholine (DMPC-dS4) in mixtures with dioleoylphosphatidylethanolamine (DOPE) were studied by 2H N M R. The quadrupolar splittings, obtained for lipid mixtures in the bilayer state, were measured as functions of temperature and PE:PC ratio and were used to obtain the approximate gauche probabilities a t a given chain position, pB. Ratios (R) ofpB for C13, C12, and C1 1 relative to that of the plateau region were used to characterize the effect of increasing P E on the gauche content of PC chains. At all temperatures studied (including the bilayer to hexagonal phase transition region), for each ratio R (e.g., RC13,P), the relative gauche content of the D M P C chains was similar over the range of 2 5 4 5 % PE. DOPE is viewed in simple terms as having a "conical" shape; if this geometry applies to the acyl chain region of the molecule, a greater lateral pressure would be expected toward the center of the bilayer as the P E content is increased, resulting in a decreased gauche content, relative to the plateau, of those methylene groups of PC. The failure to observe the predicted increase in lateral pressure has ramifications for the cone-shape molecular model. The overall "cone shape" of P E is seen to arise from the smaller size of the head-group relative to the acyl chains; however, the acyl chain region itself is not rigidly cone-shaped and is better represented by a flexible ''balloon''. These results were supported by small-angle X-ray diffraction, which showed a decreasing trend in the area per molecule with increasing P E content.

Research paper thumbnail of Surface diffusion in human serum lipoproteins

Biochemical and Biophysical Research Communications, 1987

From the viscosity dependence of the 31P RMR signals, the diffusion coefficients DT of phospholip... more From the viscosity dependence of the 31P RMR signals, the diffusion coefficients DT of phospholipid molecules in the surface monolayer of HDL, LDL and VLDL have been determined. DT for HDL, and HDL, are found to be 2.3~10-~ cm>/5 and 1.8~10-~ cm'/s, respectively. These values are similar to values reported for diffusion of phosphoiipid molecules in phospholipid bilayers above the gel to liquid crystalline phase transition temperature. Viscosity dependence of [16,16,16-ZH,]phosphatidylcholine incorporated into HDL, yielded a value similar to that determined by 31P (D 3 = 1.9x1O-8 cm2/s). Slower diffusion coefficients were measured for LDL, an VLDL. VLDL had a value DT = 9.1x10-' cm2/s. The diffusion coefficient for LDL, was 1.4~10-~ cml/s. Thus, diffusion of phospholipids in LDL, is a full order of magnitude slower at 25OC than diffusion of phospholipids in the HDLs. B

Research paper thumbnail of Deuterium magnetic resonance study of the interaction between chondroitin sulfate and human plasma low density lipoproteins

Biochemical and Biophysical Research Communications, 1984

Research paper thumbnail of Glycosphingolipid interdigitation in phospholipid bilayers examined by deuterium NMR and EPR

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990

Glycosphingolipid fatty acids commonly have up to eight methylene carbons more than do their surr... more Glycosphingolipid fatty acids commonly have up to eight methylene carbons more than do their surrounding phospholipid-attached counterparts. The resultant 'extra' segment may very well modulate glycosphingolipid function as receptor and structural element. As part of an investigation of this phenomenon, galactosylceramide was prepared with a deuterated 18-carbon fatty acid chain. Deuterium-labelled galactosylceramide was assembled at 10 tool% into unsonlcated phosphatidylcholine bilayers having all 14-carbon or all 18-carbon saturated fatty acid chains (DMPC and DSPC, respectively). The systems were studied by ZH-NMR spectroscopy above and below the phase transition temperatures, Tin, of the host matrices. At comparable reduced temperatures in fluid membranes the degree of motional order exhibited by the glycolipid fatty acid was significantly higher in the phospholipid host matrix that was four carbons shorter. The fatty acid chain segment least affected by the change from long to short chain host matrix was the terminal (deutero)methyl group (an increase of 8% in quadrupolar splitting for the terminal methyl vs. 16% for deuterons at C tT and 23-28% for the remainder of the chain). Order parameter profiles for galactosylceramide were qualitatively very similar in the two host membranes, arguing against any major conformational difference between the arrangement of the 18-carbon glycolipid fatty acid in the 18-carbon vs. 14-carbon host matrices. Similarly a nitroxide spin probe covalently attached to carbon-12 of the galactosylceramide fatty acid gave clear indication of greater order in the fluid 14-carbon fatty acid phospholipid bilayer. These results are consistent with 'tethering' of the extra length of fatty acid via interdigitation into the opposing monolayer. There was no spectroscopic evidence of any intrinsic difference in glycolipid behaviour in the two fluid host matrices. 2H-NMR specWa of galactosylceramide at comparable reduced temperatures below T m of the phospholipid bilayer were very different for 14-carbon vs. 18-carbon host matrices. The glycolipid fatty acid showed evidence of relatively reduced mobility in the shorter chain matrix.

Research paper thumbnail of Effects of fatty acid alpha-hydroxylation on glycosphingolipid properties in phosphatidylcholine bilayers

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1992

The role of glycosphingolipid fatty ~-~d a-hyd.,'oxylation as a moduli:*er of glycolipJd organiza... more The role of glycosphingolipid fatty ~-~d a-hyd.,'oxylation as a moduli:*er of glycolipJd organization and dynamics was considered by 2H-NMR in bilayer tr, embranes. For these experiments, galactosylceramides were prepared in which the notural fatty, acid mixture was ~placed with perdeuterated 18-earhen hydroxylated or non-hydroxylated stzaric acid. The L-stereoisomer of N-(a-O.q-stearoyl-d~4)gnlactosylceramide and its naterally-accureing t~a-Otl analogue, were isolated for independent study. Bilayers were formed using 10 tool% gnlactosylceramide in n shorter chain phospholi#d, dim.vristoyiphosphatidylcholine, in an attempt to reproduce several features of glyculipid-phospholipid interactions typical of c¢il membranes. Spectra of deuterated gnlactosylceramide in gel pha~ phospholipid membranes indicated Chat a-hydr~xyla*..~.-. !ed to greater motional freedom and/or conformational di~rder, .~.lh no measurable difference hetwcen n-and L-a-OH fatty a:,M derivatives, in fluid phosphatidylcholine bilayer~ the effects were modest Glycolipid fatty acid hydroxylation led to broadening of the range of order pnramett.,s assueiated with ra~zhy|cne groups near the membrane surface (frequently referred to as the 'plateau re#n#)-this ergo: being .'ue~ mad,-ed for the naturally.occurring (n) slt,reoisomer. The dctWee of overall molectdar order sensed by the glycolipid fatty acid chain in a fluid host matrix was minil:.mlly affecled by ¢~bydroxylation; although the plateau region of the n isomer was slightly i~ore ordered than that ef the t. isomer and the non-bydroxylated species. These results suggest that a significant aspect of the a-hydrexy grou.9 effect on glycosphingolipid 6ehavlour in bilayer membranes with levy glyeolipid content was interference with glycolipid packing amongst host phospholipids in the upper portion of the acyl cimins. For the u ster,'elsomer, there was some evidence titat the hydroxy group led to strengthening of interlipid interr .'tion near the membrane surface.

Research paper thumbnail of Medical Applications of Lipid Nanoparticles

From Basic Research to Application, 2014