Effect of lamellarity and size on calorimetric phase transitions in single component phosphatidylcholine vesicles.

Nano-differential scanning calorimetry (nano-DSC) is a powerful tool in the investigation of unilamellar (small unilamellar, SUVs, or large unilamellar, LUVs) vesicles, as well as lipids on supported bilayers, since it measures the main gel-to-liquid phase transition temperature (Tm), enthalpies and entropies. In order to assign these transitions in single component systems, where Tm often occurred as a doublet, nano-DSC, dynamic light scattering and cryo-transmission electron microscopy (cryo-TEM) data were compared. The two Tms were not attributable to decoupled phase transitions between the two leaflets of the bilayer, i.e. nano-DSC measurements were not able to distinguish between the outer and inner leaflets of the vesicle bilayers. Instead, the two Tms were attributed to mixtures of oligolamellar and unilamellar vesicles, as confirmed by cryo-TEM images. Tm for the oligolamellar vesicles was assigned to the peak closest to that of the parent multilamellar vesicle (MLV) peak. The other transition was higher than that of the parent MLVs for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and increased in temperature as the vesicle size decreased, while it was lower in temperature than that of the parent MLVs for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and decreased as the vesicle size decreased. These subtle shifts arose due to small differences in the values of ΔH and ΔS, since Tm is determined by their ratio (ΔH/ΔS). It was not possible to completely eliminate oligolamellar structures for MLVs extruded with the 200 nm pore size filter, even after 120 passes, while these structures were eliminated for MLVs extruded through the 50 nm pore size filter.

Lipid exchange and transfer on nanoparticle supported lipid bilayers: effect of defects, ionic strength, and size.

Lipid exchange/transfer has been compared for zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (DMPC) small unilamellar vesicles (SUVs) and for the same lipids on silica (SiO2) nanoparticle supported lipid bilayers (NP-SLBs) as a function of ionic strength, temperature, temperature cycling, and NP size, above the main gel-to-liquid crystal phase transition temperature, Tm, using d- and h-DMPC and DPPC. Increasing ionic strength decreases the exchange kinetics for the SUVs, but more so for the NP-SLBs, due to better packing of the lipids and increased attraction between the lipid and support. When the NP-SLBs (or SUVs) are cycled above and below Tm, the exchange rate increases compared with exchange at the same temperature without cycling, for similar total times, suggesting that defects provide sites for more facile removal and thus exchange of lipids. Defects can occur: (i) at the phase boundaries between coexisting gel and fluid phases at Tm; (ii) in bare regions of exposed SiO2 that form during NP-SLB formation due to mismatched surface areas of lipid and NPs; and (iii) during cycling as the result of changes in area of the lipids at Tm and mismatched thermal expansion coefficient between the lipids and SiO2 support. Exchange rates are faster for NP-SLBs prepared with the nominal amount of lipid required to form a NP-SLB compared with NP-SLBs that have been prepared with excess lipids to minimize SiO2 patches. Nanosystems prepared with equimolar mixtures of NP-SLBs composed of d-DMPC (d(DMPC)-NP-SLB) and SUVs composed of h-DMPC (h(DMPC)-SUV) show that the calorimetric transition of the "donor" h(DMPC)-SUV decreases in intensity without an initial shift in Tm, indicating that the "acceptor" d(DMPC)-NP-SLB can accommodate more lipids, through either further fusion or insertion of lipids into the distal monolayer. Exchange for d/h(DMPC)-NP-SLB is in the order 100 nm SiO2 > 45 nm SiO2 > 5 nm SiO2.

Formation of lipid sheaths around nanoparticle-supported lipid bilayers.

High-surface-area nanoparticles often cluster, with unknown effects on their cellular uptake and environmental impact. In the presence of vesicles or cell membranes, lipid adsorption can occur on the nanoparticles, resulting in the formation of supported lipid bilayers (SLBs), which tend to resist cellular uptake. When the amount of lipid available is in excess compared with that required to form a single-SLB, large aggregates of SLBs enclosed by a close-fitting lipid bilayer sheath are shown to form. The proposed mechanism for this process is one where small unilamellar vesicles (SUVs) adsorb to aggregates of SLBs just above the gel-to-liquid phase transition temperature, T(m) , of the lipids (as observed by dynamic light scattering), and then fuse with each other (rather than to the underlying SLBs) upon cooling below T(m) . The sacks of SLB nanoparticles that are formed are encapsulated by the contiguous close-fitting lipid sheath, and precipitate below T(m) , due to reduced hydration repulsion and the absence of undulation/protrusion forces for the lipids attached to the solid support. The single-SLBs can be released above T(m) , where these forces are restored by the free lipid vesicles. This mechanism may be useful for encapsulation/release of drugs/DNA, and has implications for the toxic effects of nanoparticles, which may be mitigated by lipid sequestration.

Effect of curvature on nanoparticle supported lipid bilayers investigated by Raman spectroscopy.

The packing of lipids on silica (SiO(2)) nanoparticles (NPs) was investigated by Raman spectroscopy for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) as a function of their size, for SiO(2) NPs of 5, 15, 25, 45, and 100 nm nominal diameter. Raman spectral indicators in the C-C and C-H stretching regions were used to determine conformational order and alkyl chain packing for these systems. As the ratio of NP to lipid size decreases, packing in a normal bilayer configuration increases free volume and decreases hydrophobic interaction between the chains. For the 15, 25, 45, and 100 nm SiO(2), for which single supported lipid bilayers (SLBs) are formed around the NPs, the Raman data indicate that there is increased interdigitation and increased lateral packing order between the chains with decreasing NP size, which improves hydrophobic association and decreases the voids that would occur for normal bilayers. For the same size NP, there is increased interdigitation and lateral packing for the DSPC compared with DPPC lipids, as expected based on the greater void volume that would be created for the longer alkyl chain lengths. Another mechanism for filling this void space is the formation of gauche kinks for the terminal methyl groups at the center of the bilayer, which can be monitored by a Raman band at 1122 cm(-1). These gauche defects are most prevalent for the largest size (100 nm) NPs but are observed for all NP sizes. For the 5 nm SLBs, which form aggregates, we hypothesize that bilayer bridging can occur between the NPs. Compared with the 15 nm NPs, the order parameter increases but there are fewer trans conformers, possibly due to chains that are loosely packed or isolated in the interstitial regions.

Stabilization of soft lipid colloids: competing effects of nanoparticle decoration and supported lipid bilayer formation.

Stabilization against fusion of zwitterionic lipid small unilamellar vesicles (SUVs) by charged nanoparticles is essential to prevent premature inactivation and cargo unloading. In the present work, we examined the stabilization of DMPC and DPPC SUVs by monolithic silica (SiO(2)) nanoparticle envelopment, for SiO(2) with 4-6, 10-20, 20-30, and 40-50 nm nominal diameter. We found that for these soft colloids stabilization is critically dependent on whether fusion occurs between the charged nanoparticles and neutral SUVs to form supported lipid bilayers (SLBs), or whether the reverse occurs, namely, nanoparticle decoration of the SUVs. While SLB formation is accompanied by precipitation, nanoparticle decoration results in long-term stabilization of the SUVs. The fate of the nanosystem depends on the size of the nanoparticles and on the ionic strength of the medium. We found that, in the case of highly charged SiO(2) nanoparticles in water, there is no SUV fusion to SiO(2) for a specific range of nanoparticle sizes. Instead, the negatively charged SiO(2) nanoparticles surround the uncharged SUVs, resulting in electrostatic repulsion between the decorated SUVs, thus preventing their aggregation and precipitation. Addition of millimolar amounts of NaCl results in rapid SLB formation and precipitation. This study has great potential impact toward better understanding the interaction of nanoparticles with biological membranes and the factors affecting their use as drug carriers or sensors.

Formation and colloidal stability of DMPC supported lipid bilayers on SiO2 nanobeads.

Supported lipid bilayers (SLBs) of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were formed on 20-100 nm silica (SiO(2)) nanobeads, and the formation was accompanied by an 8 nm increase in diameter of the SiO(2), consistent with single nanobeads surrounded by a DMPC bilayer. Complete SLBs were formed when the nominal surface areas of the DMPC matched that of the silica, SA(DMPC)/SA(SiO2) = 1, and required increasing ionic strength and time to form on smaller size nanobeads, as shown by a combination of nano-differential scanning calorimetry (nano-DSC), dynamic light scattering (DLS), and zeta potential (zeta) measurements. For 5 nm SiO(2), where the nanoparticle and DMPC dimensions were comparable, DMPC fused and formed SLBs on the nanobeads, but it did not form single bilayers around them. Instead, stable agglomerates of 150-1000 nm were formed over a wide surface ratio range (0.25 < or = SA(DMPC)/SA(SiO2) < 2) in 0.75 mM NaCl. At ionic strengths > 1 mM NaCl, charge shielding, as measured by zeta potential measurements (zeta --> 0), resulted in precipitation of the SLBs.

Effect of high surface curvature on the main phase transition of supported phospholipid bilayers on SiO2 nanoparticles.

Investigation of the physical properties of highly curved membranes is important in biology, for example, in fusion intermediates, and in pharmaceutical or chromatographic applications, where nanoscale features may affect substrate binding. However, vesicle fusion below 40 nm precludes study of this size regime. In this investigation, the effect of high surface curvature on the adsorption and morphology of phosphotidylcholine lipids with alkyl chain lengths of 14 (DMPC), 16 (DPPC), and 18 (DSPC) onto silica (SiO2) nanobeads was investigated by thermogravimetric analysis (TGA), high sensitivity nanocalorimetry, and vibrational spectroscopy. The SiO2 beads ranged in size from 5 to 100 nm. Stable supported bilayers were formed on all bead sizes by vesicle fusion of the parent MLVs at temperatures above the main phase transition temperature (T(m)) of the lipids. A downward shift in T(m), and a broadening (deltaT1/2) of the transition with respect to the parent MLVs, was observed for the 100 nm beads. With decreasing bead size, T(m) first decreased, but then increased. On the smallest bead size, whose dimensions were comparable to those of the adsorbed lipids, T(m)'s were higher than those of the parent MLVs. The increase in T(m) indicated a stiffening of the supported bilayer, which was confirmed by Raman spectroscopic data. Narrowing of the phase transition or the appearance of peak doublets occurred at the smaller bead sizes. The results were consistent with a model in which the high free volume and increased outer headgroup spacing of lipids on highly curved surfaces induced interdigitation in the supported lipids.