On the Influence of Nucleic Acid Backbone Modifications on Lipid Nanoparticle Morphology.

Nucleic acid therapeutics represent a major advance toward treating diseases at their root cause. However, nucleic acids are prone to degradation by serum endonucleases, clearance through the immune system, and rapid degradation in complex medium. To overcome these barriers, nucleic acids frequently include chemical modifications to improve stability or decrease immune responses. Lipid nanoparticles (LNPs) have enabled a dramatic reduction in the dose required to achieve a therapeutic effect by protecting these nucleic acids and improving their intracellular delivery. It has been assumed thus far that nonspecific ionic interactions drive LNP formation and chemical modifications to the nucleic acid backbone to confer improved stability do not impact LNP delivery in any way. Here, we demonstrate that these chemical modifications do impact LNP morphology substantially, and phosphorothioate modifications produce stronger interactions with ionizable amino lipids, resulting in enhanced entrapment. This work represents a major first step toward greater understanding of the interaction between the lipid components and nucleic acids within an LNP.

Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity.

Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD's localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of INa by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.

Measuring molecular order for lipid membrane phase studies: Linear relationship between Laurdan generalized polarization and deuterium NMR order parameter.

Two dimensional phase separation in lipid membranes and cell membranes is of interest to biology because of the idea of membrane rafts - compositionally heterogeneous liquid crystal domains with cellular functions. Few quantitative tools exist for characterizing and differentiating coexisting phases on a molecular scale. Lipid acyl chain order can be measured directly using deuterium nuclear magnetic resonance spectroscopy (2H NMR), or inferred using fluorescence microscopy along with the environment-sensitive probe Laurdan. We found a linear relationship between the 2H NMR order parameter and Laurdan generalized polarization. This observed correlation supports the idea that lipid chain order is tightly associated with the amount and dynamics of water molecules at the glycerol backbone level of the membrane.

Lamellar Phases Composed of Phospholipid, Cholesterol, and Ceramide, as Studied by 2H NMR.

Sphingolipids constitute a significant fraction of cellular plasma membrane lipid content. Among sphingolipids, ceramide levels are usually very low. However, in some cell processes like apoptosis, cell membrane ceramide levels increase markedly because of the activation of enzymes like acid sphingomyelinase. This increase can change the physical state of the membrane by promoting molecular order and inducing solid-ordered (So) phase domains. This effect has been observed in a previous 2H NMR study on membranes consisting of palmitoyl sphingomyelin (PSM) and palmitoyl ceramide (PCer). Cholesterol (Chol), too, is present at high concentrations in mammalian plasma membranes and has a favorable interaction with sphingomyelin (SM), together forming domains in the liquid-ordered phase in model membranes. There are reports that Chol is able to displace ceramide (Cer) in SM bilayers and abolish the So phase domains formed by SM:Cer. This ability of Chol appears to be concentration dependent; in membranes with low Chol and high Cer contents, So phase domains rich in Cer coexist with the continuous fluid phase of the membrane. Here, we studied the effect of increasing PCer concentration in PSM:Chol bilayers, using 2H NMR. Chol:PCer mole ratios were 3:1, 3:2, and 3:3, at a fixed 7:3 phospholipid:cholesterol mol ratio. Both PSM and PCer were monitored in separate samples for changes in their physical state by introducing a perdeuterated palmitoyl chain in either molecule. Moreover, the effect of replacing PSM with DPPC was investigated to test the impact on membrane phase behavior of replacing the sphingosine with a palmitoylated glycerol backbone. We found that PCer can increase acyl chain order in both PSM:Chol and DPPC:Chol bilayers. Especially in bilayers with Chol:PCer 1:1 molar ratios, PCer induces highly stable So phase domains in both PSM and DPPC bilayers near 37°C. However, PCer has a more pronounced ordering effect on PSM compared to DPPC bilayers.

On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable Cationic Lipids and siRNA.

Lipid nanoparticles (LNPs) containing short interfering RNA (LNP-siRNA) and optimized ionizable cationic lipids are now clinically validated systems for silencing disease-causing genes in hepatocytes following intravenous administration. However, the mechanism of formation and certain structural features of LNP-siRNA remain obscure. These systems are formed from lipid mixtures (cationic lipid, distearoylphosphatidylcholine, cholesterol, and PEG-lipid) dissolved in ethanol that is rapidly mixed with siRNA in aqueous buffer at a pH (pH 4) where the ionizable lipid is positively charged. The resulting dispersion is then dialyzed against a normal saline buffer to remove residual ethanol and raise the pH to 7.4 (above the p Ka of the cationic lipid) to produce the finished LNP-siRNA systems. Here we provide cryogenic transmission electron microscopy (cryo-TEM) and X-ray evidence that the complexes formed between siRNA and ionizable lipid at pH 4 correspond to tightly packed bilayer structures with siRNA sandwiched between closely apposed monolayers. Further, it is shown that ionizable lipid not complexed to siRNA promotes formation of very small vesicular structures at pH 4 that coalesce to form larger LNP structures with amorphous electron dense cores at pH 7.4. A mechanism of formation of LNP-siRNA systems is proposed whereby siRNA is first sandwiched between closely apposed lipid monolayers at pH 4 and subsequently trapped in these structures as the pH is raised to 7.4, whereas ionizable lipid not interacting with siRNA moves from bilayer structure to adopt an amorphous oil phase located in the center of the LNP as the pH is raised. This model is discussed in terms of previous hypotheses and potential relevance to the design of LNP-siRNA systems.

The Phase Behavior and Organization of Sphingomyelin/Cholesterol Membranes: A Deuterium NMR Study.

We have studied the dependence of the phase and domain characteristics of sphingomyelin (SM)/cholesterol model membranes on sterol content and temperature using deuterium nuclear magnetic resonance. NMR spectra of N-palmitoyl(D31)-D-erythro-sphingosylphosphorylcholine (PSM-d31) were taken for temperatures from 25 to 70°C and cholesterol concentrations of 0-40%. Analogous experiments were performed using 1-palmitoyl,2-palmitoyl(D31)-sn-glycero-3-phosphocholine (DPPC-d31)/cholesterol membranes to carefully compare the data obtained using palmitoyl chains that have similar "kinked" conformations. The constructed phase diagrams exhibit both solid-ordered (so) + liquid-ordered (lo) and liquid-disordered (ld) + lo phase-coexistence regions with a clear three-phase line. Macroscopic (micron-sized) coexistence of ld and lo phases was not observed; instead, line-broadening in the ld+lo region was characterized by intermediate exchange of lipids between the two types of domains. The length scales associated with the domains were estimated to be 75-150 nm for PSM-d31/cholesterol and DPPC-d31/cholesterol model membranes.

Constrained Versus Free Cholesterol in DPPC Membranes: A Comparison of Chain Ordering Ability Using Deuterium NMR.

We report here the first exploration of the nature of the hydrophobic region of bilayer membranes formed from sterol-modified phospholipids [Huang, Z.; Szoka, F. C., Sterol-Modified Phospholipids: Cholesterol and Phospholipid Chimeras with Improved Biomembrane Properties. J. Am. Chem. Soc. 2008, 130 (46), 15702-15712] & [Ding, J.; Starling, A. P.; East, J. M.; Lee, A. G., Binding Sites for Cholesterol on Ca(2+)-ATPase Studied by Using a Cholesterol-Containing Phospholipid. Biochemistry 1994, 33 (16), 4974-4979]. Using 2H NMR spectroscopy, we present our results for the phase behavior and acyl chain ordering of multilamellar vesicles (MLVs) of a sterol-modified phospholipid, 1-cholesterylhemisuccinoyl-2-palmitoyl(d31)-sn-glycero-3-phosphocholine (hereafter referred to as CholPPC-d31). We compared our results with the conformational order induced by cholesterol at various concentrations in 1-palmitoyl,2-palmitoyl(d31)-sn-glycero-3-phosphocholine (DPPC-d31)/cholesterol membranes. On the basis of the existing literature [Foglia, F.; Barlow, D. J.; Szoka, F. C.; Huang, Z.; Rogers, S. E.; Lawrence, M. J., Structural Studies of the Monolayers and Bilayers Formed by a Novel Cholesterol-Phospholipid Chimera. Langmuir 2011, 27 (13), 8275-8281], we expected to find that the deuterated palmitoyl chain in CholPPC-d31 membranes had an order parameter profile similar to the deuterated palmitoyl chain of sn-2 labeled DPPC-d31 in MLVs of a mixture of DPPC-d31 with 40 mol % unconstrained cholesterol. Our data indicate that the ordering ability of cholesterol in CholPPC is significantly reduced compared to free cholesterol in DPPC. This result emphasizes that cholesterol molecules must be free to move in the bilayers to reach their maximum ordering ability. In other words, when compared to unconstrained cholesterol, the constrained cholesterol moiety in CholPPC causes nonoptimal chain packing.

Link between Fluorescent Probe Partitioning and Molecular Order of Liquid Ordered-Liquid Disordered Membranes.

Fluorescence microscopy is an important technique for studying lipid membranes and is increasingly being used for examining liquid ordered-liquid disordered phase coexistence. Liquid-liquid phase coexistence is a phenomenon of biological interest because it led to the lipid raft hypothesis, which postulates the existence of lateral heterogeneities in cell membranes. Observation of membrane heterogeneity relies on differential distribution of fluorescent membrane markers, but this can also modify the phase behavior, complicating the observation. We have used 2H NMR to measure the physical changes to 35:35:30 (mol/mol) DOPC/DPPC-D62/chol membranes introduced by fluorescent probes Laurdan and naphthopyrene. We measured miscibility transition temperature (Tmix) and DPPC-D62 chain order for a range of probe concentrations. We found that up to 0.5 mol% of the equipartitioning probe Laurdan does not influence DPPC-D62 acyl chain order or phase behavior. In contrast, 2.0 mol% Laurdan slightly increases the fraction of DPPC-D62 in the liquid disordered phase below the Tmix and increases Tmix by 1 °C. Conversely, the nominally liquid ordered phase preferring probe naphthopyrene slightly perturbs the membrane even at concentrations as low as 0.3 mol%. This suggests that the strength of fluorescent probe partitioning between liquid ordered and liquid disordered phases correlates with the degree of perturbation to membrane phase behavior.

Biophysical experiments and simulation in nanoparticle-based drug delivery systems.

We are on the cusp of a new era in therapeutics which will be characterized by drugs that can be individually tailored to a patient to be more effective and less likely to cause side effects. One of the most successful ways to deliver such drugs to the physiological site of interest involves embedding them in lipid nanoparticles. Here we give a brief history of the development of lipid-based drug carriers, emphasizing the role that biophysical characterization played in their development. We further argue that in the future, the revolutionary gain in power of computer simulation techniques will enable researchers to efficiently identify optimal lipid nanoparticle compositions and physical characteristics for experimental validation.

Effect of Sterol Structure on the Physical Properties of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Membranes Determined Using (2)H Nuclear Magnetic Resonance.

The effect of a series of phytosterols on lipid chain ordering in 1-palmitoyl((2)H31)-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31) multibilayer vesicles was examined by (2)H NMR spectroscopy at 25 °C. These results, along with existing data for other sterols, indicate that the ordering power of sterols in POPC-d31 depends on subtle aspects of sterol structure. Cholesterol, 7-dehydrocholesterol (7-DHC), campesterol, ß-sitosterol, ergosterol, brassicasterol, and stigmasterol all increase the lipid chain order as sterol concentration is increased. However, saturation of the ordering occurs at different sterol concentrations for ergosterol (as previously reported), brassicasterol, ß-sitosterol, and stigmasterol. Here our interest lies in finding which part of the sterol structure is responsible for the observed saturation of the palmitoyl chain order as a function of sterol concentration. In particular, we propose that the saturation of the ordering of POPC-d31/brassicasterol and POPC-d31/stigmasterol membranes at quite low sterol concentrations is due to the presence of a double bond at C22. We also discuss how the structural differences between the sterols affect their ability to intercalate between the POPC acyl chains. Furthermore, the effective solubility of sterols in POPC is discussed in relation to the dependence of maximum POPC-d31 chain order vs sterol concentration.

Insights into sphingolipid miscibility: separate observation of sphingomyelin and ceramide N-acyl chain melting.

Ceramide produced from sphingomyelin in the plasma membrane is purported to affect signaling through changes in the membrane's physical properties. Thermal behavior of N-palmitoyl sphingomyelin (PSM) and N-palmitoyl ceramide (PCer) mixtures in excess water has been monitored by ²H NMR spectroscopy and compared to differential scanning calorimetry (DSC) data. The alternate use of either perdeuterated or proton-based N-acyl chain PSM and PCer in our ²H NMR studies has allowed the separate observation of gel-fluid transitions in each lipid in the presence of the other one, and this in turn has provided direct information on the lipids' miscibility over a wide temperature range. The results provide further evidence of the stabilization of the PSM gel state by PCer. Moreover, overlapping NMR and DSC data reveal that the DSC-signals parallel the melting of the major component (PSM) except at intermediate (20 and 30 mol %) fractions of PCer. In such cases, the DSC endotherm reports on the presumably highly cooperative melting of PCer. Up to at least 50 mol % PCer, PSM and PCer mix ideally in the liquid crystalline phase; in the gel phase, PCer becomes incorporated into PSM:PCer membranes with no evidence of pure solid PCer.

Ethanol perturbs lipid organization in models of stratum corneum membranes: An investigation combining differential scanning calorimetry, infrared and (2)H NMR spectroscopy.

Ethanol is used in a variety of topical products. It is known to enhance the permeability of the skin by altering the ability of the stratum corneum (SC) intercellular membranes to form an effective barrier. In addition, ethanol and other alcohols are key components of antiseptic gels currently used for hand wash. Using infrared and deuterium NMR spectroscopy as well as calorimetry, we have investigated the effect of ethanol on a model membrane composed of lipids representing the three classes of SC lipids, an equimolar mixture of N-palmitoylsphingosine (ceramide), palmitic acid and cholesterol. Ethanol is found to influence the membrane in a dose dependent manner, disrupting packing and increasing lipid motion at low concentrations and selectively extracting lipids at moderate concentrations.

A novel approach to modelling water transport and drug diffusion through the stratum corneum.

BACKGROUND: The potential of using skin as an alternative path for systemically administering active drugs has attracted considerable interest, since the creation of novel drugs capable of diffusing through the skin would provide a great step towards easily applicable -and more humane- therapeutic solutions. However, for drugs to be able to diffuse, they necessarily have to cross a permeability barrier: the stratum corneum (SC), the uppermost set of skin layers. The precise mechanism by which drugs penetrate the skin is generally thought to be diffusion of molecules through this set of layers following a "tortuous pathway" around corneocytes, i.e. impermeable dead cells. RESULTS: In this work, we simulate water transport and drug diffusion using a three-dimensional porous media model. Our numerical simulations show that diffusion takes place through the SC regardless of the direction and magnitude of the fluid pressure gradient, while the magnitude of the concentrations calculated are consistent with experimental studies. CONCLUSIONS: Our results support the possibility for designing arbitrary drugs capable of diffusing through the skin, the time-delivery of which is solely restricted by their diffusion and solubility properties.

Deuterium NMR study of the effect of ergosterol on POPE membranes.

We study the effect of ergosterol on the physical properties of 1-[(2)H(31)]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) multibilayers using deuterium nuclear magnetic resonance. NMR spectra were taken as a function of temperature and ergosterol concentration up to 70 mol %. The spectral first moments show that there is a dramatic difference in the ability of ergosterol to disorder the gel phase and to order the liquid-crystalline phase of POPE membranes, an unusual behavior among lipid/sterol systems studied up to now. Further investigation of the liquid-crystalline phase shows that ergosterol (erg) increases the chain order of POPE-d31, but that this effect saturates at 10 mol % ergosterol. This is in marked contrast to the effect of cholesterol (chol) on POPE membranes: the chain order of POPE increases with cholesterol to at least 45 mol %. Moreover, we found that at higher ergosterol concentrations (>40 mol %) ergosterol decreases the POPE-d31 chain order, which, to our knowledge, has not been directly observed in other lipid/sterol systems. The temperature-composition phase diagram is presented. Finally, at all ergosterol concentrations, the chain order of liquid-crystalline-phase POPE is much smaller than that of comparable POPE/chol membranes. This implies that there is no liquid-ordered phase behavior for POPE/erg membranes.

Interaction of a charged polymer with zwitterionic lipid vesicles.

The interaction between polyethylenimine (PEI) and phospholipid bilayers plays an important role in several biophysical applications such as DNA transfection of target cells. Despite considerable investigation into the nature of the interaction between PEI and phospholipid bilayers, the physical process remains poorly understood. In this paper, we study the impact of PEI on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles as a function of salt concentration using several techniques including dynamic (DLS) and static (SLS) light scattering, differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). At low salt concentration, vesicles aggregate, leading to the formation of stable clusters whose final size depends on the PEI concentration. At high salt concentration the system does not aggregate; DSC and NMR data reveal that the PEI penetrates into the bilayer, and SLS measurements are consistent with PEI crossing the bilayer. The transfectional ability of PEI is discussed in terms of these results.

The functional roles of poly(ethylene glycol)-lipid and lysolipid in the drug retention and release from lysolipid-containing thermosensitive liposomes in vitro and in vivo.

Triggered release of liposomal contents following tumor accumulation and mild local heating is pursued as a means of improving the therapeutic index of chemotherapeutic drugs. Lysolipid-containing thermosensitive liposomes (LTSLs) are composed of dipalmitoylphosphatidylcholine (DPPC), the lysolipid monostearoylphosphatidylcholine (MSPC), and poly(ethylene glycol)-conjugated distearoylphosphatidylethanolamine (DSPE-PEG(2000)). We investigated the roles of DSPE-PEG(2000) and lysolipid in the functional performance of the LTSL-doxorubicin formulation. Varying PEG-lipid concentration (0-5 mol%) or bilayer orientation did not affect the release; however, lysolipid (0-10 mol%) had a concentration-dependent effect on drug release at 42 degrees C in vitro. Pharmacokinetics of various LTSL formulations were compared in mice with body temperature controlled at 37 degrees C. As expected, incorporation of the PEG-lipid increased doxorubicin plasma half-life; however, PEG-lipid orientation (bilayer vs. external leaflet) did not significantly improve circulation lifetime or drug retention in LTSL. Approximately 70% of lysolipid was lost within 1 h postinjection of LTSL, which could be due to interactions with the large membrane pool of the biological milieu. Considering that the present LTSL-doxorubicin formulation exhibits significant therapeutic activity when used in conjunction with mild heating, our current study provided critical insights into how the physicochemical properties of LTSL can be tailored to achieve better therapeutic activity.

Phase behavior of an equimolar mixture of N-palmitoyl-D-erythro-sphingosine, cholesterol, and palmitic acid, a mixture with optimized hydrophobic matching.

The phase behavior and lipid mixing properties of an equimolar mixture of nonhydroxylated palmitoyl ceramide (Cer16), palmitic acid (PA), and cholesterol have been investigated using 2H NMR and vibrational spectroscopy. This mixture is formed by the three main classes of lipids found in the stratum corneum (SC), the top layer of the epidermis, and provides an optimized hydrophobic matching. Therefore, its behavior highlights the role played by hydrophobic matching on the phase behavior of SC lipids. We found that, below 45 degrees C, the mixture is essentially formed of coexisting crystalline domains with a small fraction of lipids (less than 20%) that forms a gel or fluid phase, likely ensuring cohesion between the solid domains. Upon heating, there is the formation of a liquid ordered phase mainly composed of PA and cholesterol, including a small fraction of Cer16. This finding is particularly highlighted by correlation vibrational microspectroscopy that indicates that domains enriched in cholesterol and PA include more disordered Cer16 than those found in the Cer16-rich domains. Solubilization of Cer16 in the fluid phase occurs progressively upon further heating, and this leads to the formation of a nonlamellar self-assembly where the motions are isotropic on the NMR time scale. It is found that the miscibility of Cer16 with cholesterol and PA is more limited than the one previously observed for ceramide III extracted from bovine brain, which is heterogeneous in chain composition and includes, in addition to Cer16, analogous ceramide with longer alkyl chains that are not hydrophobically matched with cholesterol and PA. Therefore, it is inferred that, in SC, the chain heterogeneity is a stronger criteria for lipid miscibility than chain hydrophobic matching.

Phase diagrams of lipid mixtures relevant to the study of membrane rafts.

The present paper reviews the phase properties of phosphatidylcholine-sphingomyelin-cholesterol mixtures, that are often used as models for membrane "raft" microdomains. The available data based on X-ray, microscopic and spectroscopic observations, surface pressure and calorimetric measurements, and detergent solubilization assays, are critically evaluated and rationalized in terms of triangular phase diagrams. The remaining uncertainties are discussed specifically and separately from the data on which a consensus appears to exist.

Fatty acids influence "solid" phase formation in models of stratum corneum intercellular membranes.

Stacked intercellular lipid membranes in the uppermost epidermal layer, the stratum corneum (SC), are responsible for skin's barrier function. These membranes are unique in composition, the major lipids being ceramides (Cer), cholesterol, and free fatty acids (FFA) in approximately equimolar proportions. Notably, SC lipids include chains much longer than those of most biological membranes. Previously we showed that Cer's small hydrophilic headgroup enabled SC model membranes composed of bovine brain ceramide (BBCer), cholesterol, and palmitic acid in equimolar proportion to solidify at pH 5.2. In order to determine the influence of FFA chain length on the phase behavior of such membranes, we used 2H NMR and FT-IR to study BBCer/cholesterol/FFA dispersions containing linear saturated FFA 14-22 carbons long. Independent of chain length, the solid phase dominated the FFA spectrum at physiological temperature. Upon heating, each dispersion underwent phase transitions to a liquid crystalline phase (only weakly evident for the membrane containing FFA-C22) and then to an isotropic phase. The phase behavior, the lipid mixing properties, and the transition temperatures are shown to depend strongly on FFA chain length. A distribution of FFA chain lengths is found in the SC and could be required for the coexistence of a proportion of solid lipids with some more fluid domains, which is known to be necessary for normal skin barrier function.