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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.

Previous work has shown that lipid nanoparticles (LNP) composed of an ionizable cationic lipid, a poly(ethylene glycol) (PEG) lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and small interfering RNA (siRNA) can be efficiently manufactured employing microfluidic mixing techniques. Cryo-transmission electron microscopy (cryo-TEM) and molecular simulation studies indicate that these LNP systems exhibit a nanostructured core with periodic aqueous compartments containing siRNA. Here we examine first how the lipid composition influences the structural properties of LNP-siRNA systems produced by microfluidic mixing and, second, whether the microfluidic mixing technique can be extended to macromolecules larger than siRNA. It is shown that LNP-siRNA systems can exhibit progressively more bilayer structure as the proportion of bilayer DSPC lipid is increased, suggesting that the core of LNP-siRNA systems can exhibit a continuum of nanostructures depending on the proportions and structural preferences of component lipids. Second, it is shown that the microfluidic mixing technique can also be extended to encapsulation of much larger negatively charged polymers such mRNA (1.7 kb) or plasmid DNA (6 kb). Finally, as a demonstration of the generality of the microfluidic mixing encapsulation process, it is also demonstrated that negatively charged gold nanoparticles (5 nm diameter) can also be efficiently encapsulated in LNP containing cationic lipids. Interestingly, the nanostructure of these gold-containing LNP reveals a "currant bun" morphology as visualized by cryo-TEM. This structure is fully consistent with LNP-siRNA structure predicted by molecular modeling.

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.

Limit size systems are defined as the smallest achievable aggregates compatible with the packing of the molecular constituents in a defined and energetically stable structure. Here we report the use of rapid microfluidic mixing for the controlled synthesis of two types of limit size lipid nanoparticle (LNP) systems, having either polar or nonpolar cores. Specifically, limit size LNP consisting of 1-palmitoyl, 2- oleoyl phosphatidylcholine (POPC), cholesterol and the triglyceride triolein were synthesized by mixing a stream of ethanol containing dissolved lipid with an aqueous stream, employing a staggered herringbone micromixer. Millisecond mixing of aqueous and ethanol streams at high flow rate ratios (FRR) was used to rapidly increase the polarity of the medium, driving bottom-up synthesis of limit size LNP systems by spontaneous assembly. For POPC/triolein systems the limit size structures consisted of a hydrophobic core of triolein surrounded by a monolayer of POPC where the diameter could be rationally engineered over the range 20−80 nm by varying the POPC/triolein ratio. In the case of POPC and POPC/cholesterol (55/45; mol/mol) the limit size systems achieved were bilayer vesicles of approximately 20 and 40 nm diameter, respectively. We further show that doxorubicin, a representative weak base drug, can be efficiently loaded and retained in limit size POPC LNP, establishing potential utility as drug delivery systems. To our knowledge this is the first report of stable triglyceride emulsions in the 20−50 nm size range, and the first time vesicular systems in the 20−50 nm size range have been generated by a scalable manufacturing method. These results establish microfluidic mixing as a powerful and general approach to access novel LNP systems, with both polar or nonpolar core structures, in the sub-100 nm size range.

The synthesis, physical properties, and transfection potencies of two representives of a new class of divalent, tetraalkyl cationic lipids is described. These cationic lipids are dimers of N,N-Dioleyl-N,N-dimethylammonium chloride (DODAC) joined by a hydrocarbon tether three or six carbons in length (TODMAC3 and TODMAC6, respectively). It is shown that TODMAC6 can display improved transfection properties in comparison to DODAC when formulated into plasmid DNA-cationic lipid complexes. These improved transfection potencies are observed at cationic lipid to DNA charge ratios of two or higher. It is also shown that TODMAC6 exhibits equivalent or improved ability (as compared to DODAC) to induce nonbilayer structure in mixtures with anionic lipid. This is consistent with the hypothesis that the ability of cationic lipids to induce nonbilayer structures when mixed with anionic lipids is correlated to their transfection potency. Complexes containing TODMAC3 on the other hand exhibit lower transfection potencies than achieved with DODAC, behavior that is consistent with steric effects limiting the formation of ion pairs with anionic lipids. It is concluded that TODMAC6 exhibits potential as a transfection agent for in vitro and in vivo use and that the design of cationic lipids according to their ability to induce nonbilayer structure provides a useful guide for synthesis of new cationic lipids.

Determination of the mechanisms by which cationic liposomes adhere to and fuse with biological membranes is important to understanding how these lipid vesicles mediate cellular transfection. To determine what role the lipid composition of "target" membranes might have in promoting fusion with cationic liposomes, we have examined the ability of large unilamellar vesicles composed of 1,2-dioleoylsn-phosphatidylethanolamine (DOPE) and N,N-dimethyl-N,N-di-9-cis-octadecenylammonium chloride (DODAC) (1:1) to fuse with target liposomes of varying composition in the absence of DNA. Membrane fusion was promoted by increased negative surface charge and, for liquid crystalline lipids, by increased acyl chain unsaturation in target liposomes. However, the presence of disaturated phospholipids promoted fusion below the gel to liquid crystalline transition temperature, an effect which was eliminated by the addition of cholesterol. It was also shown that DOPE/DODAC (1:1) LUVs fused with erythrocyte ghosts and that this fusion was blocked by the presence of serum. Membrane fusion was determined by a quantitative fluorescent lipid mixing assay and qualitatively by freeze-fracture electron microscopy and fluorescence microscopy.

The influence of poly(ethylene glycol)-lipid conjugates on phospholipid polymorphism has been examined using 31P-NMR and freeze--fracture electron microscopy. An equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and cholesterol adopts the hexagonal (HII) phase when hydrated under physiological conditions but can be stabilized in a bilayer conformation when a variety of PEG-lipid conjugates are included in the lipid mixture. These PEG conjugates produced an increase in the bilayer to hexagonal (HII) phase transition temperature and a broadening of the temperature range over which both phases coexisted. Further, the fraction of phospholipid adopting the bilayer phase increased with increasing mole fraction of PEG-lipid such that at 20 mole % DOPE--PEG2000 no HII phase phospholipid was observed up to a least 60 degrees C. Increasing the size of the PEG moiety from 2000 to 5000 Da (while maintaining the PEG--lipid molar ratio constant) increased the proportion of lipid in the bilayer phase. In contrast, varying the acyl chains of the PE anchor had no effect on polymorphic behavior. PEG--lipid conjugates in which ceramide provides the hydrophobic anchor also promoted bilayer formation in DOPE:cholesterol mixtures but at somewhat higher molar ratios compared to the corresponding PEG--PE species. The slightly greater effectiveness of the PE conjugates may result from the fact that these derivatives also possess a net negative charge. Phosphorus NMR spectroscopy indicated that a proportion of the phospholipid in DOPE:cholesterol:PEG--PE mixtures experienced isotropic motional averaging with this proportion being sensitive to both temperature and PEG molecular weight. Surprisingly, little if any isotropic signal was observed when PEG--ceramide was used in place of PEG--PE. Consistent with the 31P-NMR spectra, freeze-fracture electron microscopy showed the presence of small vesicles (diameter

Liposome aggregation is a major problem associated with the covalent attachment of proteins to liposomes. This report describes a procedure for coupling proteins to liposomes that results in little or no change in liposome size. This is achieved by incorporating appropriate levels of poly(ethy1ene glycol)-modified lipids into the liposomes. The studies employed thiolated avidin-D coupled to liposomes containing the thio-reactive lipid N-( 4-(p-maleimidophenyl)butyryl)dipalmitoyl phosphatidylethanolamine (1 mol % of total lipid) and various amounts of MePEG-S-POPE (monomethoxypoly(ethy1ene glycol) linked to phosphatidylethanolamine via a succinate linkage). The influence of PEG chain length and density was also assessed. The presence of PEG on the surface of liposomes is shown to provide an effective method of inhibiting aggregation and the corresponding increase in liposome size during the covalent coupling of avidin-D. A balance between the size of the PEG used and the amount of PEG-lipid incorporated into the liposome had to be achieved in order to maintain efficient coupling. Optimal coupling efficiencies in combination with minimal aggregation effects were achieved using 2 mol % MePEGzooo-S-POPE (PEG of 2000 MW) or 0.8 mol % MePEG5000-S-POPE (PEG of 5000 MW). At these levels, the presence of PEG did not affect the biotin binding activity of the covalently attached avidin. The ability of the resulting liposomes to specifically target to biotinylated cells is demonstrated.

The ability of lipid asymmetry to regulate Ca(2+)-stimulated fusion between large unilamellar vesicles has been investigated. It is shown that for 100-nm-diameter LUVs composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylinositol, and dioleoylphosphatidic acid (DOPC/DOPE/PI/DOPA; 25:60:5:10) rapid and essentially complete fusion is observed by fluorescent resonance energy transfer techniques when Ca2+ (8 mM) is added. Alternatively, for LUVs with the same lipid composition but when DOPA was sequestered to the inner monolayer by incubation in the presence of a pH gradient (interior basic), little or no fusion is observed on addition of Ca2+. It is shown that the extent of Ca(2+)-induced fusion correlates with the amount of exterior DOPA. Further, it is shown that LUVs containing only 2.5 mol % DOPA, but where all the DOPA is in the outer monolayer, can be induced to fuse to the same extent and with the same rate as LUVs containing 5 mol % DOPA. These results strongly support a regulatory role for lipid asymmetry in membrane fusion and indicate that the fusogenic tendencies of lipid bilayers are largely determined by the properties of the monolayers proximate to the fusion interface.

The influence of a transmembrane pH gradient on the Ca2+-induced fusion of phospholipid vesicles, containing free fatty acids, has been investigated. Large unilamellar vesicles composed of an equimolar mixture of cardiolipin, dioleoylphosphatidylcholine, and cholesterol, containing 20 mol 7% oleic acid, were employed. Fusion was measured using a kinetic assay for lipid mixing, based on fluorescence resonance energy transfer. At pH 7.5, but not at pH 6.0, in the absence of a pH gradient, oleic acid stimulates the fusion of the vesicles by shifting the Ca2+ threshold concentration required for aggregation and fusion of the vesicles from about 13 mM to 10 mM. In the presence of a pH gradient (at an external pH of 7.5 and a vesicle interior pH of 10.5), the vesicles exhibit fusion characteristics similar to vesicles that do not contain oleic acid at all, consistent with an effective sequestration of the fatty acid to the inner monolayer of the vesicle bilayer induced by the imposed pH gradient. The kinetics of the fusion process upon simultaneous generation of the pH gradient across the vesicle bilayer and initiation of the fusion reaction show that the inward movement of oleic acid in response to the pH gradient is extremely fast, occurring well within 1 s. Conversely, dissipation of an imposed pH gradient, by addition of a proton ionophore during the course of the fusion process, results in a rapid enhancement of the rate of fusion due to reequilibration of the oleic acid between the two bilayers leaflets. Membrane fusion is a fundamental process in cell biology. It plays a key role in cell-cell fusion phenomena such as fertilization and myogenesis. It is also the basis of intracellular trafficking and sorting processes, involving fusion of shuttle vesicles derived from one cellular compartment with the lim- iting membrane of another compartment or, as in the process of exocytosis, with the plasma membrane of the cell. Obvi- ously, these membrane fusion processes must be highly specific and strictly controlled, at the level of the initial recognition and attachment of the interacting membranes as well as that of the actual fusion reaction. However, very little is known about the molecular mechanisms involved. Much of our current knowledge of the molecular mecha- nisms of membrane fusion has been derived from investigation of fusion in lipid vesicle (liposome) systems (for reviews, see

The polymorphic phase behavior of mixtures of synthetic dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) and the influence of cholesterol on these phase preferences have been investigated by employing nuclear magnetic resonance (NMR) techniques. In particular, 31P NMR procedures are utilized to study the overall phase preferences of these mixed systems, whereas 2H NMR is employed to monitor the structural preferences of individual components of these systems by using versions of DOPE and DOPC which are deuterium (2H) labeled at the C11 position of the acyl chains. The results obtained show that DOPE-DOPC systems containing as little as 20 mol % DOPC initially assume lamellar structure at 40 degrees C, even though DOPE in isolation prefers the hexagonal (HII) organization at this temperature. However, this lamellar organization appears to represent a metastable state, as incubation for extended periods at 40 degrees C results in formation of a structure, possibly the cubic phase, in which the phospholipids experience isotropic motional averaging. The addition of cholesterol induces hexagonal (HII) phase organization. 2H NMR studies of appropriately labeled versions of these systems indicate that cholesterol does not produce such effects by associating preferentially with either DOPE or DOPC. Further, in situations where bilayer, hexagonal, or "isotropic" phases coexist in the same sample, the phospholipids exhibit apparently ideal mixing behavior.

The influence of temperature and ionic strength on the vesiculation properties of large multilamellar vesicles containing various proportions of dimyristoylphosphatidylglycerol has been investigated. It is shown that at low ionic strengths preformed large multilamellar vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (7:3) on incubation at the gel to liquid-crystalline transition temperature (Tc approximately 23 degrees C) spontaneously vesiculate to form predominantly unilamellar systems with a mean diameter of 120 nm. Such vesiculation is not observed for incubations at temperatures appreciably above or below Tc, and is also inhibited by higher ionic strengths. Stable large multilamellar vesicles are formed, however, in systems containing the dioleoyl species of phosphatidylcholine or phosphatidylglycerol and also for dimyristoylphosphatidylcholine/dimyristoylphosphatidylserine mixtures. The vesiculation properties of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol mixtures, therefore, appear to reflect an instability in the region of the Tc driven by surface potential effects which are specific for the glycerol headgroup.

The polymorphic phase behavior of aqueous dispersions of 1-oleoyl-, 1-linoleoyl-, and 1 - linolenoyl-sn-3-glycerophosphoethanolamine ( 1 -C 18: l,-PE, 1 -C 18:2,-PE, and 1 -C 18:3,-PE, respectively) has been investigated by 31P NMR, small-angle and wide-angle X-ray diffraction, and freeze-fracture techniques in response to changes in temperature and pH. Between -20 and 0 "C at pH 7, NMR and X-ray data indicate that 1-C18:lC-PE adopts a lamellar phase. Above 20 OC, the X-ray diffraction from 1-C18:lC-PE reveals no long-range lattice order, whereas the NMR data indicate lamellar structure to 90 OC. Freeze-fracture electron microscopy shows that 1-C18: 1,-PE at pH 8.2 forms closed multilamellar vesicles upon dispersion and also that large unilamellar vesicles produced by extrusion techniques (LUVETs) can be made from 1-C18:lC-PE at pH 7. Such LUVETs can trap (3H)inulin and support a K+ diffusion potential for up to 4 h. At pH 8.5 and above, 1-C18:lC-PE forms optically clear, fluid dispersions with NMR and X-ray characteristics consistent with a micellar (noninverted) phase structure. Attempts to prepare LUVETs from 1-C18:lC-PE at pH 9 result in structures that can neither trap (3H)inulin nor support a membrane potential. At temperatures below -10 OC at pH 7, both 1-C18:2,-PE and l-C18:3,-PE form a lamellar phase whereas at temperatures of 20 OC and above, both lipids form optically clear, gellacious phases with NMR and X-ray characteristics consistent with a phase (such as inverted micellar) in which the phospholipid head groups are not in intimate contact with the bulk aqueous phase. For both l-C18:2,-PE and 1-C18:3,-PE at 0 OC, there is X-ray and NMR evidence, respectively, of a hexagonal phase, putatively HII, occurring as an intermediate in the lamellar-inverted phase transition.

The uptake of dibucaine into large unilamellar vesicles in response to proton gradients (delta pH; inside acidic) or membrane potentials (delta psi; inside negative) has been investigated. Dibucaine uptake in response to delta pH proceeds rapidly in a manner consistent with permeation of the neutral (deprotonated) form of the drug, reaching a Henderson-Hasselbach equilibrium where [dibucaine]in/[dibucaine]out = [H+]in/[H+]out and where the absolute amount of drug accumulated is sensitive to the buffering capacity of the interior environment. Under appropriate conditions, high absolute interior concentrations of the drug can be achieved (approximately 120 mM) in combination with high trapping efficiencies (in excess of 90%). Dibucaine uptake in response to delta psi proceeds more than an order of magnitude more slowly and cannot be directly attributed to uptake in response to the delta pH induced by delta psi. This induced delta pH is too small (less than or equal to 1.5 pH units) to account for the transmembrane dibucaine concentration gradients achieved and does not come to electrochemical equilibrium with delta psi. Results supporting the possibility that the charged (protonated) form of dibucaine can be accumulated in response to delta psi were obtained by employing a permanently positively charged dibucaine analogue (N-methyldibucaine). Further, the results suggest that delta psi-dependent uptake may depend on formation of a precipitate of the drug in the vesicle interior. The uptake of dibucaine into vesicles in response to ion gradients is of direct utility in drug delivery and controlled release applications and is related to processes of drug sequestration by cells and organelles in vivo.

The ability of calcium to induce phase separation in multicomponent lipid mixtures containing various unsaturated species of acidic and neutral phospholipids has been investigated by 31P NMR, 3H NMR, and small-angle X-ray diffraction techniques. It is shown that, in unsaturated (dioleoyl-) phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) (1:1) and phosphatidic acid (PA)/phosphatidylcholine (PC) (1:1) mixtures, calcium is unable to induce lateral phase separation of the acidic and neutral lipids and that all the lipids adopt a hexagonal (HII) phase in the presence of calcium. In multicomponent mixtures containing one or more acidic species the presence of cholesterol either facilitates calcium-induced lamellar to hexagonal (HII) transitions for all the lipid components or, in systems already in a hexagonal (HII) phase, mitigates against calcium-induced lateral phase separations. Further, cholesterol is shown to exhibit no preferential interaction on the NMR time scale with either PC, PE, or phosphatidylserine (PS) when the lipids are in the liquid-crystal state. The ability of cholesterol to directly induce HII phase formation in PC/PE mixtures is also shown to be common to various other sterols including ergosterol, stigmasterol, coprostanol, epicoprostanol, and androstanol.

The influence of membrane pH gradients on the transbilayer distribution of some common phospholipids has been investigated. We demonstrate that the transbilayer equilibrium of the acidic phospholipids egg phosphatidylglycerol (EPG) and egg phosphatidic acid (EPA) can be manipulated by membrane proton gradients, whereas phosphatidylethanolamine, a zwitterionic phospholipid, remains equally distributed between the inner and outer monolayers of large unilamellar vesicles (LUVs). Asymmetry of EPG is examined in detail and demonstrated by employing three independent techniques: ion-exchange chromatography, 13C NMR, and periodic acid oxidation of the (exterior) EPG headgroup. In the absence of a transmembrane pH gradient (delta pH) EPG is equally distributed between the outer and inner monolayers of LUVs. When vesicles composed of either egg phosphatidylcholine (EPC) or DOPC together with 5 mol % EPG are prepared with a transmembrane delta pH (inside basic, outside acidic), EPG equilibrates across the bilayer until 80-90% of the EPG is located in the inner monolayer. Reversing the pH gradient (inside acidic, outside basic) results in the opposite asymmetry. The rate at which EPG equilibrates across the membrane is temperature dependent. These observations are consistent with a mechanism in which the protonated (neutral) species of EPG is able to traverse the bilayer. Under these circumstances EPG would be expected to equilibrate across the bilayer in a manner that reflects the transmembrane proton gradient. A similar mechanism has been demonstrated to apply to simple lipids that exhibit weak acid or base characteristics [Hope, M. J., & Cullis, P. R. (1987) J. Biol. Chem 262, 4360-4366]