Margaret Lawrence

Nonionic oil-in-water microemulsions: the effect of oil type on phase behaviour

The formation of oil-in-water (o/w) microemulsions stabilized by the nonionic surfactants, polyoxyethylene-10-dodecyl ether, polyoxyethylene-10-oleyl ether, N,N-dimethyldodecylamine-N-oxide and N,N-dimethyloleylamine-N-oxide and containing a variety of pharmaceutically acceptable oils, namely ethyl butyrate, ethyl caprylate, ethyl oleate and the triglycerides, soybean oil, Miglyol 812 and tributyrin, has been examined at 298 K. The effect on microemulsion formation of replacing water with phosphate buffered saline (PBS) and complete PBS has been established. In addition, the effect of changing temperature (from 298 to 310 K) on the phase behaviour of microemulsions formulated using PBS as continuous phase has been determined. Although some small differences in phase behaviour were noted when altering the continuous phase, the greatest difference in phase behaviour was observed when changing the experimental temperature, particularly for microemulsions stabilized by polyoxyethylene-10-oleyl ether. Regardless of the temperature and aqueous phase used, however the larger molecular volume oils (soybean oil, Miglyol 812 and ethyl oleate) were solubilized to a lower extent than the smaller molecular volume oils (namely, ethyl butyrate and ethyl caprylate). The only exception to this rule was when polyoxyethylene-10-oleyl ether was used as surfactant, particularly at 298 K, where it was the larger molecular volume oils that were solubilized to the greatest extent. Cloud point/phase inversion temperature experiments suggested that the higher molecular volume oils were incorporated into the microemulsions prepared using the polyoxyethylene-based surfactants in a different way than the smaller molecular volume oils and suggest that the smaller molecular volume oils are acting in much the same way as a cosurfactant in that they interchelate with their hydrophilic group interspersed in the surfactant head group region. As N,N-dimethyldodecylamine-N-oxide does not exhibit a cloud point it was not possible to determine the mode of oil incorporation in microemulsions prepared with this surfactant.

Investigations into the formation and characterization of phospholipid microemulsions. I. Pseudo-ternary phase diagrams of systems containing water-lecithin-alcohol-isopropyl myristate

Abstract Pseudo-ternary phase diagrams have been constructed for systems comprising of water-lecithin-alcohol-isopropyl myristate. Two types of lecithin were used in this study, namely soybean (Epikuron 200) and egg lecithin (Ovothin 200). Seven short chain alcohols (i.e., n -propanol, isopropanol, n -butanol, sec -butanol, isobutanol, tert -butanol and n -pentanol) were investigated as cosurfactants. In each system studied, a large monophasic, isotropic, non-birefringent area was seen to occur along the surfactant/oil axis; while at low oil concentrations, a second isotropic, non-birefringent area, usually associated with a liquid crystalline phase, was observed in many systems. Both isotropic regions were stable at room temperature at least for 3 months. Although no significant difference was observed between the phase diagrams produced by the two types of lecithin, the extent of the isotropic regions was dependent upon both the nature of the cosurfactant and lecithin/cosurfactant mixing ratio ( K m ).

Investigations into the formation and characterization of phospholipid microemulsions. IV. Pseudo-ternary phase diagrams of systems containing water-lecithin-alcohol and oil; The influence of oil

Phase studies have been performed for quaternary systems composed of egg lecithin, cosurfactant, water and oil. The lecithin used was the commercially available egg lecithin Ovothin 200 (which comprises ≥ 92% phosphatidylcholine). The cosurfactants employed were propanol and butanol, and these were used at lecithin/cosurfactant mixing ratios (Km) of 1:1 and 1.94:1 (weight basis). Six polar oils were investigated, including the alkanoic acids, octanoic and oleic, their corresponding ethyl esters and the medium and long chain triglycerides, Miglyol 812 and soybean oil. All oils, irrespective of the alcohol and the Km used, gave rise to systems that produced a stable isotropic region along the surfactant/oil axis (designated as a reverse microemulsion system). In addition, the systems incorporating propanol at both Km and butanol at a Km of 1.94: 1, generally gave rise to a liquid crystalline region and, in some cases, a second isotropic non-birefingent area (designated as a normal microemulsion system). The phase behaviour observed was largely dependent upon the alcohol and Km used and the size and the polarity of the oil present.

Investigations into the formation and characterization of phospholipid microemulsions. III: Pseudo-ternary phase diagrams of systems containing water-lecithin-isopropyl myristate and either an alkanoic acid, amine, alkanediol, polyethylene glycol alkyl ether or alcohol as cosurfactant

The phase behaviour of quaternary systems composed of lecithin/isopropyl myristate/water/cosurfactant, at a lecithin: cosurfactant mixing ratio (Km) of 1:1 (on a weight basis) have been investigated by the construction of phase diagrams. The lecithin used in this study was the commercially available soybean lecithin, Epikuron 200 (purity greater than 94% phosphatidylcholine) and the cosurfactants examined were either short (alkyl chain length 4–6) straight-chain alkanoic acids, amines, alkanediols, diethylene glycol alkyl ethers or acohols. With the exception of the amines which appeared to react with lecithin, all the systems showed the area of existence of a stable isotropic region along the surfactant/oil axis (i.e., reverse microemulsion area; L2). In no case was a second isotropic region (i.e., normal microemulsion area; L1) observed, although in certain systems a single clear isotropic region covered virtually the whole of the phase diagram and may have included an L1 region. A liquid crystalline (LC) region was observed only in systems containing either an alkanediol or polyethylene glycol alkyl ether as cosurfactant. It was concluded that the area of existence of the various phase regions was very dependent upon the nature of the cosurfactant used.

Disposition of ceramide in model lipid membranes determined by neutron diffraction.

The lipid matrix present in the uppermost layer of the skin, the stratum corneum, plays a crucial role in the skin barrier function. The lipids are organized into two lamellar phases. To gain more insight into the molecular organization of one of these lamellar phases, we performed neutron diffraction studies. In the diffraction pattern, five diffraction orders were observed attributed to a lamellar phase with a repeat distance of 5.4 nm. Using contrast variation, the scattering length density profile could be calculated showing a typical bilayer arrangement. To obtain information on the arrangement of ceramides in the unit cell, a mixture that included a partly deuterated ceramide was also examined. The scattering length density profile of the 5.4-nm phase containing this deuterated ceramide demonstrated a symmetric arrangement of the ceramides with interdigitating acyl chains in the center of the unit cell.

Investigations into the formation and characterization of phospholipid microemulsions. II. Pseudo-ternary phase diagrams of systems containing water-lecithin-isopropyl myristate and alcohol: influence of purity of lecithin

Abstract The phase behaviour of quaternary systems composed of lecithin/isopropyi myristate/ water/and one of seven short-chain alcohols at various surfactant/cosurfactant mixing ratios ( K m ) has been investigated by the construction of phase diagrams. A commercially available soybean lecithin (namely, Epikuron 170, phosphatidylcholine purity 68–72%) was used in the study. Phase diagrams showed the area of existence of a stable isotropic region along the surfactant/oil axis (i.e., reverse microemulsion area; L 2 ) in all systems regardless of the K m . The existence of a second water-rich isotropic region (i.e., normal microemulsion area; l 1 ) was, however, seen to be very dependent upon the K m and occurred in only a few instances. This second isotropic region, L 1 , always occurred in conjunction with a liquid crystalline domain, although in some cases, particularly at the lower K m , a liquid crystalline region was seen to occur without the presence of an L 1 phase. Comparison of the results with those obtained using higher purity lecithins (greater than 92% phosphatidylcholine content) indicated that while the phase diagrams were qualitatively similar, significant differences occurred at oil levels below 50%. It was found that the existence of an L 1 and LC region and the extent of the L 2 region were dependent upon both the purity of surfactant and the surfactant/cosurfactant mixing ratios ( K m ).

A comparison of the effect of chitosan and chitosan-coated vesicles on monolayer integrity and permeability across Caco-2 and 16HBE14o-cells.

The effects of chitosan-coated vesicles on the paracellular permeability of Caco-2 and 16HBE14o-cell lines, and their toxicity towards the same cell lines, have been compared to equivalent concentration of chitosan in solution. Chitosan-coated phospholipid vesicles and the same concentration of chitosan in solution were found to reduce the transepithelial electrical resistance (TER) of monolayers of 16HBE14o- and Caco-2 cells to a comparable extent. Upon removal of the vesicle suspension and the chitosan solution, TER had completely recovered within 24 h for Caco-2 cells and to about 50% of its original value for the 16HBE14o-cells. The extent of enhancement of transport across 16HBE14o-cell monolayers of hydrophilic markers of varying molecular weight was found to be comparable in the presence of chitosan-coated phospholipid vesicles and the same concentration of chitosan and was dependent upon the molecular weight of the hydrophilic marker. Chitosan (either bound to vesicles or free in solution) did not display a significant toxicity towards the Caco-2 cell line whereas chitosan-coated phospholipid vesicles were less toxic towards the 16HBE14o-cell line than the equivalent chitosan concentration. These data suggest that chitosan-coated vesicles are good candidates for the delivery of drugs and other biomolecules across epithelial barriers.

Stratum corneum lipid matrix: Location of acyl ceramide and cholesterol in the unit cell of the long periodicity phase.

The extracellular lipid matrix in the skins outermost layer, the stratum corneum, is crucial for the skin barrier. The matrix is composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) and involves two lamellar phases: the short periodicity phase (SPP) and the long periodicity phase (LPP). To understand the skin barrier thoroughly, information about the molecular arrangement in the unit cell of these lamellar phases is paramount. Previously we examined the molecular arrangement in the unit cell of the SPP. Furthermore X-ray and neutron diffraction revealed a trilayer arrangement of lipids within the unit cell of the LPP [D. Groen et al., Biophysical Journal, 97, 2242-2249, 2009]. In the present study, we used neutron diffraction to obtain more details about the location of lipid (sub)classes in the unit cell of the LPP. The diffraction pattern revealed at least 8 diffraction orders of the LPP with a repeating unit of 129.6±0.5Å. To determine the location of lipid sub(classes) in the unit cell, samples were examined with either only protiated lipids or selectively deuterated lipids. The diffraction data obtained by means of D2O/H2O contrast variation together with a gradual replacement of one particular CER, the acyl CER, by its partly deuterated counterpart, were used to construct the scattering length density profiles. The acyl chain of the acyl CER subclass is located at a position of ~21.4±0.2Å from the unit cell centre of the LPP. The position and orientation of CHOL in the LPP unit cell were determined using tail and head-group deuterated forms of the sterol. CHOL is located with its head-group positioned ~26±0.2Å from the unit cell centre. This allows the formation of a hydrogen bond with the ester group of the acyl CER located in close proximity. Based on the positions of the deuterated moieties of the acyl CER, CHOL and the previously determined location of two other lipid subclasses [E.H. Mojumdar et al., Biophysical Journal, 108, 2670-2679, 2015], a molecular model is proposed for the unit cell of the LPP. In this model CHOL is located in the two outer layers of the LPP, while CER EOS is linking the two outer layers with the central lipid layers. Finally the two other lipid subclasses are predominantly located in the central layer of the LPP.

The effect of neutral helper lipids on the structure of cationic lipid monolayers

Successful drug delivery via lipid-based systems has often been aided by the incorporation of ‘helper lipids’. While these neutral lipids enhance the effectiveness of cationic lipid-based delivery formulations, many questions remain about the nature of their beneficial effects. The structure of monolayers of the cationic lipid dimethyldioctadecylammonium bromide (DODAB) alone, and mixed with a neutral helper lipid, either diolelyphosphatidylethanolamine or cholesterol at a 1 : 1 molar ratio was investigated at the air–water interface using a combination of surface pressure–area isotherms, Brewster angle microscopy (BAM) and specular neutron reflectivity in combination with contrast variation. BAM studies showed that while pure DODAB and DODAB with cholesterol monolayers showed fairly homogeneous surfaces, except in the regions of phase transition, monolayers of DODAB with diolelyphosphatidylethanolamine were, in contrast, inhomogeneous exhibiting irregular bean-shaped domains throughout. Neutron reflectivity data showed that while the thickness of the DODAB monolayer increased from 17 to 24 Å as it was compressed from a surface pressure of 5–40 mN m−1, the thickness of the helper lipid-containing monolayers, over the same range of surface pressures, was relatively invariant at between 25 and 27 Å. In addition, the monolayers containing diolelyphosphatidylethanolamine were found to be more heavily hydrated than the monolayers of cationic lipid, alone or in combination with cholesterol, with hydration levels of 18 molecules of water per molecule of lipid being recorded for the diolelyphosphatidylethanolamine-containing monolayers at a surface pressure of 30 mN m−1 compared with only six and eight molecules of water per molecule of lipid for the pure DODAB monolayer and the cholesterol-containing DODAB monolayer, respectively.

Analysis and Optimization of the Cationic Lipid Component of a Lipid/Peptide Vector Formulation for Enhanced Transfection In Vitro and In Vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin®, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.