Triggered release of doxorubicin following mixing of cationic and anionic liposomes

In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 degrees C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [31P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H(II)) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.     

Novel fluorescent cationic phospholipid, O-4-napthylimido-1-butyl-DOPC, exhibits unusual foam morphology, forms hexagonal and cubic phases in mixtures, and transfects DNA

The novel cationic triester of phosphatidylcholine, O-4-napthylimido-1-butyl-dioleoylphosphatidylcholine (NB-DOPC), has been synthesized: 1-amino-4-butanol was reacted with napthylic anhydride to form 4-hydroxybutyl-1-napthylamide, which was then reacted with triflic anhydride; the resultant triflate was reacted with dioleoylphosphatidylcholine so as to transfer the napthylimido-butyl group to the unsubstituted phosphate oxygen. The resultant compound is thus not only positively charged, but also has a bulky hydrophobic moiety attached to the headgroup. This novel cationic phospholipid exhibits a peculiar long-living foam-like morphology upon hydration, which could have applications in encapsulation and delivery. It is characterized by high adhesiveness to hydrophobic surfaces. X-ray diffraction showed that it forms a lamellar structure of rather short repeat period, indicative of an unusually small interlamellar separation and low hydration level. It readily incorporates DNA and organizes into lamellar lipoplexes. These DNA-lipid complexes effectively transfect DNA into cells. In an equimolar mixture of this lipid with the anionic dioleoylphosphatidylglycerol lamellar arrays coexist with disordered uncorrelated structures, however, these transform into a bicontinuous cubic phase, Pn3m, upon addition of DNA. When mixed with another anionic lipid, cardiolipin, at a NB-DOPC/ cardiolipin 2:1 molar ratio, it forms the inverted hexagonal phase which is of potential interest for nanotechnology applications.     

Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: A measure for transfection efficiency

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by ∼ 10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L_α → H_II, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L_α → H_II phase transition and promoted formation of an inverted cubic phase between the L_α and H_II phases. In contrast, moderate and weak transfection agents retained the direct L_α → H_II transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.     

Induction of apoptosis in macrophages by cationic liposomes

The effects of liposomes on apoptosis in macrophages were evaluated from DNA content and DNA fragmentation. Cationic liposomes composed of different kinds of cationic lipids induced apoptosis in mouse splenic macrophages and the macrophage-like cell line, RAW264.7 cells. Generation of reactive oxygen radicals from macrophages treated with cationic liposomes was detected using flow cytometry, and further apoptosis was inhibited by the addition of oxidant scavenger, N-acetylcysteine. From these findings, the production of reactive oxygen species may be important in the regulation of apoptosis induced by cationic liposomes.     

Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient

This study examines a new method for the remote loading of doxorubicin into liposomes. It was shown that doxorubicin can be loaded to a level of up to 98% into large unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3 mol/mol) with a transmembrane phosphate gradient. The different encapsulation efficiencies which were achieved with ammonium salts (citrate 100%, phosphate 98%, sulfate 95%, acetate 77%) were significantly higher as compared to the loading via sodium salts (citrate 54%, phosphate 52%, sulfate 44%, acetate 16%). Various factors, including pH-value, buffer capacity, solubility of doxorubicin in different salt solutions and base counter-flow, which likely has an influence on drug accumulation in the intraliposomal interior are taken into account. In contrast to other methods, the newly developed remote loading method exhibits a pH-dependant drug release property which may be effective in tumor tissues. At physiological pH-value doxorubicin is retained in the liposomes, whereas drug release is achieved by lowering the pH to 5.5 (approximately 25% release at 25 °C or 30% at 37 °C within two h). The DXR release of liposomes which were loaded via a sulfate gradient showed a maximum of 3% at pH 5.5.     

Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes

Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes.     

Enhancement of immune responses by DNA vaccination through targeted gene delivery using mannosylated cationic liposome formulations following intravenous administration in mice

The present study investigated the potency of the mannosylated cationic liposomes (Man liposomes) that we have developed in novel DNA vaccine carrier. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. The potency of the Man liposome/pCMV-OVA complex was compared with naked pCMV-OVA and that complexed with DC-Chol liposomes. In cultured mouse peritoneal macrophages, MHC class I-restricted antigen presentation of the Man liposome/pCMV-OVA complex was significantly higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. After intravenous administration, OVA mRNA expression and MHC class I-restricted antigen presentation on CD11c+ cells and inflammatory cytokines, such as TNF-alpha, IL-12, and IFN-gamma, that can enhance the Th1 response of the Man liposome/pCMV-OVA complex were higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. Also, the spleen cells from mice immunized by intravenous administration of the Man liposome/pCMV-OVA complex showed the highest proliferation response and IFN-gamma secretion. These findings suggest that the targeted delivery of DNA vaccine by Man liposomes is a potent vaccination method for DNA vaccine therapy.     

In vitro stability and content release properties of phosphatidylglyceroglycerol containing thermosensitive liposomes

Recently, we reported that 1,2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPGOG) prolongs the circulation time of thermosensitive liposomes (TSL). Since the only TSL formulation in clinical trials applies DSPE-PEG2000 and lysophosphatidylcholine (P-lyso-PC), the objective of this study was to compare the influence of these lipids with DPPGOG on in vitro stability and heat-induced drug release properties of TSL. The content release rate was significantly increased by incorporating DPPGOG or P-lyso-PC in TSL formulations. DPPC/DSPC/DPPGOG 50:20:30 (m/m) and DPPC/P-lyso-PC/DSPE-PEG2000 90:10:4 (m/m) did not differ significantly in their release rate of carboxyfluorescein with > 70% being released within the first 10s at their phase transition temperature. Furthermore, DPPC/DSPC/DPPGOG showed an improved stability at 37 °C in serum compared to the PEGylated TSL. The in vitro properties of DPPGOG-containing TSL remained unchanged when encapsulating doxorubicin instead of carboxyfluorescein. The TSL retained 89.1 ± 4.0% of doxorubicin over 3 h at 37  °C in the presence of serum. The drug was almost completely released within 120s at 42 °C. In conclusion, DPPGOG improves the in vitro properties in TSL formulations compared to DSPE-PEG2000, since it not only increases the in vivo half-life, it even increases the content release rate without negative effect on TSL stability at 37 °C which has been seen for DSPE-PEG2000/P-lyso-PC containing TSL.     

Study of the release of a microencapsulated acid dye in polyamide dyeing using mixed cationic liposomes

The main objective of this work was to increase the retarding effect of the acid dye Telon^® Blue RR (C.I. Acid Blue 62; DyStar, Frankfurt, Germany) release on polyamide fibres dyeing by encapsulation of the dye in liposomes as an alternative to synthetic auxiliaries, in order to reduce effluent pollution. The retarding effect achieved with the use of mixed cationic liposomes of dioctadecyldimethylammonium bromide (DODAB)/soybean lecithin (containing a 10% molar fraction of DODAB) was better in comparison with either pure soybean lecithin liposomes or synthetic auxiliaries. The retarding effect of liposomes on the dye release was analysed through changes in the absorption and fluorescence spectra of the acid dye at different conditions. The effect of temperature (in the range of 25 °C - 70 °C) on the spectroscopic behaviour of the dye in the absence and in presence of polyamide was also studied, in order to simulate the dyeing conditions. Exhaustion curves obtained in dyeing experiments showed that, below 45 °C, the retarding effect of the mixed liposomes (lecithin/DODAB (9:1)) was similar to that of the auxiliaries, but better than the one of pure lecithin liposomes. At higher temperatures (above 45 °C), the system lecithin/DODAB presents a better performance, achieving a higher final exhaustion level when compared with the commercial leveling agent without losing the smoothing effect of lecithin.     

The synthesis of cholesterol-based cationic lipids with trimethylamine head and the effect of spacer structures on transfection efficiency

Five cholesterol-based cationic lipids were newly synthesized based on cholest-5-en-3β-oxyethane-N,N,N-trimethylammonium bromide (Chol-ETA) structure where the cholesterol backbone is linked to cationic head via various lengths of ether-linked carbon spacer. The transfection efficiency of these compounds was increased in order of three (Chol-PRO) < four (Chol-BTA) < two (Chol-ETA) methylene unit in their spacer, and was decreased by an addition of isomethyl group to Chol-PRO spacer. In case of the presence of multiple bonds in the spacer, it required the more cationic lipids in liposome formulation than single bond in the spacer to present similar transfection efficiency.     

Anionic and cationic components from protein aggregates in bovine seminal plasma and their effects of sperm motility

Bovine seminal plasma proteins are in an aggregated form of high molecular weight in their native state. By immobilisation on a cation exchanger with exposure to disaggregating conditions (i.e., acetonitrile and low pH), the high-molecular-weight aggregates could be dissociated to slowly release the low-molecular-weight components. The anionic component released from the cation exchanger during disaggregation was collected by adsorption on a hydrophobic interaction column. The cationic component remaining on the cation exchanger was eluted with NaOH. Both components were found on gel permeation chromatography to be <5 kDa. SDS—PAGE of the various fractions showed that component of low molecular weight were still in an aggregated form. These components resulting from the disaggregation process have detrimental effects on sperm motility and the effects were more substantial compared with that of whole seminal plasma. All the cationic components were significantly detrimental to sperm motility, especially the fractions of low molecular weight. The anionic fractions reduced sperm motility when in an aggregated state. The isolated anionic peptide was not detrimental in its free form. In all fractions the peptides tended to re-aggregate to a higher molecular weight under neutral conditions, however, the isolated anionic peptide (molecular weight <1,500) failed to do so. © Wiley-Liss, Inc.     

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-l-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.     

Development of wrapped liposomes: Novel liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids

Novel wrapped liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids were prepared using an efficient, innovative procedure. In this study, dextran fluorescein anionic (DFA) was used as an example of a polyanionic compound. During the process, neutral lipids accumulated around the complexes and eventually covered the complexes. The resulting liposomes were 120-140 nm in diameter and the encapsulation efficiency was up to 90%. In fetal bovine serum, DFA/cationic lipid complexes degraded rapidly but the wrapped liposomes were considerably more stable. Following intravenous administration to rats, DFA/cationic lipid complexes were rapidly eliminated whereas the wrapped liposomes exhibited a much longer blood half-life. These data suggest that DFA is located on the surface of the complexes, but DFA is present inside the wrapped liposomes. The drug-delivery properties of the wrapped liposomes established in the present study suggests that formulations based on this technology could offer important advantages for the administration of many types of drug including antisense oligonucleotides, plasmids and siRNAs which may therefore lead to improved therapeutic effectiveness of this range of drugs. The method of preparation of the wrapped liposomes is so simple that it should be straightforward to adapt to a manufacturing scale.     

pH-sensitive cationic guar gum/poly (acrylic acid) polyelectrolyte hydrogels: Swelling and in vitro drug release

Novel polyelectrolyte hydrogels (coded as GA) based on cationic guar gum (CGG) and acrylic acid monomer by photoinitiated free-radical polymerization were synthesized with various feed compositions. Fourier transform infrared spectra (FTIR), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) confirmed that the formation of the polyelectrolyte hydrogels was attributed to the strong electrostatic interaction between cationic groups in CGG and anionic groups in poly (acrylic acid) (PAA). Swelling experiments provided important information on drug diffusion properties, which indicated the GA hydrogels were highly sensitive to pH environments. Potential applications of the hydrogels matrices in controlled drug delivery were also examined. The ketoprofen-loaded CGG/PAA matrices were prepared by hydrogels and directly compressed tablets, respectively. Release behavior of ketoprofen relied on the preparative methods of matrices, ratios of CGG/AA and pH environments. The release mechanism was studied by fitting experimental data to a model equation and calculating the corresponding parameters. The result showed that the kinetics of drug release from the hydrogels in pH 7.4 buffer solution was mainly non-Fickian diffusion. However, for tablets, the drug release in pH 7.4 buffer solution was mainly affected by polymer erosion. The pH of the dissolution medium appeared to have a strong effect on the drug transport mechanism. At more basic pH values, Case II transport was observed, indicating a drug release mechanism highly influenced by macromolecular chain relaxation. The ketoprofen release is also tested in the conditions chosen to simulate gastrointestinal tract conditions. The results implied that the GA hydrogels can be exploited as potential carriers for colon-specific drug delivery.     

Sensitization of multidrug-resistant malignant cells by liposomes co-encapsulating doxorubicin and chloroquine through autophagic inhibition

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters play a key role in the development of multidrug resistance (MDR) in cancer cells. P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) are important proteins in this superfamily which are widely expressed on the membranes of multidrug resistance (MDR) cancer cells. Besides, upregulation of cellular autophagic responses is considered a contributing factor for MDR in cancer cells. We designed a liposome system co-encapsulating a chemotherapeutic drug (doxorubicin hydrochloride, DOX) and a typical autophagy inhibitior (chloroquine phosphate, CQ) at a weight ratio of 1:2 and investigated its drug resistance reversal mechanism. MTT assay showed that the IC₅₀ of DOX/CQ co-encapsulated liposome in DOX-resistant human breast cancer cells (MCF7/ADR) was 4.7?±?0.2?μM, 5.7-fold less than that of free DOX (26.9?±?1.9 μM), whereas it was 19.5-fold in doxorubicin-resistant human acute myelocytic leukemia cancer cells (HL60/ADR) (DOX/CQ co-encapsulated liposome 1.2?±?0.1?μM, free DOX 23.4?±?2.8?μM). The cellular uptake of DOX increased upon addition of free CQ, indicating that CQ may interact with P-gp and MRP1; however, the expressions of P-gp and MRP1 remained unchanged. In contrast, the expression of the autophagy-related protein LC3-II increased remarkably. Therefore, the mechanism of MDR reversal may be closely related to autophagic inhibition. Evaluation of anti-tumor activity was achieved in an MCF-7/ADR multicellular tumor spheroid model and transgenic zebrafish model. DOX/CQ co-encapsulated liposome exerted a better anti-tumor effect in both models than that of liposomal DOX or DOX alone. These findings suggest that encapsulating CQ with DOX in liposomes significantly improves the sensitivity of DOX in DOX-resistant cancer cells.     

D-甘露糖修饰黄芩苷阳离子脂质体的制备及其对肺癌A549细胞增殖的抑制作用

目的:制备一种D-甘露糖修饰的黄芩苷阳离子脂质体[Baicalin cationic liposome,BC-Lipo(+)],并考察其对肺癌A549细胞增殖的抑制效果。方法:用乙醇注入法制备BC-Lipo(+),并考察其药剂学性质;以肺癌A549细胞为模型,采用MTT法考察其对肿瘤细胞的抑制效果。结果:透射电镜下可见BC-Lipo(+)呈圆形或类圆形,平均粒径(111.3±2.7)nm,Zeta电位(9.6±0.3)m V,包封率(95.4±0.8)%;体外释药行为符合Ritger-Peppas方程(lnQ=0.3497lnt-1.6611,r=0.9924);A549细胞的增殖抑制率可达(88.3±5.7)%。结论:处方和制备工艺合理,BC-Lipo(+)包封率较高,粒径分布均匀,带一定的正电荷,具有明显的体外缓释特性,抑制肺癌A549细胞增殖效果显著,为深入研究肺靶向BC纳米脂质体制剂奠定了基础。     

Reversal of P-glycoprotein-mediated multidrug resistance by doxorubicin and quinine co-loaded liposomes in tumor cells

Multidrug resistance (MDR) is a major obstacle to successful clinical cancer chemotherapy. Currently, there is still unsatisfactory demand for innovative strategies as well as effective and safe reversing agent to overcome MDR. In this study, we developed a novel nanoformulation, in which doxorubicin hydrochloride (DOX) and quinine hydrochloride (QN) were simultaneously loaded into liposomes by a pH-gradient method for overcoming MDR and enhancing cytotoxicity in a doxorubicin-resistant human breast cancer cell line (MCF-7/ADR). The various factors were investigated to optimize the formulation and manufacturing conditions of DOX and QN co-loaded liposomes (DQLs). The DQL showed uniform size distribution and high encapsulation efficiency (over 90%) for both the drugs. Furthermore, DQLs significantly displayed high intracellular accumulation and potential of MDR reversal capability in MCF-7/ADR cells through the cooperation of DOX with QN, in which QN played the role as a MDR reversing agent. The IC₅₀ of DQL0.5:1 with the DOX/QN/SPC weight ratio of 0.5:1:50 was 1.80?±?0.03?μg/mL, which was 14.23 times lower than that of free DOX in MCF-7/ADR cells. And the apoptotic percentage induced by DQL0.5:1 was also increased to 62.2%. These findings suggest that DQLs have great potential for effective treatment of MDR cancer.     

Spectroscopic characterization of DMPC/DOTAP cationic liposomes and their interactions with DNA and drugs

Gene and synthetic drug-delivery vectors have been developed and characterized to treat several genetic diseases and cancers. Our study aims at characterizing cationic liposomes containing the zwitterionic phospholipid DMPC and the cationic lipid DOTAP as well as their interactions with two types of DNA and a new class of antineoplastic agents derived from arylchloroethylureas (CEU). Results obtained using FTIR spectroscopy as well as ³¹P and ²H NMR indicate that DMPC and DOTAP form cationic liposomes in a highly disordered fluid phase at a molar ratio of 1:1. In addition, the FTIR results indicate that the presence of DNA or CEUs within the liposomes does not significantly affect the conformational order of both the DMPC and DOTAP acyl chains. Our results therefore provide a detailed characterization of complexes between cationic liposomes and both DNA and drugs and indicate that these complexes are stable and fluid assemblies.     

The thermotropic phase behaviour and phase structure of a homologous series of racemic beta-D-galactosyl dialkylglycerols studied by differential scanning calorimetry and X-ray diffraction

The thermotropic phase behaviour of aqueous dispersions of some synthetic 1,2-di-O-alkyl-3-O-(beta-D-galactosyl)-rac-glycerols (rac-beta-D-GalDAGs) with both odd and even hydrocarbon chain lengths was studied by differential scanning calorimetry (DSC), small-angle (SAXS) and wide-angle (WAXS) X-ray diffraction. DSC heating curves show a complex pattern of lamellar (L) and nonlamellar (NL) phase polymorphism dependent on the sample's thermal history. On cooling from 95 degrees C and immediate reheating, rac-beta-D-GalDAGs typically show a single, strongly energetic phase transition, corresponding to either a lamellar gel/liquid-crystalline (L(beta)/L(alpha)) phase transition (N< or =15 carbon atoms) or a lamellar gel/inverted hexagonal (L(beta)/H(II)) phase transition (N> or =16). At higher temperatures, some shorter chain compounds (N=10-13) exhibit additional endothermic phase transitions, identified as L/NL phase transitions using SAXS/WAXS. The NL morphology and the number of associated intermediate transitions vary with hydrocarbon chain length. Typically, at temperatures just above the L(alpha) phase boundary, a region of phase coexistence consisting of two inverted cubic (Q(II)) phases are observed. The space group of the cubic phase seen on initial heating has not been determined; however, on further heating, this Q(II) phase disappears, enabling the identification of the second Q(II) phase as Pn3 m (space group Q(224)). Only the Pn3 m phase is seen on cooling. Under suitable annealing conditions, rac-beta-D-GalDAGs rapidly form highly ordered lamellar-crystalline (L(c)) phases at temperatures above (N< or =15) or below (N=16-18) the L(beta)/L(alpha) phase transition temperature (T(m)). In the N< or =15 chain length lipids, DSC heating curves show two overlapping, highly energetic, endothermic peaks on heating above T(m); corresponding changes in the first-order spacings are observed by SAXS, accompanied by two different, complex patterns of reflections in the WAXS region. The WAXS data show that there is a difference in hydrocarbon chain packing, but no difference in bilayer dimensions or hydrocarbon chain tilt for these two L(c) phases (termed L(c1) and L(c2), respectively). Continued heating of suitably annealed, shorter chain rac-beta-D-GalDAGs from the L(c2) phase results in a phase transition to an L(alpha) phase and, on further heating, to the same Q(II) or H(II) phases observed on first heating. On reheating annealed samples with longer chain lengths, a subgel phase is formed. This is characterized by a single, poorly energetic endotherm visible below the T(m). SAXS/WAXS identifies this event as an L(c)/L(beta) phase transition. However, the WAXS reflections in the di-16:0 lipid do not entirely correspond to the reflections seen for either the L(c1) or L(c2) phases present in the shorter chain rac-beta-D-GalDAGs; rather these consist of a combination of L(c1), L(c2) and L(beta) reflections, consistent with DSC data where all three phase transitions occur within a span of 5 degrees C. At very long chain lengths (N> or =19), the L(beta)/L(c) conversion process is so slow that no L(c) phases are formed over the time scale of our experiments. The L(beta)/L(c) phase conversion process is significantly faster than that seen in the corresponding rac-beta-D-GlcDAGs, but is slower than in the 1,2-sn-beta-D-GalDAGs already studied. The L(alpha)/NL phase transition temperatures are also higher in the rac-beta-D-GalDAGs than in the corresponding rac-beta-D-GlcDAGs, suggesting that the orientation of the hydroxyl at position 4 and the chirality of the glycerol molecule in the lipid/water interface influence both the L(c) and NL phase properties of these lipids, probably by controlling the relative positions of hydrogen bond donors and acceptors in the polar region of the membrane.