Conformational state diagram of DOPC/DPPCd62/cholesterol mixtures

Raman spectra of aqueous suspensions of vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), deuterated 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC_d62), and cholesterol (Chol) were studied at room temperature to determine the conformational states of the phospholipid hydrocarbon chains. Deuteration of DPPC_d62 allowed us to characterize the conformational states of DOPC and DPPC_d62 independently. The parameters of Raman peaks, which are sensitive to the conformational order, were studied in a wide range of compositions. It was found that the DOPC molecules are conformationally disordered for all compositions. The conformational state of the DPPC_d62 molecules changes with composition. Their conformational state is influenced by cholesterol-induced partial disordering and DOPC solvation, transforming the DPPC molecules into the disordered state. The conformational state diagram from the Raman experiment was compared with outcomes from the differential scanning calorimetry (DSC) experiment. The Raman spectra also revealed that the DPPC molecules coexist in the disordered and all-trans ordered states for the DOPC/DPPC_d62/Chol mixtures except for the pure liquid-disordered phase.     

Characterization of the interaction between liposomal formulations and Pseudomonas aeruginosa

The interactions between three liposomal formulations and Pseudomonas aeruginosa cells were evaluated by a lipid mixing assay and electron paramagnetic resonance (EPR) spectroscopy. The effect of the bacteria on the liposomal phase characteristics, the release of the liposomes’ content, and the uptake rate of gentamicin by bacteria were monitored as a function of time, using EPR spectroscopy. The [16-DSA uptake]_Total from DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) liposomes reached 93?±?12% over a 3-hour assay period, of which 9% crossed the bacterial inner membrane. A small amount of 16-DSA uptake from DPPC/Chol (cholesterol) vesicles was found throughout the 3-hour period of time. Although DPPC/DMPG (dimyristoylphosphatidylglycerol) vesicles showed a smaller value of [16-DSA uptake]_Total with respect to that of DPPC vesicles, they appeared to be effective in disrupting the bacterial membrane, resulting in a greater accumulation of 16-DSA inside the inner membrane. Exposure to bacteria caused the DPPC/Chol, DPPC, and DPPC/DMPG formulations to release 4.6?±?1.5, 17.6?±?1.2, and 34?±?3.7% of their content, respectively. Time-dependent fluid regions were developed within the vesicles when mixed with bacteria, and their growth over time depended on liposomal formulations. Incubation of gentamicin with bacteria for 3 hours resulted in 87?±?3% of the drug crossing the bacterial inner membrane. In conclusion, interaction between the liposome drug carriers and the bacterial cells result in vesicle fusion, disruption of the bacterial membrane, release of the liposomal content in the close vicinity of the bacteria cells, and the subsequent intracellular uptake of the released liposomal content.     

Degradation of bacterial DNA by a natural antimicrobial agent with the help of biomimetic membrane system

The antimicrobial efficacy of methylglyoxal (MG) against several gram-negative bacteria including Escherichia coli has been reported. To determine the mechanism of action of MG, molecular interactions between lipid and MG within the liposomal membrane were also investigated. Multilamellar and unilamellar vesicles were prepared from 1, 2-dipalmitoyl-snglycero-3-phosphocholine (DPPC). The effect of MG on DPPC liposomal membrane was studied by fluorescence spectroscopy and differential scanning calorimetry. The results indicate that MG interacts mainly with the DPPC head group that produces a significant increase in the fluidity of liposomal vesicles, which could be the cause of a fusion/aggregation effect in microbial cells. The agarose gel electrophoresis study with the genomic DNA extracted from E. coli ATCC 25922 revealed that addition of MG could completely degrade this DNA within 1 h, pointing out to their distinctly high degree of sensitivity towards MG. Further, the drug was able to cross the cell membranes, penetrating into the interior of the cell and interacting with DNA for demonstrating antibacterial activity of MG.     

Effects of ursolic acid on the structural and morphological behaviours of dipalmitoyl lecithin vesicles

Effects of ursolic acid on the structural and morphological characteristics of dipalmitoyl lecithin(DPPC)-water system was studied by using differential scanning calorimetry (DSC), small- and wide-angle X-ray scattering (SWAXS), freeze-fracture method combined with transmission electron-microscopy (FF-TEM) and infrared spectroscopy (FT-IR). The surface of the uncorrelated lipid system is rippled or grained and a huge number of small, presumably unilamellar vesicles are present if the UA/DPPC molar ratio is 0.1 mol/mol or higher. Besides the destroyed layer packing of regular multilamellar vesicles, non-bilayer (e.g. cubic or hexagonal) local structures are evidenced by SAXS and FF-TEM methods. The ability of UA to induce non-bilayer structures in hydrated DPPC system originates from the actual geometry form of associated lipid and UA molecules as concluded from the FT-IR measurements and theoretical calculations. Beside numerous beneficial e.g. chemopreventive and chemotherapeutic effect of ursolic acid against cancer, their impact to modify the lipid bilayers can be utilized in liposomal formulations.     

Physicochemical study of the protein–liposome interactions: influence of liposome composition and concentration on protein binding

Abstract

The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and foetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution, and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio), and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and CH both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing CH and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), CH, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (F_s), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when F_s is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.     

Oxidized phospholipids induce phase separation in lipid vesicles

The thermal behaviour of phospholipid multilamellar vesicles (MLV) made of various molar percentages of DPPC and LPPC, containing also oxidized LPPC (LPPCox), was studied by use of EPR spectroscopy and n-DSPC spin label in order to determine variations in the membrane fluidity brought about by lipid oxidation. Experimental variables were temperature, ranging from 4 to 44 degrees C, and molar percentage composition of DPPC/LPPC/LPPCox ternary mixture. We found that the presence of LPPCox in a percentage higher than both normal phospholipids' heavily hindered membrane formation, while lower percentage of the oxidized lipid with higher DPPC percentages yielded two-components EPR spectra, showing the presence of two different fluidity domains, indicative of membrane phase separation. When LPPC was the dominant lipid in the ternary mixture, simple EPR spectra were observed, indicating homogeneity of MLV membranes. Phase separation observed in the presence of LPPCox was better visible at lower temperature (12 degrees C or less), and almost disappeared with increasing temperature (36 degrees C or more). Furthermore, the correlation time of 16-DSPC in ternary mixture MLVs with higher LPPC percentage (homogeneous membranes) was not affected by the presence of LPPCox, while it normally increased upon DPPC percentage increase, as readily calculated from the EPR spectra featuring simple bands at 24 degrees C. It is concluded that oxidized lipid induces phase separation in more rigid DPPC-rich membranes, while leaving fluidity unaffected in more fluid LPPC-rich membranes, and at higher temperature.     

The effects of temperature,salinity, concentration and PEGylated lipid on the spontaneous nanostructures of bicellar mixtures

The self-assembling morphologies of low-concentration (mostly 1 and 10 mg/mL) bicellar mixtures composed of zwitterionic dipalmitoyl (di-C₁₆) phosphatidylcholine (DPPC), dihexanoyl (di-C₆) phosphatidylcholine (DHPC), and negatively charged dipalmitoyl (di-C₁₆) phosphatidylglycerol (DPPG) were investigated using small angle neutron scattering, dynamic light scattering and transmission electron microscopy. A polyethylene glycol conjugated (PEGylated) lipid, distearoyl phosphoethanolamine-[methoxy (polyethyleneglycol)-2000] (PEG2000-DSPE), was incorporated in the system at 5 mol% of the total lipid composition. The effects of several parameters on the spontaneous structures were studied, including temperature, lipid concentration, salinity, and PEG2000-DSPE. In general, nanodiscs (bicelles) were observed at low temperatures (below the melting temperature, T_M of DPPC) depending on the salinity of the solutions. Nanodisc-to-vesicle transition was found upon the elevation of temperature (above T_M) in the cases of low lipid concentration in the absence of PEG2000-DSPE or high salinity. Both addition of PEG2000-DSPE and high lipid concentration stabilize the nanodiscs, preventing the formation of multilamellar vesicles, while high salinity promotes vesiculation and the formation of aggregation. This study suggests that the stability of such nanodiscs is presumably controlled by the electrostatic interactions, the steric effect induced by PEG2000-DSPE, and the amount of DHPC located at the disc rim.     

Temperature-dependent osmotic permeability in glycoproteion containing liposomes

The osmotic water outflow of large multilamellar liposomes containing ₁-acid glycoprotein was measured at a temperature near the lipid's phase transition temperature. The liposomes were formed from a mixture of DPPC, cholesterol and glycoprotein in molar ratios 100:20:1, by continuous sucrose density gradient centrifugation. These liposomes captured 35% of the radiolabeled glycoprotein. The temperature-dependent experiments showed that near phase transition temperature the initial rate of water outflow increased drastically in comparison with glycoprotein free liposomes incubated in buffer containing glycoprotein. We suggested that eventual a channel mechanism may be involved due to spontaneous incorporation of glycoprotein into the bilayer.     

The use of fluorescence energy transfer to distinguish between poly(ethylene glycol)-induced aggregation and fusion of phospholipid vesicles

Two fluorescence energy transfer assays for phospholipid vesicle-vesicle fusion have been developed, one of which is also sensitive to vesicle aggregation. Using a combination of these assays it was possible to distinguish between vesicle aggregation and fusion as induced by poly(ethylene glycol) PEG 8000. The chromophores used were 1-(4′-carboxyethyl)-6-diphenyl-trans-1,3,5-hexatriene as fluorescent ‘donor’ and 1-(4′-carboxyethyl)-6-(4″-nitro)diphenyl-trans-1,3,5-hexatriene as ‘acceptor’. These acids were appropriately esterified giving fluorescent phospholipid and triacylglycerol analogues. At 20°C poly(ethylene glycol) 8000 (PEG 8000) caused aggregation of l-α-dipalmitoylphosphatidylcholine (DPPC) vesicles without extensive fusion up to a concentration of about 35% (w/w). Fusion occurred above this poly(ethylene glycol) concentration. The triacylglycerol probes showed different behaviour from the phospholipids: while not exchangeable through solution in the absence of fusogen, they appeared to redistribute between bilayers under aggregating conditions. DPPC vesicles aggregated with < 35% poly(ethylene glycol) could not be disaggregated by dilution, as monitored by the phospholipid probes. However, DPPC vesicles containing approx. 5% phosphatidylserine which had been aggregated by poly(ethylene glycol) could be disaggregated by either dilution or sonication. Phospholipid vesicles aggregated by low concentrations of poly(ethylene glycol) appear to fuse to multilamellar structures on heating above the lipid phase transition temperature.     

Spin labeling EPR studies of the properties of oxidized phospholipid-containing lipid vesicles

This study aims at characterizing the structure and some properties of phospholipid multi-lamellar vesicles (MLVs) containing the oxidized species γ-palmitoyl-β-(9-hydroperoxy-10,12-octadecanedienoyl)-lecithin (HPPLPC), γ-palmitoyl-β-(9-hydroxy-10,12-octadecanedienoyl)-lecithin (HOPLPC), γ-palmitoyl-β-glutaroyl-lecithin (GlPPC) and γ-palmitoyl-β-azelaoyl-lecithin (AzPPC). Sepharose 4B gel-chromatography was used to ensure and check that only MLVs are used in EPR measurements. Gel-solid to gel-liquid transition temperature (T_m), lateral phase separation, fluidity gradient and polarity profile were studied by use of EPR spectroscopy of enclosed n-doxylstearoyl lecithin spin labels. Contrarily to conjugate dienes and normal phospholipids, pure carboxyacyl species yielded aqueous suspensions showing gel-chromatography elution profile resembling that of lysolecithin micelles. Conjugate dienes/DPPC MLVs showed lateral phase separation at room temperature and T_m value lower than pure DPPC MLVs. Pure conjugate dienes MLVs resembled more PLPC MLVs and displayed free miscibility with PLPC in mixed MLVs. Pure HPPLPC MLV bilayer appeared to be slightly more rigid, while that of HOPLPC and the polarity profile of MLVs made of the pure conjugate dienes species were similar to those of normal PLPC. It is concluded that carboxyacyl lecithins in MLVs tend to disrupt vesicle structure, while conjugated dienes lecithins are more able to affect some physical properties of the bilayer, and that DPPC in MLVs enhances these effects while PLPC shows a better compatibility with the lipoperoxides.     

Interaction of Doxorubicin and Dipalmitoylphosphatidylcholine Liposomes

The interaction between doxorubicin (DOX), an anthracycline antibiotic frequently used in chemotherapy, and zwitterionic dipalmitoylphosphatidylcholine (DPPC) was investigated using Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and rheological measurements. FTIR results showed that DOX shifted the wavenumber of the PO₂ ⁻ band for pure DPPC to a higher wavenumber. This may have been because of the strong interactions between the NH₃ ⁺ group in DOX and the phosphate (PO₂ ⁻) group in the polar head of DPPC. The main transition temperature of DPPC liposomes was slightly shifted to a lower temperature for DPPC liposome-encapsulated DOX. This suggested that DOX had a significant effect on the acyl chains in the DPPC bilayers, and that its presence decreased the transition cooperativity of lipid acyl chains. There was also the appearance of an additional transition peak at nearly 136°C for the DPPC/DOX sample. These interactions between DOX and DPPC phospholipid would cause a decrease in the DPPC liposomes plastic viscosity and increase membrane fluidity. A better understanding of the interactions between DOX and lipid bilayers could help in the design and development of improved liposomal drug delivery systems.     

Tobacco mosaic virus protein induces fusion of liposome membranes

The fusogenic properties of tobacco mosaic virus (TMV) coat protein were investigated. Tobacco mosaic virus protein induces membrane fusion of a population of L-alpha-dimyristoylphosphatidylcholine (DMPC) and DL-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles giving rise to larger particles as seen by a drastic absorbance increase of the liposomal solution. Differential scanning calorimetry spectra demonstrate complete mixing of the acyl chains of the lipids during fusion. Electron micrographs indicate that the fused entities are multilamellar.     

Amyloglucosidase enzymatic reactivity inside lipid vesicles

Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG) (EC 3.2.1.3) from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, V _max and K _m , were determined by initial velocity measurements, and K _i was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose) formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations.     

Change in membrane fluidity induced by lectin-mediated phase separation of the membrane and agglutination of phospholipid vesicles containing glycopeptides

Changes in membrane fluidity induced by lectin addition to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles containing synthetic glycopeptides were measured by depolarization of the fluorescent probes 8-anilino-1-naphthalenesulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH). In the present synthesized glycopeptides, N-acetylglucosamine (GlcNAc) and a tripeptide were connected by aliphatic chains of different lengths. A pyrenyl group, which is introduced to the peptide moiety, acted as a probe to characterize the distribution of glycopeptides in the membrane on the basis of its excimer formation. The glycopeptide was shown to be distributed to DPPC vesicles with the peptide moiety buried in the hydrophobic core of the lipid bilayer and the glyco moiety exposed to the outside of the membrane. By the addition of wheat germ agglutinin (WGA) to the vesicles containing the glycopeptides, intravesicular cross-linking of glycopeptides in the membrane and aggregation of vesicles were observed. The intravesicular cross-linking was antagonized by GlcNAc above the phase transition temperature. However, the dissociation of aggregation required the addition of a stronger antagonist, N,N'-diacetylchitobiose. The addition of the glycopeptide to DPPC vesicles above the phase transition temperature decreased the membrane fluidity. However, a succeeding addition of WGA caused a large increase of membrane fluidity at either the surface or the hydrophobic core of the lipid bilayer membrane. This increase of membrane fluidity was attributed to two factors by use of two kinds of antagonists having different potencies: one is a WGA-mediated cross-linking of glycopeptides in the membrane, and the other is a close contact of vesicles on aggregation.     

Interaction of the cholesterol reducing agent simvastatin with zwitterionic DPPC and charged DPPG phospholipid membranes

Simvastatin is a lipid-lowering drug in the pharmaceutical group statins. Interaction of a drug with lipids may define its role in the system and be critical for its pharmacological activity. We examined the interactions of simvastatin with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) and anionic dipalmitoyl phosphatidylglycerol (DPPG) multilamellar vesicles (MLVs) as a function of temperature at different simvastatin concentrations using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The FTIR results indicate that the effect of simvastatin on membrane structure and dynamics depends on the type of membrane lipids. In anionic DPPG MLVs, high simvastatin concentrations (12, 18, 24 mol%) change the position of the CH₂ antisymmetric stretching mode to lower wavenumber values, implying an ordering effect. However, in zwitterionic DPPC MLVs, high concentrations of simvastatin disorder systems both in the gel and liquid crystalline phases. Moreover, in DPPG and DPPC MLVs, simvastatin has opposite dual effects on membrane dynamics. The bandwidth of the CH₂ antisymmetric stretching modes increases in DPPG MLVs, implying an increase in the dynamics, whereas it decreases in DPPC MLVs. Simvastatin caused broadening of the phase transition peaks and formation of shoulders on the phase transition peaks in DSC curves, indicating multi-domain formations in the phospholipid membranes. Because physical features of membranes such as lipid order and fluidity may be changed with the bioactivity of drugs, opposing effects of simvastatin on the order and dynamics of neutral and charged phospholipids may be critical to deduce the action mechanism of the drug and estimate drug-membrane interactions.     

Phase transition behavior of single phosphatidylcholine bilayers on a solid spherical support studied by DSC, NMR and FT-IR

For the first time, the chain melting transition from the gel phase to the liquid crystalline phase of a single DPPC bilayer on a solid, spherical support (silica beads) is observed by differential scanning calorimetry (DSC). This transition is remarkably cooperative, exhibits a transition temperature T_m which is 2°C lower than usually found for DPPC multilamellar vesicles and its excess enthalpy is about 25% less than in DPPC multilayers. 31P- and 2H-NMR data as well as FT-IR data provide evidence that despite the highly asymmetric characteristic of the model system, the whole single bilayer undergoes the transition at T_m, i.e., there is no decoupling of the two monolayer leaflets at the main phase transition. Furthermore, our results show that the formation of the ripple (P_β') phase is inhibited in single bilayers on a solid support. This result confirms a conclusion which we reached previously on the basis of neutron scattering data obtained on planar supported bilayers. The most likely reason for this inhibition as well as for the above mentioned thermodynamic differences between multilamellar vesicles and supported membranes is a significantly higher lateral stress in the latter. Moreover, the exchange of lipids between two populations of spherical supported vesicles (DMPC and chain perdeuterated DMPC) is studied by DSC. It is shown that this exchange process is symmetric and its half-time is a factor of 3-4 higher than observed for small sonicated DMPC vesicles.     

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.     

Liposome-based formulations for the antibiotic nonapeptide Leucinostatin A: Fourier transform infrared spectroscopy characterization and in vivo toxicologic study

Leucinostatin-A is a nonapeptide isolated fromPaecilomyces marquandii Paecilomyces lilacinus A257, andAcremonium sp., exerting remarkable phytotoxic, antibacterial (especially against Gram-positive) and antimycotic activities. With the aim to find alternative formulation for in vivo administration, a number of Leucinostatin-A—loaded liposomal formulations have been prepared and characterized. Both large unilamellar vesicles and multilamellar vesicles consisting of synthetic and natural lipids were evaluated. In addition, to determine the nature of peptide-membrane interactions and the stability of liposomes loaded with Leucinostatin-A, a Fourier Transform Infrared Spectroscopy study was performed. The results suggest that the mode of interaction of the peptide is dependent on its concentration, on bilayer fluidity, and on liposome type. Finally, the LD₅₀ of both free and liposome-delivered Leucinostatin-A was determined in mice. These results suggest that the incorporation of Leucinostatin-A into liposomes may result in decreased Leucinostatin-A toxicity, as the intraperitoneal administration of Leucinostatin-A—loaded liposomes reduced the LD₅₀ of Leucinostatin-A 15-fold.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Characteristics of photosensitization of Pheophorbide a in liposomal media

Pheophorbide a (PPa), a decomposition product of chlorophyll a, is a photosensitizer. The photosensitization mechanisms (Type Ⅰ and Type Ⅱ) of PPa in simple buffer solutions and in buffer solutions containing double-layered DPPC liposomes have been studied using techniques of ESR, spin-trapping, spin-counteraction and laser flash photolysis. The results showed that adding DPPC liposomes to the buffer solution caused an increase of efficiency of generating ~1O_2 and PPa~- by photoactivating PPa. The increase could be ascribed to the disaggregation of hydrophobic PPa caused by the addition of liposomes and the protective effect of liposomal media on the triplet state of PPa. It is concluded that the photosensitization of PPa in liposomal systems is different from that in simple aqueous solutions, and shows higher efficacy. The results will be useful to elucidating the mechanisms of photodynamic therapy of cancer.