Effect Of Vesicle Size Research Articles

Effect of Vesicle Size on the Cytolysis of Cell-Penetrating Peptides (CPPs).

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the mechanism of cytolysis as a temporal and local phase transfer of membrane lipid caused by positive membrane curvature generation. Moreover, we showed how to control the CPP cytolysis. Here, we investigate the phospholipid vesicle’s size effect on CPP cytolysis because this is the most straightforward way to control membrane curvature. Contrary to our expectation, we found that the smaller the vesicle diameter (meaning a higher membrane curvature), the more cytolysis was suppressed. Such controversial findings led us to seek the reason for the unexpected results, and we ended up finding out that the mobility of membrane lipids as a liquid crystal is the key to cytolysis. As a result, we could explain the cause of cytolysis suppression by reducing the vesicle size (because of the restriction of lipid mobility); osmotic pressure reduction to enhance positive curvature generation works as long as the membrane is mobile enough to modulate the local structure. Taking all the revealed vital factors and their effects as a tool, we will further explore how to control CPP cytolysis for developing a DDS system combined with appropriate cargo selection to be tagged with CPPs.

Effect of Vesicle Size on the Prodan Fluorescence in Diheptadecanoylphosphatidylcholine Bilayer Membrane under Atmospheric and High Pressures

The bilayer phase behavior of diheptadecanoylphosphatidylcholine (C17PC) with different vesicle sizes (large multilamellar vesicle (LMV) and giant multilamellar vesicle (GMV)) was investigated by fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan under atmospheric and high pressures. The difference in phase transitions and thermodynamic quantities of the transition was hardly observed between LMV and GMV used here. On the contrary, the Prodan fluorescence in the bilayer membranes changed depending on the size of vesicles as well as on the phase states. From the second derivative of fluorescence spectra, the three-dimensional image plots in which we can see the location of Prodan in the bilayer membrane as blue valleys were constructed for LMV and GMV under atmospheric pressure. The following characteristic behavior was found: (1) the Prodan molecules in GMV can be distributed to not only adjacent glycerol backbone region, but also near bulk-water region in the lamellar gel or ripple gel phase; (2) the blue valleys of GMV became deeper than those of LMV because of the greater surface density of the Prodan molecules per unit area of GMV than LMV; (3) the liquid crystalline phase of the bilayer excludes the Prodan molecules to a more hydrophilic region at the membrane surface with an increase in vesicle size; (4) the accurate information as to the phase transitions is gradually lost with increasing vesicle size. Under the high-pressure condition, the difference in Prodan fluorescence between LMV and GMV was essentially the same as the difference under atmospheric pressure except for the existence of the pressure-induced interdigitated gel phase. Further, we found that Prodan fluorescence spectra in the interdigitated gel phase were especially affected by the size of vesicles. This study revealed that the Prodan molecules can move around the headgroup region by responding not only to the phase state but also to the vesicle size, and they become a useful membrane probe, detecting important membrane properties such as the packing stress.

Pressure study on symmetric and asymmetric phospholipid bilayers: effect of vesicle size on Prodan fluorescence

The bilayer phase behavior of symmetric and asymmetric phosphatidylcholines (PCs), 1,2-diheptadecanoyl-PC (C17PC), 1-palmitoyl-2-stearoyl-PC (PSPC), and 1-stearoyl-2-palmitoyl-PC (SPPC), with different vesicle sizes were investigated by a high-pressure fluorescence method using the polarity-sensitive fluorescent probe Prodan. The second derivative of fluorescence spectra for all the PCs of small-sized vesicle showed four minima characteristic of four membrane states on the spectra irrespective of the acyl-chain symmetry, whereas those of large-sized vesicle had one more minimum originating from the most hydrophilic site at the membrane surface. These findings indicate that Prodan molecules can distribute into multiple sites in the bilayer and move around the head-group region depending on the vesicle size. The behavior of the spectra in the SPPC bilayer suggested that the interdigitated gel phase had a less polar "pocket" formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains. It turned out that the curvature of vesicles affects the distribution of the Prodan molecules in all phases, more particularly in the interdigitated gel phase.

Kinetic Analysis of Arf GAP1 Indicates a Regulatory Role for Coatomer

Arf GAPs are a family of enzymes that catalyze the hydrolysis of GTP bound to Arf. Arf GAP1 is one member of the family that has a critical role in membrane traffic at the Golgi apparatus. Two distinct models for the regulation of Arf GAP1 in membrane traffic have been proposed. In one model, Arf GAP1 functions in a ternary complex with coat proteins and is inhibited by cargo proteins. In another model, Arf GAP1 is recruited to a membrane surface that has defects created by the increased membrane curvature that accompanies transport vesicle formation. Here we have used kinetic and mutational analysis to test predictions of models of regulation of Arf GAP1. We found that Arf GAP1 has a similar affinity for Arf1.GTP as another Arf GAP, ASAP1, but the catalytic rate is approximately 0.5% that of ASAP1. Coatomer stimulated Arf GAP1 activity; however, different from that predicted from the current model, coatomer affected the K(m) and not the k(cat) values. Effects of most mutations in Arf GAP1 paralleled those in ASAP1. Mutation of an arginine that aligned with an arginine presumed to be catalytic in ASAP1 abrogated activity. Peptide from the cytoplasmic tail of cargo proteins inhibited Arf GAP1; however, the unrelated Arf GAP ASAP1 was also inhibited. The curvature of the lipid bilayer had a small effect on activity of Arf GAP1 under the conditions of our experiments. We conclude that coatomer is an allosteric regulator of Arf GAP1. The relevance of the results to the two models of Arf GAP1-mediated regulation of Arf1 is discussed.

Interactions between cationic liposomes and bacteria: the physical-chemistry of the bactericidal action

The bactericidal effect of dioctadecyldimethylammonium bromide (DODAB), a liposome forming synthetic amphiphile, is further evaluated for Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus in order to establish susceptibilities of different bacteria species towards DODAB at a fixed viable bacteria concentration (2.5 × 107 viable bacteria/mL). For the four species, susceptibility towards DODAB increases from E. coli to S. aureus in the order above. Typically, cell viability decreases to 5% over 1 h of interaction time at DODAB concentrations equal to 50 and 5 μm for E. coli and S. aureus, respectively. At charge neutralization of the bacterial cell, bacteria flocculation by DODAB vesicles is shown to be a diffusion-controlled process. Bacteria flocculation does not yield underestimated counts of colony forming units possibly because dilution procedures done before plating cause deflocculation. The effect of vesicle size on cell viability demonstrates that large vesicles, due to their higher affinity constant for the bacteria (45.20 m-1) relative to the small vesicles (0.14 m-1), kill E. coli at smaller DODAB concentrations. For E. coli and S. aureus, simultaneous determination of cell viability and electrophoretic mobility as a function of DODAB concentration yields a very good correlation between cell surface charge and cell viability. Negatively charged cells are 100% viable whereas positively charged cells do not survive.▪The results show a clear correlation between simple adsorption of entire vesicles generating a positive charge on the cell surfaces and cell death.—Campanhã, M. T. N., E. M. Mamizuka, and A. M. Carmona-Ribeiro. Interactions between cationic liposomes and bacteria: the physical-chemistry of the bactericidal action. J. Lipid Res. 1999. 40: 1495–1500.

Effect of vesicle size on their interaction with class A amphipathic helical peptides

Throughout the life span of a lipoprotein particle, the type and number of exchangeable apolipoproteins on its surface varies with particle size, suggesting a role of surface curvature on the lipid-binding properties of these proteins. Peptides 18A, Ac-18A-NH2, Ac-18R-NH2, 37pA, and 37aA have been designed to investigate the lipid-binding properties of the amphipathic alpha-helix structural motif that appears to modulate the lipid-binding properties of the exchangeable plasma apolipoproteins. We report here the results of a quantitative thermodynamic characterization of the effects of modifying helix length and of varying both the location of charged residues about the polar face of the peptides and vesicle size on the lipid affinity and depth of bilayer penetration for model amphipathic alpha-helices. Partition coefficients, Kp, were determined by fluorescence spectroscopy, and binding enthalpies, deltaH, by titration calorimetry. The results indicate that Kp values are on the order of 10(5), with similar deltaG(o) values for the interactions of the peptides with vesicles of various sizes. It appears that a class A motif and increased alpha-helical content optimize binding for 18-residue peptides. The interactions of the model peptides with 20 nm SUV are enthalpically driven with small, negative entropy changes; however, interactions for larger vesicles are entropically driven, likely due to disordering of bilayer hydrocarbon chains. Thermodynamic data indicate that 37pA and 37aA induce greater disordering of bilayer hydrocarbon chains than Ac-18A-NH2. The results of this study suggest that the type of interaction, i.e., enthalpically or entropically driven, may be modulated by the lateral compressibility of the bilayer membrane.

Interaction, Lipid Exchange, and Effect of Vesicle Size in Systems of Oppositely Charged Vesicles

The interactions between charged unilamellar vesicles of different size prepared from mixtures of egg lecithin (EPC1), cholesterol, and a charged component, either stearylamine or dicetyl phosphate, were investigated. Several techniques, i.e. fluorescence spectroscopy, 133Cs-NMR, light scattering measurements, and electron and optical microscopies in dilute aqueous solutions, provided evidence that vesicles bearing opposite charges interact via contact followed by lipid exchange; the progressive charge neutralization is shown by 133Cs-NMR to occur without fusion of vesicle internal pools. Vesicle size determines as diverse features as contact duration, extent of lipid exchange, and final distribution of surface charge among the vesicle mixture.

Effect of vesicle size and lipid composition on the in vivo tumor selectivity and toxicity of the non-cross-resistant anthracycline annamycin incorporated in liposomes.

Annamycin (Ann) is a non-cross-resistant lipophilic anthracycline antiobiotic optimally suited for liposome delivery. We studied how vesicle size and presence of phospholipids with a high phase transition temperature and monosialoganglioside (GM I) in the liposome bilayers affect the pharmacokinetics, tumor selectivity and toxicity of Ann. Entrapment of Ann in multilamellar vesicles (L-Ann) resulted in a 20% lower heart AUC and a 30-40% higher tumor and liver AUC. Reduction of the liposome size from 1.6 to 0.03 microns increased Ann plasma circulation time and tumor AUC by 2-fold, enhanced Ann tumor selectivity and decreased Ann subacute toxicity by 2-fold. The presence of phospholipids with a high phase transition temperature and GMI in the liposome bilayers further prolonged Ann plasma circulation time by 2- to 4-fold, did not increase Ann tumor AUC and moderately increased Ann subacute toxicity. The anti-tumor activity of Ann correlated with the tumor AUC achieved with each particular formulation. Our results strongly suggest that vesicle size may be an important determinant of the therapeutic index of liposomal Ann, but they fail to demonstrate a beneficial tumor-targeting effect of liposomes composed of GMI and phospholipids with a high phase transition temperature, as has been reported for the hydrophilic parent compound doxorubicin.

Effect of vesicle size on in vivo release of daunorubicin from hydrogenated egg phosphatidylcholine-based liposomes into blood circulation.

The effect of the vesicle size on in vivo release of daunorubicin, an anthracycline anticancer drug, from liposomes into the circulation was studied in rats. The liposomes were prepared from hydrogenated egg phosphatidylcholine (HEPC) or egg phosphatidylcholine (EPC), cholesterol and dicetylphosphate at a molar ratio of 5:4:1, and their mean vesicle sizes were adjusted to about 50 and 100 nm. The drug retained in the liposomes and the drug that leaked were separated from plasma by gel filtration. On the assumption that the lipid content does not change, the drug release from each liposome preparation was estimated from the latency (%) calculated from the drug/lipid concentration ratio of the liposome preparation. From HEPC-liposomes with a diameter of 50 nm, the drug was released gradually after intravenous administration, and the cumulative percent release of the drug reached 40% after 240 min. However, from EPC-liposomes with the same size, 50% of the drug was released within 5 min, and more than 90% of the drug was released within 60 min. From HEPC-liposomes with a diameter of 100 nm, no drug release was observed for 240 min after administration. These findings indicate that the vesicle size and the lipid composition of liposome preparation affect the in vivo drug release.

Direct Enthalpy Measurements of Factor X and Prothrombin Association with Small and Large Unilamellar Vesicles

Isothermal titration calorimetry was used to determine the enthalpy for the calcium-dependent protein conformation change and subsequent interaction of blood clotting factor X and prothrombin with phospholipid vesicles. The effect of vesicle size was also determined. The protein conformation change was accompanied by -12 +/- 1 and -7 to -15 kcal/mol for factor X and prothrombin, respectively. The range of values for prothrombin arose from use of different protein preparations and may be due to non-ideal behavior of this protein when calcium was added. The apparent enthalpy of association (delta H(assoc)) of both factor X and prothrombin with phosphatidylserine (PS)/phosphatidylcholine (PC) large unilamellar vesicles (LUVs, 120 nm diameter) was shown to be near 0 kcal/mol. In comparison, delta H(assoc) for interaction with PS/PC small unilamellar vesicles (SUVs, 40 nm diameter) was -9 +/- 3 and -7 +/- 2 kcal/mol for factor X and prothrombin, respectively. This difference appeared complementary to delta H(assoc) for calcium binding to these vesicles. That is, the interaction of calcium was athermic with SUVs and exothermic with LUVs. While such properties might suggest a considerable difference in the manner of calcium binding to LUVs versus SUVs, little difference in the quantity of calcium bound to SUVs and LUVs was detected by equilibrium dialysis. In any event, the results indicate that protein binding to LUVs was primarily entropy driven whereas binding to SUVs was primarily enthalpy driven. The exothermic process for calcium-dependent factor X or prothrombin binding to SUVs may result from protein-induced changes in the phospholipid packing/calcium interaction, possibly related to changes in how calcium is bound to the phospholipid.

Effect of vesicle size on the polymerization of a diacetylene lipid

The effect of vesicle size on the photopolymerization products of the polymerizable diacetylene lipid 1,2-bis(tricose-10,12-dignoyl)-sn-glycero-3-phosphocholine (DC 8,9 PC) has been examined using SEC and mass spectrometry. Lipid polymers from 50-, 100-, 300-, end 600-nm vesicles have the seme molecular weight range with a prominent polystyrene equivalent molecular weight peak between 1.6×10 6 end 2.2×10 4 . The observed intensity of this peak increased as vesicle size was increased from 50- through 300-nm diameter, but was decreased in 600-nm diameter vesicles

Effect of vesicle size, polydispersity and multilayering on solid state 31P- and 2H-NMR spectra

In this paper, we investigate upon the effect of isotropic motions on 31P- and 2H-NMR spectral lineshapes. The effect of size, polydispersity and number of bilayers in tumbling vesicles is taken into account by considering both the rotational diffusion of the small structure and the lateral diffusion of a molecule on the curved surface. Spectral simulations considering quadrupolar or chemical shift anisotropy interactions show that spectral lineshapes are very sensitive to the above parameters and may be used to probe size and polydispersity of unilamellar vesicles or to determine the number of bilayers and the size of the inner water core of multilamellar vesicles.

13C-NMR determination of the molecular dynamics of cholesterol in dimyristoylphosphatidylcholine vesicles

Using 13C-NMR measurements of T 1, T 2 and the nuclear Overhauser enhancement factor at 50.32, 90.56 and 150.87 MHz, we have measured the dynamics of cholesterol in dimyristoylphosphatidylcholine (DMPC) vesicles from 28 to 50°C. Using the model-free approach of Lipari and Szabo, we have found that at 37°C the motion of the rigid steroid ring can be described by an equal contribution from two effective motions with correlation times of 63 and 0.85 ns. The C 26 and C 27 carbon atoms of cholesterol were found to have an effective correlation time of 8±2 ps and a value for the square of the generalised order parameter of 0.03±0.01. The corresponding values for the C 25 carbon atom were 17±4 ps and 0.09±0.02, showing slower motion and greater order for this carbon atom, which is nearer to the rigid steroid ring. Apart from the effect of vesicle size on T 2, no concentration dependence of the dynamics of cholesterol was detected over the cholesterol concentration range 2–30 mol%. The order parameters and correlation times from the present 13C-NMR experiments are shown to be compatible with those from 2H-NMR experiments. This establishes the validity of the present approach, which we are currently extending to low concentrations of cholesteryl oleate in DMPC vesicles.

The design of liposomal paramagnetic MR agents: effect of vesicle size upon the relaxivity of surface-incorporated lipophilic chelates.

The 1/T1 NMRD profiles of lipid vesicles with the paramagnetic ion Gd attached via a chelate covalently linked to the membrane surface show a peak at approximately 20 MHz indicating that fluctuations of approximately 10(-8) s contribute to the form of the dispersion profile. If the correlation time for fluctuations of the paramagnetic chelate on the membrane surface is much less than the correlation time for rotation of the lipid vesicle, it would be expected that the measured 1/T1 relaxation rate for solvent protons should be invariant with vesicle size above a certain minimum vesicle diameter. We show that this is indeed the case for vesicles in the size range 50 to 400 nm average diameter and discuss general design considerations for the preparation of vesicle-associated MR contrast agents based upon paramagnetic chelates either trapped within the vesicle interior or attached to the membrane surface.

Complex carbohydrate-lectin interaction at the interface: a model for cellular adhesion. I. Effect of vesicle size on the kinetics of aggregation between a fatty acid conjugate of lectin and a liposomal asialoganglioside

Two types of phospholipid vesicles capable of mutual recognition have been tailor-made to serve as a model system for the study of carbohydrate-mediated cellular adhesion. One of the vesicles contained a fatty acid conjugate of a galactose specific lectin (lectin vesicle_ and the other an asialoganglioside with a reactive terminal galactose residue (galactose vesicle). The kinetics of aggregation of these two types of vesicles was followed by monitoring time-dependent change in turbidity. A 10–100-fold enhancement in the forward rate constant ( k f ranging from 7.1 · 10 5 to 4.5 · 10 7 M −1 · s −1 at 27° C) was observed when compared with that for the lectin-galactose system in solution ( k t) being 4.5 · 10 5 M −1 · s −1), reported in the literature. A study of the influence of vesicle size on the rate of aggregation showed that enhancement depended on the curvature of the galactose vesicle rather than the density of asialoganglioside suggesting a possible diffusion in the plane of the membrane. The ratio, k f/ k d is found to be approx. 10 10 M −1 indicating that the formation of multiple bonds plays a role for stable adhesion.

A flourescent probe study of the effect of size on the properties of dioctadecyldimethylammonium chloride vesicles

Fluorescence techniques are used to study the effect of size on the properties of dioctadecyldimethylammonium chloride vesicles. The solubilization site and the deactivation rates of excited pyrene by bromide counterions and oxygen are markedly dependent on vesicle size at low temperature and strongly affected by the gel-liquid crystalline phase transition. The results obtained are accounted for by considering the structural differences between large and small vesicles. It is shown that the temperature dependence of the interaction rate between the probe and oxygen can used to monitor the changes in vesicle fluidity accompanying the gel-liquid cystalline phase transition and the sensitivity of the fluidity and phase transition temperature to the incorporation of additives ( n-octanol). The effect of vesicle size upon the capacity to incorporate n-alkanols (from hexanol to nonanol) and carbon tetrachloride was also examined. From the measurement of the partition constants in both vesicles it was found that, at 22°C, the small vesicles are better solvents for all the solutes studied. The solvent capacity of the large vesicles toward heptanol improves with the amount of alkanol incorporated. These results can also be rationalized in terms of the more ordered packing of the large vesicles below the phase transition temperature.

Liposomal sustained-release delivery systems for intravenous injection. II. Design of liposome carriers and blood disposition of lipophilic mitomycin C prodrug-bearing liposomes.

Various types of unilamellar liposomes possessing a narrow size distribution were prepared by controlled dialysis and their blood clearance was studied in mice and rats to assess their suitability as drug carriers for intravenously injectable liposomal sustained-release delivery systems. Also, the utility of these liposomal carrier systems combined with lipophilic prodrugs of mitomycin C (MMC) was evaluated. The fate of the liposomes was monitored uisng N-4-nitrobenzo-2-oxa-1, 3-diazole-dipalmitoylphosphatidylethanolamine (NBD-PE), a liposomal membrane marker. The effect of vesicle size on the blood clearance was investigated for neutral liposomes. Small-sized (S-) liposomes (90±15 nm) were cleared slowly compared with medium-sized (M-) liposomes (181±31 nm) and large-sized liposomes (281±38 nm). Surface charge was also an important determinant of the disposition of small-sized liposomes. S-Liposomes were retained in the circulation longer than positively (S+-) and negatively (S--) charged liposomes. The integrity of S-liposomes in the circulation was examined by simultaneous determination of NBD-PE and carboxyfluorescein (CF) entrapped in the liposomal membrane and liposomal aqueous phase, respectively. CF administered in the liposome-encapsulated form was cleared slowly in a fashion similar to NBD-PE, while free CF, administered as an aqueous solution, was rapidly removed from the circulation. These results reveal that S-liposomes show the best pharmacokinetic properties as a carrier vehicle for intravenously injectable sustained-release delivery systems. S-Liposomes loaded with the lipophilic MMC prodrug, N-(cholesteryloxycarbonyl)glycyl MMC or cholesteryloxyacetyl MMC, successfully gave sustained blood levels of the parent drug following intravenous injection. Thus, the potential utility of MMC prodrug-bearing S-liposomes as an intravenously injectable MMC sustained-release dosage form was demonstrated.

Effect of vesicle size on the clearance, distribution, and tumor uptake of temperature-sensitive liposomes.

The blood clearance, tissue distribution, and tumor uptake of temperature-sensitive liposomes containing the anticancer drug cytosine [3H]-1-beta-D-arabino-furanoside (Ara-C) or [3H]-methotrexate (MTX) were measured. Large unilamellar liposomes composed of a mixture of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (4:1 by weight) were formed by reverse-phase evaporation and sequentially extruded through polycarbonate membranes with pore sizes of 0.4, 0.2, 0.1, 0.08, and 0.05 micron. The liposomes were injected intravenously via the tail vein into B6D2F1 mice containing solid tumors (M5076 ovarian carcinoma) implanted in both hind feet. Drug release, organ distribution, and tumor uptake were studied during and following local hyperthermia treatments (42 degrees C, 60 minutes). Local hyperthermia increased the relative amount of liposome encapsulated drug delivered to a heated solid tumor compared with the unheated control. The clearance studies showed that liposome blood clearance was inversely proportional to liposome size. However, tumor uptake was not significantly affected by reducing liposome size. In addition, for the range of liposome sizes studied, the majority of the liposome encapsulated drug was found in the liver, spleen, and kidney; not in the heated tumor.

NMR studies on phospholipid bilayers. Some factors affecting lipid distribution

1. 1. 1H-NMR and 31P-NMR are used to measure the outside/inside distribution of phospholipids in mixed vesicles. 2. 2. Ferricyanide is a suitable shift reagent for measuring the outside/inside ratio of lecithin using 1H-NMR even when the phopholipid mixture contains negative lipids 3. 3. 31P-NMR can be used to measure the distribution of all phospholipids present provided the resonances are separated. 4. 4. At 36.4 MHz the inside and outside phosphorus in lecithin vesicles have different chemical shifts. The separation at room temperature is 4–5 Hz and the individual linewidths are about 4Hz. 5. 5. In a mixture of lecithin with phosphatidylethanolamine the latter has preference for the inside layer of the bilayer. The same holds for mixtures of lecithin with phosphatidylserine, phophatidylinositol and phosphatidic acid. 6. 6. In Mixture of lecithin and phosphatidylserine the preference of the latter for the inside is increased at lower pH under which conditions the negative charge of the phosphatidylserine is decreased. 7. 7. In mixtures of lecithin with sphingomyelin the lecithin has a higher concentration at the inside. 8. 8. The effect of vesicle size on the 31P-NMR linewidth and the temperature dependence of this linewidth is in agreement with the conclusion of Berden et al. (FEBS Lett. (1974), 46, 55–58) that the chemical shift anisotropy, modulated by the isotropic tumbling of the vesicles, makes a contribution to the linewidth. The chemical shift difference between outside and inside phosphorus can be used as a parameter for the measurement of the packing density at the inside and of the size of the vesicles. 9. 9. It is concluded that both charge and the packing properties of the headgroup are major factors in determining the distribution of phospholipids in mixed vesicles.