MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Cholesterol-free phospholipid domains may be the membrane feature selected by N epsilon-dansyl-L-lysine and merocyanine 540

We have used N epsilon-dansyl-L-lysine as a fluorescent membrane probe, to study cells taken from tissues concerned with immune function. There is a striking similarity between the staining selectivity of this compound and that reported by others for merocyanine 540. Both compounds stain leukemic, human, peripheral leukocytes, an erythroleukemia line, and some mouse bone marrow cells, suggesting common selectivity for a membrane feature of hemopoietic cells. Both compounds fail to stain red blood cells, normal human leukocytes, mouse spleen and thymus cells. We have recently reported that dansyl-lysine apparently selects for cholesterol-free phospholipid domains in liposomes and now report similar selectivity for merocyanine 540 staining of liposomes.     

The effect of chloroplast coupling factor ATP synthetase (CF1 · CF0) reconstitution on fluidity properties of isolated thylakoid lipid vesicles

The reconstitution of chloroplast coupling factor ATP synthetase (CF₁ · CF₀) with thylakoid lipids by cholate dialysis produced vesicles that displayed higher steady-state anisotropy (r_s) values for both 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenyl hexatriene fluorescence than the pure lipid alone. This is interpreted as meaning that the insertion of protein into the lipid bilayer brings about an increase in the ordering of acyl chains. This ordering effect became more obvious as the protein-to-lipid ratio was increased. Time-resolved decay analyses of DPH fluorescence anisotropy confirmed the conclusion drawn from the steady-state measurements, but further indicated that the dynamic motion of the probe was also slightly restricted after CF₁ · CF₀ incorporation. The restriction of DPH motion and the change in the half-angle for its cone of rotation was observed at relatively low protein-to-lipid ratios as compared with other reconstituted or biological membranes, suggesting that perhaps lipid-protein interactions occur with the inserted CF₁ · CF₀ complex.     

Properties of bilayer membranes in the phase transition or phase separation region

The increase in passive permeability of bilayer membranes near the phase transition temperature is usually explained as caused by either the increase in the amount of ‘boundary lipid’ present in the membrane, or by the increase in lateral compressibility of the membrane. Since both the amount of ‘boundary lipid’ and the lateral compressibility show a similar anomaly near the transition temperature, it is difficult to distinguish experimentally between the two proposed mechanisms.We have examined some details of both of the proposed pictures. The fluid-solid boundary energy, neglected in previous work, has been computed as a function of the domain size. For a single component uncharged lipid bilayer, the results rule out the existence of even loosely defined solid domains in a fluid phase, or vice versa. Thermodynamic fluctuations, which are responsible for anomalous behaviour near the phase transition temperature, are not intense enough to approximate the formation of a domain of the opposite phase.Turning next to lateral compressibility of bilayer membranes we have considered two-component mixtures in the phase separation region. We present the first calculation of lateral compressibility for such systems. The behaviour shows interesting anomalies, which should correlate with existing and future data on transport across membranes.     

Functional group contributions to the partitioning of phenols between liposomes and water

The temperature dependency of the partitioning of p-alkylphenols and p-halophenols has been determined between dimyristoyl phosphatidylcholine liposomes and 0.15 M NaCl. Partition coefficients increased as a function of temperature below the endothermic phase transition temperature (T_c) of the phospholipid but decreased above this temperature. The transfer process was found to be entropy-dominated below and enthalpy-dominated above the T_c, although large negative entropy changes were observed. Regular changes in the thermodynamic functions, partition coefficients and functional group free energies occurred as a function of the alkyl chain length or size of the halogen substituent below but not above the T_c. This has tentatively been attributed to increased phenol-phospholipid interaction at the higher temperatures. The partitioning of p-fluorophenol behaved in a manner expected of fluorinated compounds, yielding relatively low partition coefficients, but it produced an additional effect of markedly lowering the T_c of dimyristoyl phosphatidylcholine. Good correlations of the partition coefficients in liposomes with those in bulk organic solvents and with molecular size of the solute have been obtained.     

Interaction of melittin with dimyristoyl phosphatidylcholine liposomes. Evidence for boundary lipid by raman spectroscopy

The interaction of melittin, a polypeptide consisting of 26 amino acid residues, with dimyristoyl phosphatidylcholine bilayers was investigated by vibrational Raman spectroscopy. Spectral peak height intensity ratios, involving vibrational transitions in both the 3000 cm^?1 acyl chain methylene carbon-hydrogen stretching mode region and the 1100 cm^?1 acyl chain carbon-carbon skeletal stretching mode interval, served as temperature profile indices for monitoring the bilayer order-disorder processes. For a lipid : melittin molar ratio of 14 : 1 two order-disorder transitions were observed. In comparison to a gel to liquid crystalline phase transition of 22.5°C for the pure lipid, the lower transition, exhibiting a 2°C width, is centered at 17°C and is associated with a depression of the main lipid phase transition of dimyristoyl phosphatidylcholine. The second thermal transition, displaying a 7°C interval, occurs at approx. 29°C and is associated with the melting behavior of approximately seven immobilized boundary lipids which surround the inserted hydrophobic segment of the polypeptide. For a lipid : melittin molar ratio of 10 : 1 two thermal transitions are also observed at 11 and 30°C. As before, they represent, respectively, the main gel to liquid crystalline phase transition and the melting behavior of approximately four boundary lipids attached to melittin. From these data alternative schemes are suggested for disposing the immobilized lipids around the hydrophobic portion of the polypeptide within the bilayer.     

Preparation of asymmetric,cerebroside sulfate-containing phospholipid vesicles

A procedure is described which inserts asymmetrically cerebroside sulfate (‘sulfatide’) into the outer leaflet of bilayered phospholipid vesicles. Cerebroside sulfate is adsorbed onto a cellulose, filter-paper support and, when incubated with phosphatidylcholine vesicles is transferred to and inserted into the outer leaflet of these vesicles. This transfer occurs at, or above the transition temperature of the phospholipid and follows a similar pattern with small or larger (‘fused’) unilamellar vesicles. The transfer is linear with time for 1–2 h and is maximal after about 6 h, when the sulfatide content reaches about 6 mol% of the total quantity of phospholipid, corresponding to about 10 mol% of the phospholipids present in the outer layer. Initial rates of sulfatide transfer were somewhat increased when the vesicles contained a positively charged lipid (e.g. stearylamine) and decreased when this lipid was negatively charged (e.g. dicetyl phosphate) or hydrophobic (e.g. cholesterol). Divalent ions markedly inhibited sulfatide transfer and monovalent ions did so to a lesser degree. Once incorporated into the outer leaflet of the vesicle, the sulfatide could not be removed by washing with buffer, 1 M NaCl or 1 M urea.     

Protein-lipid interactions and differential scanning calorimetric studies of bacteriorhodopsin reconstituted lipid-water systems

Bacteriorhodopsin has been reconstituted at various molar concentrations into liposomes of dimyristoyl- and also of dipalmitoylphosphatidylcholine. Differential scanning calorimetry indicates that as the protein concentration within the lipid bilayer increases, the cooperativity of the lipid phase transition is reduced, i.e. the transition is broadened, while the midpoint transition temperature remains virtually unchanged. Freeze-fracture electron microscopy of our preparation shows, in agreement with previous data from other laboratories, that extensive protein aggregation occurs when the liposome is cooled below the T_c transition temperature of the lipid. Laser flash photolysis measurements of protein rotation of the bacteriorhodopsin show, especially in the case of protein-rich recombinants, that protein aggregates exist even above T_c. The perturbation caused by the presence of bacteriorhodopsin in the lipid bilayer is similar to that produced by other intrinsic proteins. The difficulty of correlating the observed calorimetric enthalpy data with a simple concept of a ‘boundary lipid layer’ based upon consideration of a single isolated protein is discussed in view of the occurrence of protein aggregates both above and below T_c. It is concluded that the reduction of enthalpy is related to the number of lipids which solvate the protein aggregates within the protein-lipid patches and are thereby removed from the cooperative melting and enthalpy of the remaining regions of pure lipid.     

Investigation of human erythrocyte ghost membranes with intramolecular excimer probes

Human erythrocyte ghost membranes have been investigated using two intramolecular excimer probes, di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether. Values for the viscosity of the direct probe environment in the ghost membranes range from 76 cP at 37°C to 570 cP at 5°C, as reported for di(1-pyrenyl)propane, with liquid paraffin as the reference solvent. For the activation energy of the excimer formation process, determined here mainly by the viscosity of the medium, a value of 37 kJ/mol is obtained. The other probe molecule reports a higher local viscosity, 133 cP at 37°C, as well as a higher activation energy of excimer formation, 54 kJ/mol. Neither thermotropic phase transitions nor temperature hysteresis effects are observed within the temperature range (0 to 40°C) studied. From the vibrational structure of the fluorescence spectrum of di(1-pyrenylmethyl) ether, a polarity of the probe environment close to that of hexanol ($\text{? = 13.3}$) results for the erythrocyte ghost membranes. The polarity measured in egg phosphatidylcholine membranes and in multibilayers of dimyristoylphosphatidylcholine is slightly larger, comparable to that of butanol ($\text{? = 17.5}$), whereas a polarity comparable to that of methanol ($\text{? = 32.7}$) is observed for aqueous micellar solutions of sodium dodecyl sulphate. Further, from the wavelength shifts in the absorption spectrum of di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether, the polarizability of the probe surroundings can be determined, leading to a surprisingly high value for the apparent refractive index. This is attributed to a high local density of the direct environment of the probe, for which a location between the membrane/water interface and the unpolar bilayer mid-plane is deduced.