Electron spin resonance studies of phosphatidylcholine interacted with cholesterol and with a hopanoid in liposomal membrane.

The effects of bacteriohopane-32-ol (Monol) on liposomal membrane composed of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (egg PC) were compared with those of cholesterol (Chol) in the change of fluidity using a spin label. The fluidity change close to the polar head groups caused by temperature increase in the DPPC membrane containing Monol was different from that of Chol. Chol had a condensing effect on DPPC membrane, whereas Monol had only a slight effect except when used at 20 mol%. Near the hydrophobic end, Chol incorporation into DPPC led to fluidization below transition temperature (Tm) and condensation above Tm. Monol incorporation into DPPC had only a fluidizing effect below Tm. On the other hand, in egg PC membrane Chol had the condensing effect at any temperature, whereas Monol had only slight effect. These results suggest that Monol may have a role in supporting constant membrane fluidity under drastic conditions.     

Effects of poly(ethylene glycol) (PEG) chain length of PEG-lipid on the permeability of liposomal bilayer membranes

The effects of poly(ethylene glycol) (PEG) chain length of PEG-lipid on the membrane characteristics of liposomes were investigated by differential scanning calorimetry (DSC), freeze-fracture electron microscopy (FFEM), fluorescence polarization measurement and permeability measurement using carboxyfluorescein (CF). PEG-liposomes were prepared from mixtures of dipalmitoyl phosphatidylcholine (DPPC) and distearoyl phosphatidylethanolamines with covalently attached PEG molecular weights of 1000, 2000, 3000 and 5000 (DSPE-PEG). DSC and FFEM results showed that the addition of DSPE-PEG to DPPC in the preparation of liposomes caused the lateral phase separation both in the gel and liquid-crystalline states. The fluidity in the hydrocarbon region of liposomal bilayer membranes was not significantly changed by the addition of DSPE-PEG, while that in the interfacial region was markedly increased. From these results, it was anticipated that the CF leakage from PEG-liposomes is accelerated compared with DPPC liposomes. However, CF leakage from liposomes containing DSPE-PEG with a 0.060 mol fraction was depressed compared with regular liposomes, and the leakage decreased with increasing PEG chain length. Furthermore, the CF leakage from liposomes containing DSPE-PEG with a 0.145 mol fraction was slightly increased compared with that of liposomes containing DSPE-PEG with a 0.060 mol fraction. It is suggested that the solute permeability from the PEG-liposomes was affected by not only properties of the liposomal bilayer membranes such as phase transition temperature, phase separation and membrane fluidity, but also the PEG chain of the liposomal surface.     

Effect of a series of essential oil molecules on DPPC membrane fluidity: a biophysical study

Monoterpenes (MTs) were known to cause biological membranes perturbation. Here, the interaction of four (eucalyptol, pulegone, terpineol, and thymol) with dipalmitoylphosphatidylcholine (DPPC) liposomes was studied by Raman spectroscopy, differential scanning calorimetry and fluorescence anisotropy. Liposomes were prepared by the thin-film hydration method, and MTs were added to DPPC at various molar percentages (from 0 to 25%). All the studied MT abolished the pre-transition of DPPC membrane and modified the intensity of the Raman peak at 715 cm^?1, proving their interaction with the choline head group of phospholipids. MTs decreased also the main transition temperature suggesting their interaction with the alkyl chains of DPPC membrane. Besides, a splitting of the main transition peak was obtained with thymol. The results of fluorescence anisotropy showed that the studied molecules fluidized the liposomal membrane at 25, 41, and 50 °C. A mixture of isomers of terpineol fluidized the membrane more than α-terpineol. The presence of a hydroxyl group in the MT structure seems to improve the membrane fluidizing effect of MTs.     

Effect of adsorption of bovine serum albumin on liposomal membrane characteristics

The effect of adsorption of bovine serum albumin (BSA) on the membrane characteristics of liposomes at pH 7.4 was examined in terms of zeta potential, micropolarity, microfluidity and permeability of liposomal bilayer membranes, where negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG)/L-alpha-dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP)/DPPC and positively charged stearylamine (SA)/DPPC mixed liposomes were used. BSA with negative charges adsorbed on negatively charged DPPG/DPPC mixed liposomes but did not adsorb on negatively charged DCP/DPPC and positively charged SA/DPPC mixed liposomes. Furthermore, the adsorption amount of BSA on the mixed DPPG/DPPC liposomes increased with increasing the mole fraction of DPPG in spite of a possible electrostatic repulsion between BSA and DPPG. Thus, the adsorption of BSA on liposomes was likely to be related to the hydrophobic interaction between BSA and liposomes. The microfluidity of liposomal bilayer membranes near the bilayer center decreased by the adsorption of BSA, while the permeability of liposomal bilayer membranes increased by the adsorption of BSA on liposomes. These results are considered to be due to that the adsorption of BSA brings about a phase separation in liposomes and that a temporary gap is consequently formed in the liposomal bilayer membranes, thereby the permeability of liposomal bilayer membranes increases by the adsorption of BSA.     

Liposome fluidization and melting point depression by pressurized CO2 determined by fluorescence anisotropy

The influence of CO2 on the bilayer fluidity of liposomes, which are representative of model cellular membranes, was examined for the first time at the elevated pressures (up to 13.9 MPa) associated with CO2-based processing of liposomes and microbial sterilization. Fluidization and melting point depression of aqueous dipalmitoylphosphatidylcholine (DPPC) liposomes by pressurized CO2 (present as an excess phase) were studied by steady-state fluorescence anisotropy using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Isothermal experiments revealed reversible, pressure-dependent fluidization of DPPC bilayers at temperatures corresponding to near-gel (295 K) and fluid (333 K) phases at atmospheric pressure, where the gel-to-fluid phase transition (Tm) occurs at approximately 315 K. Isobaric measurements (PCO2 =1.8, 7.0, and 13.9 MPa) of DPH anisotropy demonstrate substantial melting point depression (DeltaTm = -4.8 to -18.5 K) and a large broadening of the gel-fluid phase transition region, which were interpreted using conventional theories of melting point depression. Liposome fluidity is influenced by CO2 accumulation in the hydrocarbon core and polar headgroup region, as well as the formation of carbonic acid and/or the presence of buffering species under elevated CO2 pressure.     

Interaction of tetraphenyl-porphyrin derivatives with DPPC-liposomes: an EPR study

The effect of the symmetry and polarity of the porphyrin molecules on their membrane localization and interaction with membrane lipids were investigated by electron paramagnetic resonance (EPR). For this purpose, two glycoconjugated tetraphenyl porphyrin derivatives were selected, respectively, symmetrically and asymmetrically substituted. Small unilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and spin labeled stearic acids were prepared. The spin probe was located at the 5th or 7th or 12th or 16th position of the hydrocarbon chain in order to monitor various regions of the lipid bilayer. EPR spectra of porphyrin-free and porphyrin-bound liposomes were recorded at various temperatures below and above the phase transition temperature of DPPC. The effect on membrane fluidity proved to be stronger with the asymmetrical porphyrin derivative than with the symmetrical one. The rigidity increased when the spin label was near lipid head groups. The difference observed between control and porphyrin-treated samples when measured below the main lipid transition temperature disappeared at higher temperature. When the spin label was near the end of the hydrophobic tails, the symmetrical porphyrin derivative caused increase in fluidity, while the asymmetrical one slightly decreased it. To explain this phenomenon we propose that the asymmetrical derivative exerts a stronger ordering effect caused by its fluorophenyl group located at the level of the lipid heads, which is attenuated to the hydrophobic tails. The perturbing effect of the symmetric derivative could not lead to similar extent of ordering at the head groups and looses the hydrocarbon chains deeper in the membrane.     

Specific effect of polyunsaturated fatty acids on the cholesterol-poor membrane domain in a model membrane

To understand more fully the effect of polyunsaturated fatty acids (PUFAs) on lipid bilayers, we investigated the effects of treatment with fatty acids on the properties of a model membrane. Three kinds of liposomes comprising dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC), and cholesterol (Ch) were used as the model membrane, and the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and detergent insolubility were determined. Characterization of the liposomes clarified that DPPC, DPPC/Ch, and DPPC/DOPC/Ch existed as solid-ordered phase (L beta), liquid-ordered phase (l o), and a mixture of l o and liquid-disordered phase (L alpha) membranes at room temperature. Treatment with unsaturated fatty acids such as oleic acid (OA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) markedly decreased the fluorescence anisotropy value and detergent insolubility. PUFAs and OA had different effects on the model membranes. In DPPC liposomes, the most prominent change was induced by PUFAs, whereas, in DPPC/Ch and DPPC/DOPC/Ch liposomes, OA had a stronger effect than PUFAs. The effect of PUFAs was strongly affected by the amount of Ch in the membrane, which confirmed a specific effect of PUFAs on the Ch-poor membrane domain. We further explored the effect of fatty acids dispersed in a water-in-oil-in-water multiple emulsion and found that unsaturated fatty acids acted on the membranes even when incorporated in emulsion form. These findings suggest that treatment with PUFAs increases the segregation of ordered and disordered phase domains in membranes.     

Stabilized liposomes with phospholipid polymers and their interactions with blood cells

To stabilize a phospholipid liposome, addition of various water-soluble polymers into a liposomal aqueous suspension was investigated. The water-soluble polymers were poly(ethylene oxide) (PEO), poly(N-vinyl pyrrolidone) (PVPy) and poly[2-methacryloyloxyethyl phosphorylcholine(MPC)], and poly[MPC-co-n-butyl methacrylate(BMA)]. The gel–liquid crystal transition temperature (T_c) of the diparmitoylphosphatidylcholine (DPPC) liposome was not changed by addition of these polymers significantly. However, membrane fluidity of DPPC liposome treated with water-soluble polymers, which was measured with fluorescence probe, depended on the chemical structure of the water-soluble polymers. In the case of PEO and PVPy, the temperature dependence of membrane fluidity was the same as that of the original DPPC liposome, on the other hand, poly(MPC) and poly(MPC-co-BMA) induced a rise in the temperature where an increase in the membrane fluidity was observed. The release of carboxy fluorescein from the DPPC liposome was suppressed by the addition of the MPC polymers. The liposomes in the MPC polymer solution were stable compared with those in water when plasma was added into the suspension. Interactions with stabilized liposome with blood cells such as platelets and erythrocytes were evaluated. Activation of platelets in contact with liposome covered with poly(MPC) or poly(MPC-co-BMA) was less than PEO-stabilized liposome. On the other hand, no hemolysis of erythrocytes was observed when every polymer-treated liposome was added in the suspension of erythrocytes. Based on these results, the MPC polymers could interact with the liposome surface, adsorb on the liposomes and stabilize them, and had no adverse effect to the blood cells even when they were in a physiological environment.     

Thermotropic behaviour of dipalmitoylphosphatidylcholine liposomes containing retinoids

Differential scanning calorimetry has been used to investigate the thermotropic behaviour of DPPC liposomes in the presence of different amounts of retinoids in a study of the sites concerned in the mutual lipid-retinoid interaction. The perturbing effect of retinal and retinol on DPPC liposome gel-liquid crystal phase transition has been related to the difference in the polar end group of retinoid. The hydrophilic polar group prevalence over the apolar tail has been evidentiated. Membrane fluidity increases by increasing the retinoid amount. These liposomes displayed a phase separation at high retinal or retinol concentrations.     

Calorimetric and spin-label ESR studies of PEG:2000-DPPE containing DPPC/lyso-PPC mixtures

The thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) multibilayers containing up to 10 mol% of lyso-palmitoylphosphatidylcholine (lyso-PPC) with and without low content of poly(ethylene glycol:2000)-grafted dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE) has been studied by high sensitivity differential scanning calorimetry (DSC) and electron spin resonance (ESR) using the spin probe di-tert-butyl-nitroxide (DTBN). The three lipids, dispersed in buffer at appropriate concentrations, form thermosensitive liposomes used as site-specific drug-delivery systems. Without polymer–lipids, the DPPC main transition temperature is downshifted of 1.2–1.3 °C at the highest lyso-PPC content. The molar enthalpy and the cooperative unit of the DPPC main transition first decrease rapidly, then more slowly and finally slightly increase with lyso-PPC content. Moreover, in the mixed dispersions, the membrane fluidity increases at any temperature. The addition up to 5 mol% of PEG:2000-DPPE to DPPC/10 mol% lyso-PPC mixtures does not affect neither the thermotropic phase behavior nor the transition cooperativity and the fluidity of the dispersions.     

Alteration in the temperature-dependent content release property of thermosensitive liposomes in plasma

The effect of plasma components on the temperature-dependent content release property of thermosensitive liposomes has been described. Temperature-sensitive liposomes containing mitomycin C (MMC) were prepared from dipalmitoylphosphatidylcholine (DPPC liposomes) and a 7 : 3 mixture of DPPC and dipalmitoylophosphatidylglycerol (DPPC/DPPG liposomes). We defined in this study the difference in the content release between 38 degrees C and 44 degrees C as an index of the temperature-dependent content release efficiency (Delta% release). In the absence of rat plasma, the Delta% release of the DPPC liposomes and the DPPC/DPPG liposomes was 83% and 71%, respectively. However, when the release study was conducted with rat plasma, the Delta% release increased to about 96% for both liposomes. In addition, while the DPPC liposomes were destabilized by rat plasma below the gel-to-liquid crystalline phase transition temperature (T(m)), MMC leakage from the DPPC/DPPG liposomes below T(m) was suppressed by rat plasma. Moreover, the plasma protein binding onto lipid bilayer was concomitant with the gel-to-liquid crystalline phase transition and then enhanced the temperature-dependent release from the DPPC/DPPG liposomes. The possible mechanism of interaction between liposomes and plasma proteins, especially serum albumin, was discussed based on differential scanning calorimetry and protein binding experiments.     

Interactions of cyclodextrins with dipalmitoyl, distearoyl, and dimyristoyl phosphatidyl choline liposomes. A study by leakage of carboxyfluorescein in inner aqueous phase of unilamellar liposomes

The interaction of cyclodextrins (CDs) with L-alpha-dipalmitoyl phopsatidyl choline (DPPC), L-alpha-distearoyl phosphatidyl choline (DSPC), and L-alpha-dimyristoyl phosphatidyl choline (DMPC) unilamellar liposomes was investigated by the leakage of carboxylfluorescein (CF) entrapped in the inner aqueous phase of liposomes, at 25 degrees C (DPPC and DSPC liposomes) and at 5 degrees C (DMPC liposomes). The efficiency of CDs for CF leakage was remarkable in the order of heptakis (2,6-di-O-methyl)-beta-CD (DOM-beta-CD) > alpha-CD > heptakis (2,3,6-tri-O-methy)-beta-CD (TOM-beta-CD) from DPPC liposomes, in the order of DOM-beta-CD > TOM-beta-CD > alpha-CD from DSPC liposomes and in the order of alpha-CD > DOM-beta-CD > TOM-beta-CD from DMPC liposomes. The other CDs used in the present studies, beta-CD, 2-hydroxylpropyl beta-CD, and gamma-CD scarcely induced the CF leakage from above the three liposomes. From the profiles of % CF leakage, together with measurements of differential scanning calorimetry, it was found that hydrophobic DOM-beta-CD penetrates the matrix of the liposomes to interact with them as well as TOM-beta-CD, and that less hydrophobic alpha-CD exists at the surface of the membrane to interact with the liposomes. Further, it was found that the interaction of CDs with liposomes changes depending not only on the length of fatty acid chain of phospholipid (condensation force and hydrophobicity) but also the hydrophobicity and the cavity size of CD.     

Temperature-sensitive nonionic vesicles prepared from Span 80 (sorbitan monooleate)

Different types of nonionic vesicles were prepared from commercial Span 80 (also called sorbitan monooleate), as an inexpensive, biocompatible alternative to conventional phospholipid-based vesicles (liposomes). The vesicles were characterized by different techniques and comparison was made with vesicles formed from POPC (1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine) or DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine). Dynamic light scattering measurements, electron microscopy analyses, and two types of fusion assays indicate that Span 80 vesicles are stable for at least 7 days at 4 or 25 degrees C, while storage at 42 degrees C causes irreversible vesicle fusion. This indicates that Span 80 vesicles are thermoresponsive with vesicle fusion occurring at elevated temperature. This property may be related to headgroup dehydration and is certainly not directly linked to the phase transition temperature (Tm) of the vesicles, since the Tm is below -30 degrees C, as determined by differential scanning calorimetry (DSC). The measured Tm value for Span 80 vesicles is lower than in the case of DOPC or POPC, correlating with a higher fluidity of Span 80 vesicles as compared to POPC or DOPC vesicles, as determined with DPH (1,6-diphenyl-1,3,5-hexatriene) as fluorescent membrane probe. High fluidity correlates with increased leakage of entrapped water-soluble dye molecules. Addition of cholesterol and soybean phosphatidylcholine lowers the extent of leakage, allowing a tuning of the bilayer permeability.     

Membrane properties of cationic liposomes composed of dipalmitoylphosphatidylcholine and dipalmityldimethylammonium bromide

Cationic liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmityldimethylammmonium bromide (DPAB) were prepared by the Bangham method and the effect of DPAB on the membrane properties was examined in terms of liposomal shape, particle size, trapping efficiency, surface potential and dispersibility. The dispersibility of the mixed DPPC/DPAB liposomes (the mole fraction of DPAB (X_DPAB)  0.05) was excellent and the dispersibility was maintained for 6 months, since the zeta-potential of the mixed liposomes was approximately +40 mV. The trapping efficiency of the mixed DPPC/DPAB liposomes (X_DPAB = 0.05) was 10 times greater than that of the DPPC liposomes, and the value was largest among the mixed liposomes (X_DPAB = 0–1.0). Freeze-fracture electron micrographs indicated that the shape of the mixed DPPC/DPAB liposomes (X_DPAB = 0.05) was that of large unilamellar vesicles (LUVs) with a diameter of approximately 2 μm, while the shape of the DPPC liposomes was that of multilamellar vesicles (MLVs). The mixed liposomes had, therefore, a high trapping efficiency. Furthermore, the shape of the mixed DPPC/DPAB liposomes (X_DPAB = 0.75) was also that of LUVs with a diameter of approximately 2 μm and these had a high trapping efficiency. Whereas, the particle size (500 nm) of the mixed DPPC/DPAB liposomes (X_DPAB = 0.25) was smaller than that of the former and had the minimum trapping efficiency. The phase transition temperature of the liposomal bilayer membranes indicated a maximum value at 0.25–0.30 mole fractions of DPAB. These facts were considered to be due to the fact that DPPC and DPAB, whose molar ratio was 7.5:2.5, were tightly packed in the liposomal bilayer membranes and that the curvature of the liposomal particle was resultantly large. Nevertheless, LUVs having a high trapping efficiency were easily obtained by mixing a small amount of DPAB with the DPPC.     

A DSC study of paeonol-encapsulated liposomes, comparison the effect of cholesterol and stigmasterol on the thermotropic phase behavior of liposomes

Thermotropic phase behaviors of paeonol-encapsulated liposomes containing stigmasterol or cholesterol have been investigated by differential scanning calorimetry. We compared the thermotropic phase behavior of pure dipalmitoylphosphatidylcholine (DPPC) liposomes, sterol/DPPC liposomes, and paeonol/sterol/DPPC liposomes increasing the ratio of paeonol to sterol from 0 to 1, by analyzing the calorimetric parameters of main phase transition of liposomes including phase transition temperature (onset temperature and peak temperature) and phase transition cooperativity. The results showed that paeonol could incorporate into the hydrophobic region of DPPC, thus, decrease phase transition temperature of DPPC. Though stigmasterol interacts with DPPC less favorably than cholesterol, thermotropic phase behavior of paeonol/cholesterol/DPPC liposomes and that of paeonol/stigmasterol/DPPC liposomes are very similar. A phase separation occurred when the molar ratio of paeonol to sterol reached 1:1 in paeonol-encapsulated liposomes, where a paeonol-rich domain coexisted with a sterol-rich domain. The packing order of acyl chains of DPPC in sterol-rich domain is a little higher than that in paeonol-rich domain.     

Membrane interactions of ternary phospholipid/cholesterol bilayers and encapsulation efficiencies of a RIP II protein

Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P(min), the bilayer lysis tensions gamma(l) were determined. The increase in P(min) and gamma(l) with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron microscopy (Cryo-TEM) images show that beside spherical, single-walled liposomes, there is a considerable fraction of discoidally deformed vesicles. Based on our results and those found in the literature, we speculate that the flattening of the vesicles is a consequence of lipid phase separation and the formation of condensed complexes and areas of different bending elasticities. This phenomenon eventually leads to agglomeration of deformed liposomal structures, becoming more pronounced with the increase in the relative amount of saturated fatty acids, presumably caused by hydrophobic interaction. For the same lipid mixture aggregation correlated linearly with the ML content. Finally, tested liposomal samples were kept at 4 degrees C to examine their stability. Only slight fluctuations in diameter and the increase in polydispersity after 3 weeks of storage occurred, with no statistically significant evidence of drug leakage during a time period of 12 days, illustrating physical stability of liposomes.     

Effects of silver nanoparticles on the fluidity of bilayer in phospholipid liposome

This paper describes the formation and characterization of liposome entrapping the silver nanoparticles in bilayer. Silver nanoparticles were entrapped in the bilayer of dipalmitoylphosphatidylcholine (DPPC) liposome, named as silver-loaded liposome. Specifically, above the gel to liquid-crystalline phase transition temperature of this lipid (i.e., 41 degrees C), it was observed that membrane fluidities of silver-loaded liposomes were increased, and fluorescence anisotropy values were reduced from 0.114 to 0.097. This might be due to the structural modifications and interactions between DPPC molecules and silver nanoparticles within the bilayer. It was also confirmed that silver nanoparticles were entrapped in hydrophobic region of lipid bilayer with transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) measurements.     

Influence of some physical properties of 5-fluorouracil on encapsulation efficiency in liposomes

The aim of this study is to encapsulate two drugs: 5-fluorouracil (5-FU) with the hydrophobic properties and 1-β-D-arabinofuranosylcytosine (Ara-C) with the amphiphilic properties into liposomes prepared by the modified reverse-phase evaporation method (mREV) from L-α-phosphatidylcholine dipalmitoyl (DPPC). We studied the thermotropic phase behavior of liposome entrapped 5-FU and Ara-C. It is known that the stability of liposomes depends not only on the method of chemical gradient loading, the use of membrane stabilizer such as sterols, but also on the phase transition temperature (T _c) of phospholipids, which undergoes an alteration after encapsulation of drugs to liposomes. The competition of these two drugs entrapped in liposomes was analyzed by the use of two spectroscopies: ¹H NMR and UV on the basis of the analysis of the signals of each drug in the liposome—drug system. The percent of encapsulation in DPPC/Ara-C/5-FU liposome obtained by the use of UV spectroscopy amounted 93.84 and 96.05% for 5-FU and Ara-C, respectively. Phase transition temperature T _c of liposomes containing Ara-C did not significantly change while for the liposomes containing 5-FU it increased in comparison with T _c of the reference liposomes formed from DPPC.     

The physicochemical/thermodynamic balance of advanced drug liposomal delivery systems

The aim of this work is to study the morphological characteristics via fractal analysis and the alterations of the thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) liposomes, caused by the incorporation of cholesterol, poly(amidoamine) (PAMAM) dendrimer, and MPOx (poly(2-methyl-2-oxazoline)-grad-poly(2-phenyl-2-oxazoline)) gradient block copolymer (9:1 molar ratio). A gamut of light scattering techniques and differential scanning calorimetry were used in order to extract information on the morphological (in different dispersion media) and thermodynamic characteristics of liposomal drug nanocarriers, respectively. The vesicles’ structure of liposomes has a different thermodynamic content, which corresponds to a different thermotropic behavior, in comparison to pure lipid bilayers. The observed metastable phase only for DPPC liposomes has been considered as a “physical impurity”, which leads to “physical incompatibility” and consequently promotes the aggregation of DPPC liposomes in aqueous media. The incorporation of biomaterials such as PAMAM G4 and MPOx, caused alterations in the thermotropic behavior of DPPC liposomes affecting only the main transition specific enthalpy ΔH. All the other calorimetric parameters remained unaltered. These findings supported the hypothesis that the exceptional stability and transition cooperativity of the chimeric liposomal membrane might be due to the reduction of the vesicle size with the smaller membrane curvature that is indicated by the fractal dimensionality of the system. In conclusion, the results from the thermal analysis of the liposomal systems were in line with the picture of their structural characteristics, as indicated by the interplay between physicochemical and thermodynamical parameters, which determines their fractal morphology.     

Freeze-fracture electron microscopic and calorimetric studies on microscopic states of surface-modified liposomes with poly(ethylene glycol) chains

The differential scanning calorimetry (DSC) and the freeze-fracture electron microscopy of dipalmitoyl phosphatidylcholine (DPPC) liposomes containing distearoyl-N-monomethoxy poly(ethylene glycol)-succinyl-phosphatidylethanolamines (PEG-DSPE) were carried out. The DSC peak of DPPC liposomes containing PEG-DSPE had a shoulder. The main phase transition temperature of DPPC bilayer membranes containing PEG-DSPE whose molecular weight of PEG is less than 3000 was slightly shifted to a higher temperature, while that containing PEG-DSPE whose molecular weight of PEG is more than 5000 was slightly shifted to a lower temperature. The electron micrographs of freeze-fracture replicas of DPPC liposomes containing PEG-DSPE quenched from 37±2°C exhibited banded and planar textures, suggesting the lateral phase separation in the bilayer membranes.