Integrity of Membrane Structures in Giant Unilamellar Vesicles as Assay for Antioxidants and Prooxidants.

We have attempted to evaluate, on the basis of optical microscopy for a single giant unilamellar vesicle (GUV), the potency of antioxidants in protecting GUV membranes from oxidative destruction. Photosensitized membrane budding of GUVs prepared from soybean phosphatidylcholine with chlorophyll a (Chl a) and ß-carotene (ß-Car) as photosensitizer and protector, respectively, were followed by microscopic imaging. A dimensionless entropy parameter, ΔE, as derived from the time-resolved microscopic images, was employed to describe the evolution of morphological variation of GUVs. As an indication of membrane instability, the budding process showed three successive temporal regimes as a common feature: a lag phase prior to the initiation of budding characterized by LP (in s), a budding phase when ΔE increased with a rate of kΔE (in s-1), and an ending phase with morphology stabilized at a constant ΔEend (dimensionless). We show that the phase-associated parameters can be objectively obtained by fitting the ΔE-t kinetics curves to a Boltzmann function and that all of the parameters are rather sensitive to ß-Car concentration. As for the efficacy of these parameters in quantifying the protection potency of ß-Car, kΔE is shown to be most sensitive for ß-Car in a concentration regime of biological significance of <1 × 10-7 M, whereas LP and ΔEend are more sensitive for ß-Car concentrations exceeding 1 × 10-7 M. Furthermore, based on the results of GUV imaging and fluorescence and Raman spectroscopies, we have revealed for different phases the mechanistic interplay among 1O2* diffusion, PC-OOH accumulation, Chl a and/or ß-Car consumption, and the morphological variation. The developed assay should be valuable for characterizing the potency of antioxidants or prooxidants in the protection or destruction of the membrane integrity of GUVs.

Subnanosecond charge recombination dynamics in P3HT/PC61BM films.

Ultrafast near-infrared absorption spectroscopy was used to investigate the influence of film morphology and excitation photon energy on the charge recombination (CR) dynamics in the initial nanosecond timescale in the P3HT/PC(61)BM blend films. With reference to the CS(2)-cast films, the solvent vapor annealed (SVA) ones show 2­3-fold improvement in hole mobility and more than 5-fold reduction in the polymer-localized trap states of holes. At Dt = 70 ps, the hole mobility (m(h)) and the bimolecular CR rate (γ(bi)) of the SVA films are µ(h) = 8.7 × 10(−4) cm2 × s(−1) × V(−1) and γ(bi) = 4.5 × 10(−10) cm3 × s(−1), whereas at Δt = 1 ns they drop to 8.7 × 10(−5) cm2 × s(−1) × V(−1) and 4.6 × 10(−11) cm3 × s(−1), respectively. In addition, upon increasing the hole concentration, the hole mobility increases substantially faster under the above-gap photoexcitation than it does under the band-gap photoexcitation, irrespective of the film morphologies. The results point to the importance of utilizing the photogenerated free charges in the early timescales.

Naturally occurring nanotube with surface modification as biocompatible, target-specific nanocarrier for cancer phototherapy.

Phototherapy has drawn increasing attention including the use of nanocarriers with high drug loading capacity and delivery efficacy for target-specific therapy. We have made use of naturally-occurring halloysite nanotubes (HNTs) to build a biomimetic nanocarrier platform for target-specific delivery of phototherapeutic agents. The HNTs were decorated with poly(sodium-p-styrenesulfonate) (PSS) to enhance the biocompatibility, and were further functionalized by lumen loading the type-II photosensitizer indocyanine green (ICG). The HNT-PSS-ICG nanocarrier, without further tethering targeting groups, was shown to associate with the membrane of giant unilamellar vesicles (GUVs) via Pickering effects. Application of HNT-PSS-ICG nanocarrier to human breast cancer cells gave rise to a cell mortality as high as 95%. The HNT-PSS-ICG nanocarrier was further coated with MDA-MB-436 cell membranes to endow it with targeting therapy performance against breast cancer, which was confirmed by in vivo experiments using breast cancer tumors in mice. The membrane-coated and biocompatible nanocarrier preferentially concentrated in the tumor tissue, and efficiently decreased the tumor volume by a combination of photodynamic and photothermal effects upon near-infrared light exposure. Our results demonstrate that the HNT-based nanocarrier by virtue of facial preparation and high loading capacity can be a promising candidate for membrane-targeting nanocarriers.

Dependence of Excited-State Properties of a Low-Bandgap Photovoltaic Copolymer on Side-Chain Substitution and Solvent.

The excited-state properties and chain conformations of a new low-bandgap copolymer based on benzo[1,2-b:4,5-b']dithiophene (BDT) and thieno[3,4-b]thiophene with meta-alkoxyphenyl-substituted side chains in solution were investigated comprehensively. Time-resolved spectroscopy suggested that the excited-state properties were sensitive to the conformations of the copolymer in solution. In addition, excited-state dynamics analyses revealed the photogeneration of triplet excited states by intersystem crossing (ISC) at a rate constant of ∼0.4×10(9)  s(-1) as a result of direct meta-alkoxyphenyl connection to the donor unit BDT irrespective to the macromolecular conformations. According to El-Sayed's rule, the fast ISC herein is correlated with the change of orbital types between singlet and triplet excited states as also shown by quantum chemical calculations. Our studies may shed light on the structure-property relationships of photovoltaic materials.

ß-Carotene as a membrane antioxidant probed by cholesterol-anchored daidzein.

ß-Carotene is found to be more effective as an antioxidant in phosphatidylcholine (PC) liposomes when protecting against hydrophilic radicals compared to lipophilic radicals, as measured by the rate of formation of conjugated dienes. Daidzein alone is without effect, but decreases the antioxidative effect of ß-carotene for hydrophilic initiation and increases the effect for lipophilic initiation. The newly synthesized 7-cholesterylglycol daidzein has the opposite effect for ß-carotene as antioxidant, with a strong enhancement for hydrophilic initiation and a slight decrease for lipophilic initiation. Redistributing ß-carotene to membrane surfaces by cholesterol-anchoring of daidzein enhances protection against aqueous radicals significantly at the expense of protection against lipid-derived radicals. Anchoring of daidzein to cholesterol is concluded to be useful as a mechanistic tool for controlling antioxidant distribution in membranes sensitive to radical damage, as supported by quantum mechanical calculation within the density function theory and further supported by fluorescence probes and fluorescence polarization probes.

Antioxidants and physical integrity of lipid bilayers under oxidative stress.

Giant unilamellar vesicles (GUVs of diameter 5-25 µm) of soy phosphatidylcholine (PC), resistant to intense light exposure (400-440 nm, ~15 mW·mm(-2)), underwent budding when containing chlorophyll a (Chla) in the lipid bilayer ([PC]:[Chla] = 1500:1). On the basis of image heterogeneity analysis using inverted microscopy, a dimensionless entropy parameter for the budding process was shown to increase linearly during an initial budding process. Lipophilic ß-carotene (ß-Car, [PC]:[ß-Car] = 500:1) reduced the initial budding rate by a factor of 2.4, while the hydrophilic glycoside rutin ([PC]:[rutin] = 500:1) had no effect. Chla photosensitized oxidation of PC to form linoleoyl hydroperoxides, further leading to domains of higher polarity in the vesicles, is suggested to trigger budding. The average dipole moment (µ) of linoleic acid hydroperoxides was calculated using density functional theory (DFT) to have the value of 2.84 D, while unoxidized linoleic acid has µ = 1.86 D. ß-Carotene as a lipophilic antioxidant and singlet-oxygen quencher seems to hamper oxidation in the lipid bilayers and delay budding in contrast to rutin located in the aqueous phase. The effect on budding of GUVs as a detrimental process for membranes is suggested for use in assays for evaluation of potential protectors of cellular integrity and functions under oxidative stress.

Chain length effects in isoflavonoid daidzein alkoxy derivatives as antioxidants: a quantum mechanical approach.

Daidzein, an isoflavonoid with known prooxidative effects in heterogeneous lipid/water systems, changes to an antioxidant for 7-n-alkoxy derivatives of daidzein. For an alkyl length increasing from 4 to 8, 12, and 16 carbons, the oxidation potential decreases gradually from 1.09 V (vs NHE) for daidzein (D) to 0.94 V for D16 in tetrahydrofuran as determined by cyclic voltammetry at 25 °C. The prooxidative effects transform into antioxidative effects from D8 with a maximal effect for D12 for aqueous phase initiation of lipid oxidation in liposomes despite a gradual decrease in Trolox equivalent antioxidant capacity (TEAC) with increasing alkyl chain length. Quantum mechanical calculations using density functional theory (DFT) showed that the bond dissociation energy of the O-H bond of the 4'-phenol is constant along the homologue series in contrast to Δµ, the change in dipole moment upon hydrogen atom donation, which increases for increasing chain length. The frontier orbital energy gap goes through a maximum for D12. The change in the A-to-B dihedral angle upon hydrogen atom donation further shows a maximum for D12 of 6.45°. The importance of these microscopic properties for antioxidative activity was confirmed by a change in liposome fluorescence anisotropy using a fluorescent probe showing maximal penetration into the lipid bilayer for D12 along the homologue series.

Comparison of flavonoids and isoflavonoids as antioxidants.

The isoflavonoid genistein was found to be a better antioxidant than the isomeric flavonoid apigenin in phosphatidyl liposomes at pH 7.4. The higher antioxidation activity of genistein compared with apigenin is in agreement with its lower oxidation potential (0.73 vs 0.86 V as determined by cyclic voltammetry in aqueous solution of pH= 7.4), lower dissociation enthalpy (87.03 vs 87.88 kcal mol(-1) as calculated for the more reducing 4'-hydroxyl group), and higher radical scavenging capacity in the TEAC assay. On the basis of quantum mechanical calculations for genistein and apigenin in comparison with the flavonoid naringenin and the isoflavonoids puerarin, daidzein, and equol, a lower dipole moment and a larger deviation for the A-to-B dihedral angle from coplanarity (39.3 degrees for genistein, 18.5 degrees for apigenin) are suggested to be important for the increased antioxidant efficiency at water/lipid interfaces among (iso) flavonoids with an equal number of phenolic groups.