IR-780-loaded polymeric micelles enhance the efficacy of photothermal therapy in treating breast cancer lymphatic metastasis in mice.

Cancer metastasis is responsible for over 90% of breast cancer-related deaths, and inhibiting lymph node metastasis is an option to treat metastatic disease. Herein, we report the use of IR-780-loaded polymeric micelles (IPMs) for effective photothermal therapy (PTT) of breast cancer lymphatic metastasis. The IPMs were nanometer-sized micelles with a mean diameter of 25.6 nm and had good stability in simulated physiological solutions. Under 808-nm laser irradiation, IPMs exhibited high heat-generating capability in both in vitro and in vivo experiments. After intravenous injection, IPMs specifically accumulated in the tumor and metastatic lymph nodes and penetrated into these tissues. Moreover, a single IPMs treatment plus laser irradiation significantly inhibited primary tumor growth and suppressed lymphatic metastasis by 88.2%. Therefore, IPMs are an encouraging platform for PTT applications in treatment of metastatic breast cancer.

Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity.

Polymerization of substrate-supported bilayers composed of dienoylphosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability; however, the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl-phosphatidylcholine (mono-SorbPC), bis-dienoyl-phosphatidylcholine (bis-DenPC), and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lß phase for each lipid. In all cases, polymerization reduced membrane fluidity; however, measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate interleaflet bonding. The D values measured after polymerization were 0.1-0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lß phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV-polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed.

Giant vesicles under oxidative stress induced by a membrane-anchored photosensitizer.

We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid headgroup. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.

X-rays destroy the lamellar structure of model membranes.

Lipid membranes undergo a dramatic lamellar-to-non-lamellar phase transformation upon being irradiated with X-rays. The structure change was evidenced by chemical breakdown of the lipid and by a change in the X-ray diffraction properties of the model membrane indicating complete disruption of lamellar stacking. Considering the importance of X-ray diffraction as a structure probe the problem of radiation damage to biological materials must be duly recognized. Because X-ray damage has been shown to be free radical-mediated these results suggest a means by which free radicals accumulating in cells during aging might compromise membrane integrity and contribute to cell death.

Raman spectroscopic study of microcosmic and photosensitive damage on the liposomes of the mixed phospholipids sensitized by hypocrellin and its derivatives.

Using Raman spectroscopy, we studied and compared the characteristics of microcosmic and photosensitive damage of the liposomes of mixed DPPE and DPPC sensitized by hypocrellin and its derivatives at the molecular level. After photosensitive damage, the structure of the liposomes of mixed phospholipids changed considerably. The trans conformation decreased and gauche conformation increased. The longitudinal order parameter in chains and the lateral order parameter between chains decreased clearly. The results suggested that the hydrocarbon chains of DPPE and DPPC were broken after the photodamage sensitized by hypocrellin B (HB) and 5-Br-hypocrellin B (5-Br-HB). Photosensitive damage on the liposomes sensitized by 5-Br-HB is stronger than that by hypocrellin A (HA) and HB, that is, 5-Br-HB > HB > HA. The results elucidated the sites of interaction or binding to HA, HB and 5-Br-HB in the liposomes and these changed with the use of drugs.

[Behavior reactions and lipids of brain synaptic membranes of rats under chronic exposure to gamma irradiation]. / Povedencheskie reaktsii i lipidy sinapticheskikh membran golovnogo mozga u krys pri khronicheskom vozdeistvii gamma-izlucheniia.

The effects of low level chronic ionising irradiation (12.9 cGy/day on the sensory attention to the stimuli of different modalities (somatosensory, visual, odor) of Wistar rats were studied. Analysis of animals behaviour was made after they had received the different doses of irradiation: 4, 6, 8, 10, 15 and 20 Gy. It was founded, that the attention and exploratory activity of rats is significantly decreased up to 20-30% after 4-6 Gy. The irradiation doses 8 Gy did not change animal behaviour as compared to control animals, but doses 10, 15 and 20 Gy decreased the exploratory activity as well as sensory attention of rats to 3-5-times as compared to previous dose. Such a wave-like way of behaviour reflects the functioning of an adaptive mechanism. Biochemical data indicated that after 5 months of the irradiation (dose 20 Gy) the level of phospholipids, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine, cholesterol were decreased.

[Influence of the antioxidant status on the characteristics of lipids in tissues of animals after acute irradiation with sublethal doses]. / Vliianie antioksidantnogo statusa na kharakteristiki lipidov v tkaniakh zhivotnykh posle ostrogo oblucheniia v subletal'nykh dozakh.

The effect of the acute exposure to sublethal doses of X-rays on the interrelation between parameters of the lipid peroxidation regulatory system (lipid antioxidative activity, AOA; peroxide amount, lipid composition) was studied in liver, spleen and blood erythrocytes of CBA and SHK mice and rats within 1 month after irradiation. The reverse correlation between the lipid AOA values and the initial peroxide amount in lipids of the CBA mice spleen was found. The coefficient of the linear regression of this correlation for the exposed mice was 1.8-fold higher as compared with control. The correlative dependence between the ratio of the sums of the more readily to more poorly oxidizable phospholipid and the ratio of phosphatidyl choline to phosphatidyl ethanolamine content in phospholipids of liver and blood erythrocytes was revealed. The direction (the phospholipids of the rat liver) or the value of the linear regression coefficient of that correlation were different for groups of the exposed and control animals, especially in the blood erythrocytes. Thus, the different sensitivity of examined characteristics of lipids and the possibility of their normalization in the dependence on the lipid AOA value cause the conversion of the lipid peroxidation regulatory system in organs and blood erythrocytes of the exposed animals to the other scale of the functioning.

Photooxidation of glycated and non-glycated phosphatidylethanolamines monitored by mass spectrometry.

Phosphatidylethanolamines (PE) are one of the major components of cells membranes, namely in skin and in retina, that are continuously exposed to solar UV radiation being major targets of photooxidation damage. In addition, due to the presence of the free amine group, PE can also undergo glycation, in hyperglycemic conditions which may increase the susceptibility to oxidation. The aim of this study is to develop a model, based on mass spectrometry (MS) analysis, to identify photooxidative degradation of selected PE (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4) and glycated PEs due to UV irradiation. Photooxidation products were analysed by electrospray ionization MS (ESI-MS) and tandem MS (ESI-MS/MS) in positive and negative mode. Emphasis is placed in the influence of glycation in the generation of distinct photooxidation products. ESI-MS spectra of PE after UV photo-irradiation showed mainly hydroperoxy derivatives, due to oxidation of unsaturated fatty acyl chains. Glycated PE gave rise to several new photooxidation products formed due to oxidative cleavages of the glucose moiety, namely between C1 and C2, C2 and C3, and C5 and C6 of this sugar unit. These new products were identified by ESI-MS/MS in positive mode showing distinct neutral loss depending on the different structure of the polar head group. These new identified advanced glycated photooxidation products may have a deleterious role in the etiology of diabetic retinopathy and in diabetic retinal microvascular complications.

Ultrasound exposure of lipoplex loaded microbubbles facilitates direct cytoplasmic entry of the lipoplexes.

Recently we reported that the transfection of cells by PEGylated lipoplexes becomes significantly better by binding the PEGylated lipoplexes to the surface of microbubbles and applying ultrasound. To further optimize this gene delivery system it is important to understand the working mechanism. This paper elucidates the cellular entry path of these lipoplexes. The results clearly show that the PEGylated lipoplexes, released from the microbubbles upon applying ultrasound, are not taken up by endocytosis, the most common route for nanoparticles to enter cells. Our data demonstrate that, upon implosion of the microbubbles, the PEGylated lipoplexes are released and are most probably able to passively diffuse through the cell membrane pores or become injected in the cytoplasm of the target cells. This is attractive as the in vivo use of PEGylated nanoparticles remains currently limited due to a decreased cellular uptake and inefficient escape of the PEGylated nanoparticles from the endosomes.

UV immobilized phospholipid bilayers.

Dinitrophenyl phosphotidylethanolamine-containing bilayers have been immobilized on carbon-shadowed support films by UV irradiation of the first monolayer transferred to the support film. The immobilized bilayer is capable of allowing bound protein (anti-DNP antibody) to organize into 2-D arrays in the presence of organic solvents such as acetonitrile and dilute concentrations of detergents such as beta-octyl glucoside. The ability of the bilayers to remain attached to supports under various conditions that include organic solvents and detergents as well as divalent ions is of potential interest in the study of protein crystallization and particularly in the study of membrane proteins.

Determination of L(alpha)-H(II) phase transition temperature for 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine.

The thermodynamic properties of fully-hydrated lipids provide important information about the stability of membranes and the energetic interactions of lipid bilayers with membrane proteins (Nagle and Scott, Physics Today, 2:39, 1978). The lamellar/inverse hexagonal (L(alpha)-H(II)) phase transition of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) water mixtures is a first-order transition and, therefore, at constant pressure, must have a thermodynamically well-defined equilibrium transition temperature. The observed transition temperature is known to be dependent upon the rate at which the temperature is changed, which accounts for the many different values in the literature. X-ray diffraction was used to study the phase transition of fully-hydrated DOPE to determine the rate-independent transition temperature, T(LH). Samples were heated or cooled for a range of rates, 0.212 < r < 225 degrees C/hr, and the rate-dependent apparent phase transition temperatures, T(A)(r) were determined from the x-ray data. By use of a model-free extrapolation method, the transition temperature was found to be T(LH) = 3.33 +/- 0.16 degrees C. The hysteresis, /T(A)(r) - T(LH)/, was identical for heating and cooling rates, +/-r, and varied as /r/beta for beta approximately 1/4. This unexpected power-law relationship is consistent with a previous study (Tate et al., Biochemistry, 31:1081-1092, 1992) but differs markedly from the exponential behavior of activation barrier kinetics. The methods used in this study are general and provide a simple way to determine the true mesomorphic phase transition temperatures of other lipid and lyotropic systems.

Photoinduced destabilization of liposomes.

The stability of two-component liposomes composed of the polymerizable 1,2-bis-[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphati dylcholine (SorbPC) and either a phosphatidylethanolamine (PE) or a phosphatidylcholine (PC) were examined via fluorescence leakage assays. Ultraviolet light exposure of SorbPC-containing liposomes forms poly-SorbPC, which phase separates from the remaining monomeric lipids. If the nonpolymerizable lipids are PE's, then the photoinduced polymerization destabilizes the liposome with loss of aqueous contents. The permeability of the control dioleoylPC/SorbPC membranes was not affected by photopolymerization of SorbPC. The photodestabilization of dioleoylPE/SorbPC (3:1) liposomes required the presence of oligolamellar liposomes. NMR spectroscopy of extended bilayers of dioleoylPE/SorbPC (3:1) showed that the photopolymerization lowers the temperature for the appearance of 31P NMR signals due to the formation of isotropically symmetric lipid structures. These observations suggest the following model for the photoinduced destabilization of liposomes composed of PE/SorbPC; photopolymerization induced phase separation with the formation of enriched domains of PE, which allows the close approach of apposed regions of enriched PE lamellae and permits the formation of an isotropically symmetric structure between the lamellae. The formation of such an interlamellar attachment (ILA) between the lamellae of an oligolamellar liposome provides a permeability pathway for the light-stimulated leakage of entrapped water-soluble reagents.

UV-induced reaction kinetics of dilinoleoylphosphatidylethanolamine monolayers.

The UV-induced reactivity of dilinoleoylphosphatidylethanolamine (DLiPE) Langmuir and Langmuir-Blodgett films has been studied by in situ measurements of the changes in the mean molecular area, UV-vis and Fourier transform infrared spectroscopy, and atomic force microscopy (AFM). Optimum orientation and packing density of the DLiPE molecules in the monolayer were achieved by adding uranyl acetate to the subphase. A first-order reaction kinetic model was successfully fitted to the experimental reaction kinetics data obtained at a surface pressure of 30 mN/m. Topographical studies of LB films by AFM were performed on bilayer structures as a function of subphase composition and UV irradiation time. The orientational effect of the uranyl ions on the monolayer molecules was observed as an enhanced homogeneity of the freshly prepared monomeric LB films. However, the long-term stability of these films proved to be bad; clear reorganization and loss of a true monolayer structure were evidenced by the AFM images. This instability was inhibited for the UV-irradiated films, indicating that the UV irradiation gave rise to a cross-linked structure.

Photoactivated enhancement of liposome fusion.

The photopolymerization of two-component large unilamellar liposomes (LUV) composed of 3:1 dioleoylphosphatidylethanolamine (DOPE) and either 1,2-bis[10-(2'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphatidylc holine (bis-SorbPC) or 1-palmitoyl-2-[10-(2'-hexadienoyloxy)decanoyl]-sn- glycero-3-phosphatidylcholine (mono-SorbPC) facilitated liposome fusion. Fusion was characterized by fluorescent assays for lipid mixing, aqueous contents mixing, and aqueous contents leakage. The rate and extent of the liposome fusion was dependent on the extent of photopolymerization, temperature, and the fusion initiation conditions, including the pH and the presence of Mg2+ ions. Examination of the temperature dependence of fusion for unpolymerized and polymerized liposomes showed that an enhancement of the rate of fusion occurred in the temperature range delta TI, which previous NMR studies have identified as the initial appearance of precursors to the formation of the inverted cubic phase [Barry, J. A., et al. (1992) Biochemistry 31, 10114]. The phase behavior and fusion characteristics of the DOPE/bis-SorbPC (3:1) membranes provide unequivocal evidence that liposome fusion is mediated via intermediates associated with the lamellar to QII phase transition rather than the HII phase. Photopolymerization of SorbPC-containing liposomes forms poly-SorbPC, which enhances the lateral separation of the liposome components. The formation of enriched domains of polymorphic lipids, e.g., DOPE, causes isothermal induction of fusion by lowering the critical fusion temperature of the membranes.

[Availability of erythrocyte phosphatidylethanolamine for labelling by trinitrobenzenesulfonic acid at different stages of radiation sickness in rats]. / Dostupnost' fosfatidilétanolamina éritrotsitov dlia mecheniia trinitrobenzolsul'foksilotoi na raznykh étapakh luchevoi bolezni u krys.

It has been found that 1 h after rats' irradiation (dose 800 rad) the incorporation of the label 2, 4, 6-trinitrobenzenesulpho-acid into phosphatidylethanolamine of erythrocyte membranes increases and 3 h and 72 h after irradiation it decreases. Concanavalin and calcium ions modify the incorporation of the label into phosphatidylethanolamine.

[Effect of changes in the lipids of mitochondrial membranes on the activity of Mg2+-dependent ATPase]. / Vliianie izmenenii v lipidakh membran mitokhondrii na aktivnost' Mg2+-zavisimoi ATFazy.

The results obtained permit to assume that irradiation causes dysfunction of the regulatory system that provides the interdependence between the antioxidative activity of lipids, their composition and the activity of membrane-bound enzymes. There is virtually no correlation between the changes in hydrolase activity of the enzyme and the quantity of phosphatidylethanolamine (PE) and cardiolipin (CL). During the first hours following irradiation the dependence between the changes in the synthetase activity of ATPase and the fluidity of the lipid component of the membrane is directly proportional (just as it is observed in normal conditions); the lesser the fluidity of the lipid component the higher the hydrolase activity of the enzyme.

Effect of gamma irradiation and cystamine on kidney lipids of rats.

Effect of whole body gamma irradiation (1200 r) and the effect of administration cystamine prior to irradiation has been studied on kidney total lipids, cholesterol, phospholipids (phosphatidylcholine and phosphatidylethanolamine). Irradiation significantly decreases kidney cholesterol and this decrease was not prevented by administration of cystamine prior to irradiation. Irradiation did not affect the incorporation of NaH232PO4 into kidney phosphatidylcholine and phosphatidylethanolamine but the incorporation of glucose-U-14C was significantly reduced in kidney total lipids and phosphatidylcholine. Administration of cystamine before irradiation was ineffective in modifying the incorporation of glucose-U-14C into kidney lipids and phosphatidylcholine.

Synthesis of photoreactive phosphatidylethanolamine and its interaction with phospholipase A2.

A photoreactive derivative of phosphatidylethanolamine, N-(4-azidobenzoyl)phosphatidylethanolamine (AB-PE), was synthesized by acylation of phosphatidylethanolamine with an N-hydroxysuccinimide ester of 4-azidobenzoic acid. The substantial photosensitivity exhibited by AB-PE correlated with a marked decrease in the absorption spectra of the compound. The compound proved sensitive to lipase and phospholipase A2 hydrolysis but resistant to phospholipase C and D activities. Photolysis of a sonicated dispersion of AB-PE containing phospholipase A2 resulted in irreversible inhibition of the enzyme. Addition of natural phosphatidylethanolamine provided protection against photoinactivation.

Free radical mediated x-ray damage of model membranes.

The damaging effects of synchrotron-derived x rays on aqueous phospholipid dispersions have been evaluated. The effect of degree of lipid hydration, phospholipid chemical structure, mesophase identity, aqueous medium composition, and incident flux on the severity and progress of damage was quantified using time-resolved x-ray diffraction and chromatographic analysis of damage products. Electron spin resonance measurements of spin-trapped intermediates generated during irradiation suggest a free radical-mediated process. Surprisingly, radiation damage effects revealed by x-ray diffraction were imperceptible when the lamellar phases were prepared under water-stressed conditions, despite the fact that x-ray-induced chemical breakdown of the lipid occurred regardless of hydration level. Of the fully hydrated lipid systems studied, saturated diacyl-phosphatidylcholines were most sensitive to radiation damage compared to the ester- and ether-linked phosphatidylethanolamines and the ether-linked phosphatidylcholines. The inclusion of buffers or inorganic salts in the dispersing medium had only a minor effect in reducing damage development. A small inverse dose-rate effect was found when the x-ray beam intensity was changed 15-fold. These results contribute to our understanding of the mechanism of radiation damage, to our appreciation of the importance of monitoring both structure and composition when evaluating biomaterials radiation sensitivity, and to the development of strategies for eliminating or reducing the severity of damage due to an increasingly important source of x rays, synchrotron radiation. Because damage is shown to be free radical mediated, these results have an important bearing on age-related accumulation of free radicals in cells and how these might compromise membrane integrity, culminating in cell death.

Augmentation of macrophage growth-stimulating activity of lipids by their peroxidation.

Previously, we reported that some kinds of lipids (cholesterol esters, triglycerides, and some negatively charged phospholipids) that are constituents of lipoproteins or cell membranes induce growth of peripheral macrophages in vitro. In this paper, we examined the effect of peroxidation of lipids on their macrophage growth-stimulating activity because lipid peroxidation is observed in many pathological states such as inflammation. When phosphatidylserine, one of the phospholipids with growth-stimulating activity, was peroxidized by UV irradiation, its macrophage growth-stimulating activity was augmented in proportion to the extent of its peroxidation. The activity of phosphatidylethanolamine was also increased by UV irradiation. On the other hand, phosphatidylcholine or highly unsaturated free fatty acids, such as arachidonic acid and eicosapentaenoic acid, did not induce macrophage growth irrespective of whether they were peroxidized. The augmented activity of UV-irradiated phosphatidylserine was not affected by the coexistence of an antioxidant, vitamin E or BHT. These results suggest that some phospholipids included in damaged cells or denatured lipoproteins which are scavenged by macrophages in vivo may induce growth of peripheral macrophages more effectively when they are peroxidized by local pathological processes.