Simultaneous Exposure to Intracellular and Extracellular Photosensitizers for the Treatment of Staphylococcus aureus Infections.

Staphylococcus aureus is a serious threat to public health due to the rise of antibiotic resistance in this organism, which can prolong or exacerbate skin and soft tissue infections (SSTIs). Methicillin-resistant S. aureus is a Gram-positive bacterium and a leading cause of SSTIs. As such, many efforts are under way to develop therapies that target essential biological processes in S. aureus. Antimicrobial photodynamic therapy is an effective alternative to antibiotics; therefore we developed an approach to simultaneously expose S. aureus to intracellular and extracellular photosensitizers. A near infrared photosensitizer was conjugated to human monoclonal antibodies (MAbs) that target the S. aureus iron-regulated surface determinant (Isd) heme acquisition proteins. In addition, the compound VU0038882 was developed to increase photoactivatable porphyrins within the cell. Combinatorial photodynamic treatment of drug-resistant S. aureus exposed to VU0038882 and conjugated anti-Isd MAbs proved to be an effective antibacterial strategy in vitro and in a murine model of SSTIs.

Cellular entry and nuclear targeting by a highly anionic molecular umbrella.

A fluorescently labeled, persulfated molecular umbrella ( 1) has been synthesized from cholic acid, lysine, spermine, and Coumarin 343 and found capable of entering live HeLa cells. The distributions of 1 throughout the cytoplasm and the nucleus were diffuse and punctate, respectively. This finding, together with its ability to cross liposomal membranes by passive diffusion, suggests that passive diffusion plays a significant role in the ability of 1 to enter cells. The fact that 1 is concentrated at the nucleus raises the possibility that molecular umbrellas of this type could be used for the nuclear targeting of drugs.

An unusual photosensitizer: dyad of eosin-tris(2,2'-bipyridine)Ru(II).

[structure: see text] A dyad of eosin and tris(2,2'-bipyridine)Ru(II) was prepared, and its photophysical properties were investigated. The photosensitization of eosin is greatly enhanced by introduction of tris(2,2'-bipyridine)Ru(II), which is verified via photooxygenation of anthracene derivatives. The electron-transfer mechanism of photosensitization is also discussed.

[Ruthenium(II)(bpy)(2)L](2+), where L are imidazo[f]-1,10-phenanthrolines: synthesis, photophysics and binding with DNA.

Three Ru(II) complexes of type as [Ru(II)(bpy)(2)L](2+) were synthesized, where L are l,10-phenanthroline derivatives of imidazole (1), having at position 2 alpha-naphthyl (2), 3-methoxy-4-hydroxy-phenyl (3). All complexes show intense MLCT transition both in acetonitrile and in water and also exhibit strong emission at room temperature, which is efficiently quenched by oxygen as well as, to some extent, by water. The binding of complexes 1-3 to calf thymus DNA was investigated by using electronic absorption, steady-state luminescence, luminescence quenching, excited-state lifetime and circular dichroism spectra. Hypochromic effect, luminescence enhancement, and quenching studies demonstrate the existence of intercalation mode. Circular dichroism spectra indicate the stereoselectivity of the binding. The binding of 1-3 with DNA is sensitive to the nature of ligands, such as planarity, pi-electron extension and hydrophobicity. Complex 3 exhibits the strongest binding with DNA, which can be attributed to hydrogen bonding.

[Diagnostic effect of bronchoalveolar lavage in early lung injury caused by enterogenic infection in rat].

OBJECTIVE: To study the diagnostic effect of bronchoalveolar lavage in early lung injury by observing changes in inflammatory mediators in early lung injury caused by enterogenic infection. METHODS: Eighty-four Sprague-Dawley rats were randomly divided into infection group and sham-operation group. Cecal ligation and perforation was utilized to produce abdominal infection in rats. Six groups were sacrificed respectively at 0, 24, 48, 72, 96, 120 hours after operation. The differential cell count in bronchoalveolar lavage fluid (BALF) was assessed. The concentrations of endotoxin, phospholipase A2 (PLA2) and tumor necrosis factor-alpha (TNF-alpha) in BALF, lung and plasma were assayed. RESULTS: The neutrophil percentage of BALF increased progressively. The concentrations of endotoxin, PLA2 and TNF-alpha in BALF, lung and plasma were significantly increased. The levels of endotoxin and PLA2 in lung tissue were respectively correlated positively with those in BALF and plasma (BALF and lung: r=0.904, P<0.05; BALF and plasma: r=0.895, P<0.05; lung and plasma: r=0.946, P<0.01). Significant positive correlation was also present between the TNF-alpha levels in BALF and lung (r=0.952 P<0.01), but not between the TNF-alpha level in plasma and that in lung or BALF (r=0.684, r=0.608, both P>0.05). CONCLUSION: The examinations of bronchoalveolar lavage may help discover early lung injury caused by enterogenic infection.

[Analysis of platelets in the monitoring of systemic inflammatory response syndrome of critically ill patients].

OBJECTIVE: To study the clinical significance of the variance of platelets in systemic inflammatory response syndrome(SIRS) of critical illness. METHODS: Two hundred and thirteen critically ill patients in ICU, who suffered from SIRS, sepsis and multiple organ dysfunction syndrome (MODS), were enrolled in this study and divided into two groups, survivor group (n=151) and non-survivor group (n=62). Platelet, white blood cell counts and acute physiology and chronic health evaluation II (APACHE II) score were performed immediately after hospitalization, 3 days, 7 days, and 10 days later. At the same time, the serum was collected and the level of tumor necrosis factor-alpha (TNF-alpha) was measured. RESULTS: APACHE II score was much higher, but no difference in the two groups immediately after the hospitalization. However, it increased markedly in non-survivor group, and lowered dramatically in survivor group 7 days after therapy. There was a significant difference between the two groups (P<0.01). Platelets were slightly lower in both groups immediately after the hospitalization. After three days' therapy, it increased to the normal range in the two groups. However, it progressively dropped in non-survivor group 7 days and 10 days later, and it was significantly different from survivor group (P<0.001). The white blood cell counts revealed that there was no significant difference between the two groups. The level of TNF-alpha in serum was much higher in both groups immediately after the hospitalization. After three days' therapy, it further increased and was maintained at the high level in the two groups. However, it progressively dropped in survivor group, while it remained in higher level in non-survivor group 7 days and 10 days later, which was significantly different from survivor group (both P<0.001). CONCLUSION: Refractory thrombocytopenia is sensitively responsive to poor prognosis and severity of SIRS in critical illness.

Detecting cross talk between two halves of a phospholipid bilayer.

One of the most challenging questions that relates to the structure and function of biological membranes is whether the two halves of the bilayer "talk" to each other. In this letter, we show how the perturbation of the lateral organization of one leaflet of a fluid phospholipid bilayer by an external agent also alters the lateral organization of the adjoining leaflet. In addition, we show that the energy involved in such "cross talk" corresponds to ca. 100 cal/mol of phospholipid. These findings provide a basis for expecting similar cross talk to exist in cell membranes.

Molecular umbrellas: a novel class of candidate topical microbicides to prevent human immunodeficiency virus and herpes simplex virus infections.

Molecular umbrella compounds may function as novel topical microbicides to prevent human immunodeficiency virus (HIV) and herpes simplex virus (HSV) infections. In a preliminary structure-activity investigation, one umbrella compound, designated Spm8CHAS, was identified which inhibited both HIV and HSV infections with no cellular toxicity. The objectives of the current studies were to define its spectrum of antiviral activity, characterize its mechanism of action, and explore the possibility of combining Spm8CHAS with HIV-specific reverse transcriptase inhibitors. Spm8CHAS inhibited infections by laboratory and clinical R5 and X4 clade B and clade C HIV strains in cell culture. Ectocervical tissue explants exposed to HIV-1(BaL) in the presence of Spm8CHAS were completely protected (50% inhibitory concentration [IC(50)], 13.6 microg/ml), and transfer of virus to target T cells via migratory cells was abolished (IC(50), 3.8 microg/ml). Spm8CHAS inhibited HSV-2 infection of epithelial cells 10,000-fold if present throughout the infection. Notably, adding Spm8CHAS to cultures following HSV entry significantly reduced viral infection, indicating that the drug also acts postentry. Subsequent studies indicated that Spm8CHAS blocks cell-to-cell spread of HSV. Confocal microscopy using a fluorescently labeled analog of Spm8CHAS demonstrated that this conjugate crosses the plasma cell membrane and is transported to the nucleus. Combinations of Spm8CHAS with UC-781 or 9-[R-2-(phosphonylmethoxy)propyl] adenine monohydrate in vitro exhibited additive anti-HIV activity with preserved anti-HSV activity. The abilities of Spm8CHAS to inhibit primary isolates of HIV, block HSV infection postentry, and cross cell membranes support the development of a combination microbicide containing Spm8CHAS with an HIV-specific reverse transcriptase inhibitor to prevent both HIV and HSV infections by multiple mechanisms.

Ethanol-induced reorganization of the liquid-ordered phase: enhancement of cholesterol-phospholipid association.

This paper records what is believed to be the first evidence for the reorganization of the liquid-ordered phase by ethanol. Specifically, ethanol has been found to significantly enhance sterol-phospholipid association in liquid-ordered bilayers derived from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) plus cholesterol and also 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) plus cholesterol. The evidence for such reorganization comes from a series of nearest-neighbor recognition (NNR) experiments that have been carried out, where low concentrations of equilibrating lipid dimers (i.e., "reporter molecules") have been used to detect changes in the phase composition of host membranes made from varying mixtures of DPPC/cholesterol, and also DSPC/cholesterol, in the presence and in the absence of ethanol. These findings have important biological implications, which are briefly discussed.

Cholesterol-phospholipid association in fluid bilayers: a thermodynamic analysis from nearest-neighbor recognition measurements.

The mixing behavior of exchangeable, disulfide-based mimics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol has been examined as a function of temperature in host membranes made from DPPC and cholesterol in the liquid-disordered phase (ld), in the liquid-ordered phase (lo), and in the liquid-disordered/liquid-ordered coexistence region (ld/lo). In the ld region, lipid mixing was found to be temperature insensitive, reflecting close to ideal behavior. In contrast, a significant temperature dependence was observed in the lo phase from 45 to 60 degrees C, when 35 or 40 mol % sterol was present. In this region, sterol-phospholipid association was characterized by DeltaHo = -2.06 +/- 0.14 kcal/mol of phospholipid and DeltaS degrees = -4.48 +/- 0.44 cal/K mol of phospholipid. From 60 to 65 degrees C, the mixing of these lipids was found to be insensitive to temperature, and sterol-phospholipid association was now entropy driven; that is, DeltaHo = -0.23 +/- 0.38 kcal/mol of phospholipid and DeltaS degrees = +1.68 +/- 1.12 cal/K mol of phospholipid. In the liquid-disordered/liquid-ordered coexistence region, changes in lipid mixing reflect changes in the phase composition of the membrane.

Transbilayer complementarity of phospholipids in cholesterol-rich membranes.

Lipid-lipid interactions across cholesterol-rich phospholipid bilayers were investigated by measuring nearest-neighbor preferences of exchangeable phospholipids derived from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), in the presence of nonexchangeable dimers (i.e., templates) made from DMPE or DSPE. When homotemplates were present, a significant preference for homophospholipid association was observed. In contrast, when the corresponding heterotemplate was present, heterodimer formation was favored. These results support a model in which the longer phospholipid in one monolayer preferentially associates with the shorter one in the adjoining monolayer. In the absence of cholesterol, transbilayer complementarity was also observed but to a lesser degree. Transbilayer complementarity of phospholipids is likely to play an important role in stabilizing biological membranes and in promoting a compositional interdependence of their two lipid leaflets.

Transbilayer complementarity of phospholipids. Proof of principle.

A series of nearest-neighbor recognition (NNR) experiments have been carried out, which provide a rigorous test of the existence of transbilayer complementarity of phospholipids, that is, the ability of phospholipids to select complementary phospholipids from an adjoining monolayer as nearest neighbors. The application of this test to membranes derived from exchangeable phospholipids bearing myristoyl groups (A), stearoyl groups (B), and one stearoyl and one n-dodecyl group (C) in the presence of analogous nonexchangeable templates made from A', B' and C' provides compelling evidence for such complementarity in the physiologically relevant fluid phase.

Molecular umbrella-assisted transport of an oligonucleotide across cholesterol-rich phospholipid bilayers.

A series of molecular umbrella conjugates, derived from cholic acid, deoxycholic acid, spermidine, lysine, and 5-mercapto-2-nitrobenzoic acid, have been synthesized and found capable of transporting an attached 16-mer oligonucleotide (S-dT16) across liposomal membranes made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyldglycerol (POPG), and cholesterol [POPC/POPG/cholesterol (65/5/30; mol/mol/mol, v/v/v)] at 37 degrees C. Those molecular umbrellas containing four choloyl (or deoxycholoyl) groups resulted in significantly faster rates of transport as compared to those containing only two such moieties. A model that accounts for these membrane transport processes is proposed.

A chemical sensor for the liquid-ordered phase.

The mixing properties of exchangeable phospholipids, derived from 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, with an exchangeable form of cholesterol have been used to monitor the transition from the liquid-disordered to the liquid-ordered phase in cholesterol-containing bilayers, made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-distearoyl-sn-glycero-3-phosphocholine, respectively.

Molecular umbrella-assisted transport of thiolated AMP and ATP across phospholipid bilayers.

Two molecular umbrella-nucleoside conjugates (1a and 1b) have been synthesized via thiolate-disulfide displacement by adenosine 5'-O-(3-thiomonophosphate) and adenosine 5'-O-(3-thiotriphosphate) on an activated dimer derived from cholic acid, spermidine, and 5,5'-dithiobis-(2-nitrobenzoic acid). Both conjugates readily enter the aqueous compartment of liposomes made from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine (POPC) and release the free nucleoside upon reaction with entrapped glutathione. Approximately 50% of the thiolated form of AMP is released within 20 min at 23 degrees C; 120 min is required for a similar release of the thiolated form of ATP. The facile cleavage of these conjugates by glutathione, together with the fact that mammalian cells contain millimolar concentrations of this tripeptide in their cytoplasm, suggest that such chemistry may be extended to the practical development of prodrugs, e.g., antisense oligonucleotides that can be delivered into cells.

Fully detachable molecular umbrellas as peptide delivery agents.

A persulfated molecular umbrella, derived from cholic acid and spermidine, has been covalently attached to H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) by use of an o-dithiobenzyl carbamate linkage. Treatment of the resulting conjugate (1) with glutathione in solution resulted in the liberation of the free form of the peptide. Addition of 1 to glutathione-entrapped liposomes, prepared from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), and cholesterol [POPC/POPG/cholesterol, 72/4/24 (mol/mol/mol)], resulted in the delivery of DADLE into their aqueous interior.

Probing the hydration of lipid bilayers using a solvent isotope effect on phospholipid mixing.

Nearest-neighbor recognition experiments, which have been carried out using exchangeable dimers derived from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,and 1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine, indicate that replacement of H2O by D2O can significantly influence phospholipid mixing, but only in bilayers that are saturated and devoid of cholesterol. These findings, together with those of previous electron spin resonance spin-labeling studies,indicate that mammalian membranes, which are rich in cholesterol and unsaturated phospholipids, are ideal hydrophobic barriers.