Biosupramolecular complexes of amphiphilic photosensitizers with human serum albumin and cucurbit[7]uril as carriers for photodynamic therapy.

In the present work, we evaluated the supramolecular interactions between three photosensitizers, namely toluidine blue O (TBO, positively charged) and two fatty acid conjugates of 6 and 14 carbon atoms chain lengths (TBOC6 and TBOC14), with human serum albumin (HSA) and the macrocycle cucurbit[7]uril (CB[7]), alone or in combination within a biosupramolecular system as potential carriers of photosensitizers for Photodynamic therapy (PDT). Binding studies were carried out using photophysical and calorimetric techniques and accompanied with molecular docking simulations. Amphiphilic photosensitizers, particularly TBOC14, showed stronger binding to HSA and (CB[7]). Comparing the different delivery systems, (CB[7]) had a marginal effect on cell uptake and phototoxicity in HeLa cells, while HSA showed enhanced cell uptake with phototoxicities that depended on the photosensitizer. Despite low cell uptake, the combination of both (CB[7]) and HSA was the most phototoxic, which illustrates the potential of combining these systems for PDT applications.

Riboflavin Surface Modification of Poly(vinyl chloride) for Light-Triggered Control of Bacterial Biofilm and Virus Inactivation.

Poly(vinyl chloride) (PVC) is the most used biomedical polymer worldwide. PVC is a stable and chemically inert polymer. However, microorganisms can colonize PVC producing biomedical device-associated infections. While surface modifications of PVC can help improve the antimicrobial and antiviral properties, the chemically inert nature of PVC makes those modifications challenging and potentially toxic. In this work, we modified the PVC surface using a derivative riboflavin molecule that was chemically tethered to a plasma-treated PVC surface. Upon a low dosage of blue light, the riboflavin tethered to the PVC surface became photochemically activated, allowing for Pseudomonas aeruginosa bacterial biofilm and lentiviral in situ eradication.

Fatty Acid Conjugates of Toluidine Blue O as Amphiphilic Photosensitizers: Synthesis, Solubility, Photophysics and Photochemical Properties†.

Toluidine blue O (TBO) is a water-soluble photosensitizer that has been used in photodynamic antimicrobial and anticancer treatments, but suffers from limited solubility in hydrophobic media. In an effort to incrementally increase TBO's hydrophobicity, we describe the synthesis of hexanoic (TBOC6) and myristic (TBOC14) fatty acid derivatives of TBO formed in low to moderate percent yields by condensation with the free amine site. Covalently linking 6 and 14 carbon chains led to modifications of not only TBO's solubility, but also its photophysical and photochemical properties. TBOC6 and TBOC14 derivatives were more soluble in organic solvents and showed hypsochromic shifts in their absorption and emission bands. The solubility in phosphate buffer solution was low for both TBOC6 and TBOC14, but unexpectedly slightly greater in the latter. Both TBOC6 and TBOC14 showed decreased triplet excited-state lifetimes and singlet oxygen quantum yields in acetonitrile, which was attributed to heightened aggregation of these conjugates particularly at high concentrations due to the hydrophobic "tails." While in diluted aqueous buffer solution, indirect measurements showed similar efficiency in singlet oxygen generation for TBOC14 compared to TBO. This work demonstrates a facile synthesis of fatty acid TBO derivatives leading to amphiphilic compounds with a delocalized cationic "head" group and hydrophobic "tails" for potential to accumulate into biological membranes or membrane/aqueous interfaces in PDT applications.

Mechanism of Visible-Light Photooxidative Demethylation of Toluidine Blue O.

Experiments and theoretical calculations by density functional theory (DFT) have been carried out to examine a self-sensitized type I photooxidation of toluidine blue O (TBO+). This study attempts to build a connection between visible-light photolysis and demethylation processes of methylamine compounds, such as TBO+. We show that controlled photoinduced mono- and double-demethylation of TBO+ can be achieved. The kinetics for the appearance rate of the mono-demethylated TBO+ and the double-demethylated TBO+ were found to fit pseudo-first-order kinetics. DFT calculations have been used to examine the demethylation of TBO+ and included N, N-dimethylaniline as a model compound for TBO+. The results show an oxygen-dependent demethylation process. The mechanism for the sequential methyl loss is proposed to be due to H• or e-/H+ transfer to 3TBO+* followed by a reaction of TBO+• with O2, yielding a C-peroxyTBO+• intermediate. Instead of aminyl radical peroxyl formation, i.e., N-peroxyTBO+•, the C-centered peroxyTBO+• is favored, that upon dimerization (Russell mechanism) leads to dissociation of formaldehyde from the methylamine site.

Photodynamic performance of zinc phthalocyanine in HeLa cells: A comparison between DPCC liposomes and BSA as delivery systems.

Comparable intracellular concentrations (≈30pmol/10(6) cells) of bovine serum albumin-ZnPc (BSA) adduct outperformed dipalmitoyl-phosphatidyl-choline (DPPC) liposomes containing ZnPc at photodynamic-killing of human cervical cancer cells (HeLa) after only 15min of irradiation using red light (λ>620nm, 30W/cm(2)). This result could not be simply explained in terms of dye aggregation within the carrier, since in the liposomes the dye was considerably less aggregated than in bovine serum albumin, formulation that was capable to induce cell apoptosis upon red light exposure. Thus, using specific organelle staining, our cumulative data points towards intrinsic differences in intra-cellular localization depending on the cargo vehicle used, being ZnPc:BSA preferentially located in the near vicinity of the nucleus and in the Golgi structures, while the liposomal formulation ZnPc:DPPC was preferentially located in cellular membrane and cytoplasm. In addition to those differences, using real-time advanced fluorescence lifetime imaging of HeLa cells loaded with the photosensitizer contained in the different vehicles, we have found that only for the ZnPc:BSA formulation, there was no significant changes in the fluorescence lifetime of the photosensitizer inside the cells. This contrasts with the in situ≈two-fold reduction of the fluorescence lifetime measured for the liposomal ZnPc formulation. Those observations point towards the superiority of the protein to preserve dye aggregation, and its photochemical activity, post-cell uptake, demonstrating the pivotal role of the delivery vehicle at determining the ultimate fate of a photosensitizer.

Short-term dynamics of indoor and outdoor endotoxin exposure: Case of Santiago, Chile, 2012.

Indoor and outdoor endotoxin in PM2.5 was measured for the very first time in Santiago, Chile, in spring 2012. Average endotoxin concentrations were 0.099 and 0.094 [EU/m(3)] for indoor (N=44) and outdoor (N=41) samples, respectively; the indoor-outdoor correlation (log-transformed concentrations) was low: R=-0.06, 95% CI: (-0.35 to 0.24), likely owing to outdoor spatial variability. A linear regression model explained 68% of variability in outdoor endotoxins, using as predictors elemental carbon (a proxy of traffic emissions), chlorine (a tracer of marine air masses reaching the city) and relative humidity (a modulator of surface emissions of dust, vegetation and garbage debris). In this study, for the first time a potential source contribution function (PSCF) was applied to outdoor endotoxin measurements. Wind trajectory analysis identified upwind agricultural sources as contributors to the short-term, outdoor endotoxin variability. Our results confirm an association between combustion particles from traffic and outdoor endotoxin concentrations. For indoor endotoxins, a predictive model was developed but it only explained 44% of endotoxin variability; the significant predictors were tracers of indoor PM2.5 dust (Si, Ca), number of external windows and number of hours with internal doors open. Results suggest that short-term indoor endotoxin variability may be driven by household dust/garbage production and handling. This would explain the modest predictive performance of published models that use answers to household surveys as predictors. One feasible alternative is to increase the sampling period so that household features would arise as significant predictors of long-term airborne endotoxin levels.

Comparison of the Superagonist Complex, ALT-803, to IL15 as Cancer Immunotherapeutics in Animal Models.

IL15, a potent stimulant of CD8(+) T cells and natural killer (NK) cells, is a promising cancer immunotherapeutic. ALT-803 is a complex of an IL15 superagonist mutant and a dimeric IL15 receptor αSu/Fc fusion protein that was found to exhibit enhanced biologic activity in vivo, with a substantially longer serum half-life than recombinant IL15. A single intravenous dose of ALT-803, but not IL15, eliminated well-established tumors and prolonged survival of mice bearing multiple myeloma. In this study, we extended these findings to demonstrate the superior antitumor activity of ALT-803 over IL15 in mice bearing subcutaneous B16F10 melanoma tumors and CT26 colon carcinoma metastases. Tissue biodistribution studies in mice also showed much greater retention of ALT-803 in the lymphoid organs compared with IL15, consistent with its highly potent immunostimulatory and antitumor activities in vivo. Weekly dosing with 1 mg/kg ALT-803 in C57BL/6 mice was well tolerated, yet capable of increasing peripheral blood lymphocyte, neutrophil, and monocyte counts by >8-fold. ALT-803 dose-dependent stimulation of immune cell infiltration into the lymphoid organs was also observed. Similarly, cynomolgus monkeys treated weekly with ALT-803 showed dose-dependent increases of peripheral blood lymphocyte counts, including NK, CD4(+), and CD8(+) memory T-cell subsets. In vitro studies demonstrated ALT-803-mediated stimulation of mouse and human immune cell proliferation and IFNγ production without inducing a broad-based release of other proinflammatory cytokines (i.e., cytokine storm). Based on these results, a weekly dosing regimen of ALT-803 has been implemented in multiple clinical studies to evaluate the dose required for effective immune cell stimulation in humans.

Morphological Effects Induced In Vitro by Propranolol on Human Erythrocytes.

Despite the extended use and well-documented information, there are insufficient reports concerning the effects of propranolol on the structure and functions of cell membranes, particularly those of human erythrocytes. Aimed to better understand the molecular mechanisms of its interactions with cell membranes, human erythrocyte and molecular models of the red cell membrane were utilized. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of propranolol to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction. Moreover, we took advantage of the capability of differential scanning calorimetry to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from propranolol interaction with DMPC and DMPE multilamellar vesicles. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy. Results indicated that propranolol induced morphological changes to human erythrocytes and interacted in a concentration-dependent manner with phospholipid bilayer.

Structure and general properties of flavins.

Flavins are a family of yellow-colored compounds with the basic structure of 7,8-dimethyl-10-alkylisoalloxazine. Riboflavin, commonly known as vitamin B2, is an essential component of living organisms and is the precursor of all biologically important flavins. In this chapter, the redox properties of flavins are described, with special emphasis in their ability to participate in both one-electron and two-electron transfer processes; hence, flavins are indispensable mediators between two-electron and one-electron processes in biological systems. The photophysical and photochemical properties of flavins are also discussed. All oxidized flavins exhibit strong absorption in the ultraviolet and visible regions and an intense yellow-green fluorescence (in their neutral oxidized form). Flavins are thermostable compounds; however, they are photosensitive. In the absence of an external reductant, the isoalloxazine ring system undergoes intramolecular photoreduction. Some flavins are efficient photosensitizers; they can induce photomodifications of compounds that are not directly modified by visible light.

Silica nanoreactors from silylated riboflavin for efficient singlet oxygen delivery.

Silylation of riboflavin allowed its incorporation into spherical SiO2 nanoparticles that were able to generate singlet oxygen and oxidize human serum albumin while conferring riboflavin remarkable photostability.

Efficacy and mechanism-of-action of a novel superagonist interleukin-15: interleukin-15 receptor αSu/Fc fusion complex in syngeneic murine models of multiple myeloma.

ALT-803, a complex of an interleukin (IL)-15 superagonist mutant and a dimeric IL-15 receptor αSu/Fc fusion protein, was found to exhibit significantly stronger in vivo biologic activity on NK and T cells than IL-15. In this study, we show that a single dose of ALT-803, but not IL-15 alone, eliminated well-established 5T33P and MOPC-315P myeloma cells in the bone marrow of tumor-bearing mice. ALT-803 treatment also significantly prolonged survival of myeloma-bearing mice and provided resistance to rechallenge with the same tumor cells through a CD8(+) T-cell-dependent mechanism. ALT-803 treatment stimulated CD8(+) T cells to secrete large amounts of IFN-γ and promoted rapid expansion of CD8(+)CD44(high) memory T cells in vivo. These memory CD8(+) T cells exhibited ALT-803-mediated upregulation of NKG2D (KLRK1) but not PD-1 (PDCD1) or CD25 (IL2RA) on their cell surfaces. ALT-803-activated CD8(+) memory T cells also exhibited nonspecific cytotoxicity against myeloma and other tumor cells in vitro, whereas IFN-γ had no direct effect on myeloma cell growth. ALT-803 lost its antimyeloma activity in tumor-bearing IFN-γ knockout mice but retained the ability to promote CD8(+)CD44(high) memory T-cell proliferation, indicating that ALT-803-mediated stimulation of CD8(+)CD44(high) memory T cells is IFN-γ-independent. Thus, besides well-known IL-15 biologic functions in host immunity, this study shows that IL-15-based ALT-803 could activate CD8(+)CD44(high) memory T cells to acquire a unique innate-like phenotype and secrete IFN-γ for nonspecific tumor cell killing. This unique immunomodulatory property of ALT-803 strongly supports its clinical development as a novel immunotherapeutic agent against cancer and viral infections.

Computational basis for risk stratification of peripheral neuropathy from thermal imaging.

The goal of this paper is to present a computer-based system for analyzing thermal images in the detection of preclinical stages of peripheral neuropathy (PN) or diabetic foot. Today, vibration perception threshold (VPT) and sensory tests with a monofilament are used as simple, noninvasive methods for identifying patients who have lost sensation in their feet. These tests are qualitative and are ineffective in stratifying risk for PN in a diabetic patient. In our system a cold stimulus applied to the foot causes a thermoregulatory and corresponding microcirculation response of the foot. A thermal video monitors the recovery of the microcirculation in the foot plantar. Thermal videos for 8 age-matched subjects were analyzed. Six sites were tracked and an average thermal emittance calculated. Characteristics of the recovery curve were extracted using coefficients from an exponential curve fitting process and compared among subjects. The magnitude of the recovery was significantly different for the two classes of subjects. Our system shows evidence of differences between both groups, which could lead to a quantitative test to screen and diagnose peripheral neuropathy.

Human cells and cell membrane molecular models are affected in vitro by the nonsteroidal anti-inflammatory drug ibuprofen.

This report presents evidence that ibuprofen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a concentration as low as 10µM; b) in isolated unsealed human erythrocyte membranes (IUM) induced mild increase in the water content or in their molecular dynamics at the hydrophobic-hydrophilic interphase, while a corresponding ordering decrease at the deep phospholipids acyl chain level; c) at physiological temperature (37°C), 300µM ibuprofen induced a significant increase in the generalized polarization (GP) of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUV), an indication that ibuprofen molecules locate in the head polar group region of DMPC; d) X-ray diffraction studies showed that ibuprofen concentrations≥300µM induced increasing structural perturbation to DMPC bilayers; e) differential scanning calorimetry (DSC) data showed that ibuprofen was able to alter the cooperativity of DMPC phase transition in a concentration-dependent manner, to destabilize the gel phase and that ibuprofen did not significantly perturb the organization of the lipid hydrocarbon chains. Additionally, the effect on the viability of both human promyelocytic leukemia HL-60 and human cervical carcinoma HeLa cells was studied.

Different cell death mechanisms are induced by a hydrophobic flavin in human tumor cells after visible light irradiation.

Riboflavin (RF) is an endogenous cell component and an efficient photosensitizer that can act by both types I and II photochemical mechanisms. Human tumor cells lines cultured in vitro, were used as model to study the effect of a photosensitizer synthesized from riboflavin, the 2',3',4',5'-riboflavin-tetrabutyrate (RTB), to increase the flavin concentration in the human promyelocytic leukemia cell line HL-60 and the human epithelial cervical cancer cell line HeLa. We demonstrate that this compound, alone or with Trp, has a toxic dose-response effect evidenced by abnormal cell morphology and a decrease in the cell proliferation rate. The mechanism of cell death was investigated and the experimental evidence indicates that it proceeds primarily via apoptosis; however, autophagy cannot be discarded. Nuclear fluorescent staining with Hoechst 33258 and transmission electron microscopy of the cells showed condensed chromatin margination at the nuclear periphery and the formation of apoptotic bodies. Furthermore, Caspase-3 activity was demonstrated in both cell lines. In addition, the characteristic apoptotic DNA ladder was observed in HL-60 cells. On the other hand, a high cytoplasmic vacuolization was observed by electron transmission and confocal microscopy. LysoTraker-red localization in the vacuoles was observed by fluorescence microscopy, and a significant decrease in the number of vacuoles and in the cell proliferation rate diminution was observed when irradiation was performed in the presence of the autophagy inhibitor 3-methyladenine. Considering that both cell death mechanisms have a dual role in the killing of tumor cells in vivo, a harmful effect that does not cause inflammation leading to tumor prophylaxis, we conclude that RTB could have potential clinical applications.

Photophysical behaviour and photodynamic activity of zinc phthalocyanines associated to liposomes.

Phthalocyanines are macrocyclic compounds that can be employed as photosensitizers in the treatment of various infections and diseases, as well as in photodynamic therapy. Nevertheless, a disadvantage for the clinical application of these compounds is their strong tendency to form oligomers (especially dimers), a phenomenon that reduces their efficiency as photosensitizers. In the present contribution, we have studied the photophysical and photochemical properties of ZnPc and ZnF(16)Pc in an organic solvent (THF) and liposomal formulations (DMPC, DPPC and DSPC). Our results show that dye incorporation into liposomes decreases its aggregation degree, as revealed by absorption spectra, triplet quantum yield, and singlet oxygen quantum yield measurements. Additionally, we studied the photodynamic activity of both phthalocyanines in liposomal formulation on human cervical carcinoma (HeLa) cells. For ZnF(16)Pc the photophysical behavior and phototoxicity in vitro correlate with the aggregation degree. The dimers are not photoactive and the photochemistry of ZnF(16)Pc depends of the fraction present as monomer. On the other hand, ZnPc aggregation is minimal and its photophysical and photochemical properties are similar in the three liposomes studied. Nevertheless, its phototoxicity in vitro is liposome dependent.

Photophysics and photochemistry of dyes bound to human serum albumin are determined by the dye localization.

The photophysics and photochemistry of rose bengal (RB) and methylene blue (MB) bound to human serum albumin (HSA) have been investigated under a variety of experimental conditions. Distribution of the dyes between the external solvent and the protein has been estimated by physical separation and fluorescence measurements. The main localization of protein-bound dye molecules was estimated by the intrinsic fluorescence quenching, displacement of fluorescent probes bound to specific protein sites, and by docking modelling. All the data indicate that, at low occupation numbers, RB binds strongly to the HSA site I, while MB localizes predominantly in the protein binding site II. This different localization explains the observed differences in the dyes' photochemical behaviour. In particular, the environment provided by site I is less polar and considerably less accessible to oxygen. The localization of RB in site I also leads to an efficient quenching of the intrinsic protein fluorescence (ascribed to the nearby Trp residue) and the generation of intra-protein singlet oxygen, whose behaviour is different to that observed in the external solvent or when it is generated by bound MB.

Inhibition of acute vascular thrombosis in chimpanzees by an anti-human tissue factor antibody targeting the factor X binding site.

Tissue factor (TF) antagonists targeting the factor VII (FVII) binding domain have been shown to interrupt acute vascular thrombus formation without impairing haemostasis in non-human primates. In this study, we evaluate whether a human/mouse chimeric monoclonal antibody (ALT-836, formerly known as Sunol-cH36) blocking the factor X/factor IX (FX/FIX) binding site of tissue factor could achieve similar clinical benefits in an arterial thrombosis model induced by surgical endarterectomy in chimpanzees. In this model, sequential surgical endarterectomies on right and left superficial femoral arteries were performed 30 days apart in five chimpanzees. A bolus (1 mg/kg) of ALT-836 was injected intravenously immediately preceding the restoration of flow in the endarterectomised femoral artery. Pre-surgical labelling of autologous platelets using (111)In-Oxine and post-surgical gamma camera imaging of (111)In-platelet deposition at endarterectomy sites was performed. The manipulated arterial segments were harvested for patency analysis 30 days following surgery. The results indicate that ALT-836 was highly effective at reducing acute vascular thrombosis, with no significant variations in surgical blood loss and template-bleeding time in the treated group compared to the control animals. These data suggest that ALT-836 is an effective and safe antithrombotic agent in preventing TF-initiated vascular thrombogenesis without compromising haemostasis.

Blocking of CD27-CD70 pathway by anti-CD70 antibody ameliorates joint disease in murine collagen-induced arthritis.

Rheumatoid arthritis (RA) is characterized by inflammation and cellular proliferation in the synovial lining of joints that result in cartilage and bone destruction. Although the etiology of RA is unclear, activated lymphocytes and proinflammatory molecules, in particular TNF superfamily members, have been implicated in the disease pathology. A TNF superfamily member, CD70, is found on activated lymphocytes and shown to be important in memory and effector responses of lymphocytes. CD70 is expressed at high levels on chronically activated T cells in patients with autoimmune disorders, including RA. The involvement of CD70 in the progression of RA, however, remains unknown. In this study, we report effects of targeting CD70 on disease pathogenesis by using an anti-mouse CD70 Ab in a murine model of collagen-induced arthritis (CIA). In addition to blocking CD70 binding to its receptor CD27, the anti-CD70 Ab used also engages Fc-dependent effector functions including Ab-dependent cellular cytotoxicity, phagocytosis, and complement fixation. Treatment of mice with anti-CD70 Ab both before the onset or after the established disease in CIA model resulted in marked improvements in disease severity and significant reduction in the production of autoantibodies. Histopathological analyses of the joints of mice revealed a substantial reduction of inflammation, and bone and cartilage destruction in response to the anti-CD70 Ab treatment. These results uncover a novel role for CD27-CD70 interactions in the regulation of in vivo inflammatory response leading to arthritis, and provide a molecular basis to support the rationale for anti-CD70 therapy for autoimmune and inflammatory diseases.

Photophysics and photochemistry of rose bengal bound to human serum albumin.

Rose bengal (RB) readily binds to human serum albumin (HSA). At low RB concentrations, 90% of the dye is associated to the protein (5 microM), This association takes place in specific binding sites I and/or II. At higher RB concentrations, unspecific binding takes place with up to 10 RB molecules bound per protein molecule. The behavior of excited RB molecules bound to HSA is widely different to that observed in aqueous solution. Furthermore, the data also show that the behavior of bound RB molecules changes with the average number of dye molecules per protein (n). In particular, when n is large, the fluorescence yield is significantly reduced and no measurable long-lived triples and free singlet oxygen formation from bound dyes is detected. These results are related to self-quenching of the singlet and, most likely, excited triplets. All results point to the relevance of intra-protein generated singlet oxygen. However, when the dye is bound to the protein, at low oxygen concentrations such as those prevailing in vivo, trapping by oxygen of the triplet becomes inefficient and type I processes could contribute to the observed photoprocesses.

Photophysics and photochemistry of zinc phthalocyanine/bovine serum albumin adducts.

Zinc phthalocyanine (ZnPc) is a well known Type II (singlet oxygen mediated) hydrophobic photosensitizer with potential use in PDT. We have found that the presence of bovine serum albumin diminishes the aggregation degree of ZnPc in aqueous solution, indicating that albumins could be potentially useful carriers for this type of photosensitizer in PDT. In order to explore the photochemical and photophysical behavior of ZnPc associated to the protein, we have evaluated triplet excited state lifetime and yield, dye bleaching, oxygen consumption, formation of carbonyls and peroxides, and the spontaneous chemiluminescence emitted after photolysis. The results show that dye association to BSA modifies the photophysics and photochemistry of ZnPC. In particular the decreased yield of long lived triplets suggests singlet state and/or static triplet quenching of the bound dye by the host protein.