Near Infrared Photoimmunotherapy Targeting EGFR Positive Triple Negative Breast Cancer: Optimizing the Conjugate-Light Regimen

Aim

Triple-negative breast cancer (TNBC) is considered one of the most aggressive subtypes of breast cancer. Near infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that employs an antibody-photosensitizer conjugate (APC) followed by exposure of NIR light for activating selective cytotoxicity on targeted cancer cells and may have application to TNBC. In order to minimize the dose of APC while maximizing the therapeutic effects, dosing of the APC and NIR light need to be optimized. In this study, we investigate in vitro and in vivo efficacy of cetuximab (cet)-IR700 NIR-PIT on two breast cancer models MDAMB231 (TNBC, EGFR moderate) and MDAMB468 (TNBC, EGFR high) cell lines, and demonstrate a method to optimize the dosing APC and NIR light.

Method

After validating in vitro cell-specific cytotoxicity, NIR-PIT therapeutic effects were investigated in mouse models using cell lines derived from TNBC tumors. Tumor-bearing mice were separated into 4 groups for the following treatments: (1) no treatment (control); (2) 300 μg of cet-IR700 i.v., (APC i.v. only); (3) NIR light exposure only, NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2 (NIR light only); (4) 300 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 after injection and 100 J/cm² of light on day 2 after injection (one shot NIR-PIT). To compare different treatment regimens with a fixed dose of APC, we added the following treatments (5) 100 μg of cet-IR700 i.v., NIR light administered at 50 J/cm² on day 1 and 50 μg of cet-IR700 i.v. immediately after NIR-PIT, then NIR light was administered at 100 J/cm² on day 2, which were performed two times every week (“two split” NIR-PIT) and (6) 100 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2, which were performed three times per week (“three split” NIR-PIT).

Result

Both specific binding and NIR-PIT effects were greater with MDAMB468 than MDAMB231 cells in vitro. Tumor accumulation of cet-IR700 in MDAMB468 tumors was significantly higher (p < 0.05) than in MDAMB231 tumors in vivo. Tumor growth and survival of MDAMB231 tumor bearing mice was significantly lower in the NIR-PIT treatment group (p < 0.05). In MDAMB468 bearing mice, tumor growth and survival was significantly improved in the NIR-PIT treatment groups in all treatment regimens (one shot NIR-PIT; p < 0.05, “two split” NIR-PIT; p < 0.01, “three split” NIR-PIT; p < 0.001) compared with control groups.

Conclusion

NIR-PIT for TNBC was effective regardless of expression of EGFR, however, greater cell killing was shown with higher EGFR expression tumor in vitro. In all treatment regimens, NIR-PIT suppressed tumor growth, resulting in significantly prolonged survival that further improved by splitting the APC dose and using repeated light exposures.     

Near-infrared theranostic photoimmunotherapy (PIT): repeated exposure of light enhances the effect of immunoconjugate

Armed antibody-based targeted molecular therapies offer the possibility of effective tumor control with a minimum of side effects. Photoimmunotherapy (PIT) employs a monoclonal antibody-phototoxic phthalocyanine dye, IR700 conjugate, that is activated by focal near-infrared (NIR) light irradiation after antibody binding to the targeted tumor cell surface leading to rapid necrotic cell death. Therapy by single NIR light irradiation was effective without significant side effects; however, recurrences were seen in most treated mice probably because of inhomogeneous distribution of panitumumab-IR700 immunoconjugate in the tumor, leading to ineffective PIT. We describe here an optimized regimen of effective PIT method for the same HER1-overexpressing tumor model (A431) with fractionated administration of panitumumab-IR700 conjugate followed by systematic repeated NIR light irradiation to the tumor based on timing of antibody redistribution into the remnant tumor under the guidance of IR700 fluorescence signal. Eighty percent of the A431 tumors were eradicated with repeated PIT without apparent side effects and survived tumor-free for more than 120 days even after stopping therapy at day 30. Therapeutic effects were monitored using IR700 fluorescent signal. PIT is a promising highly selective and clinically feasible theranostic method for treatment of mAb-binding tumors with minimal off-target effects.     

Imaging modalities for monitoring acute therapeutic effects after near-infrared photoimmunotherapy in vivo

Near-infrared photoimmunotherapy (NIR-PIT) induces immediate cell death after irradiation with near-infrared (NIR) light. Acute therapeutic effects caused by NIR-PIT before the change of tumor size is essential to be monitored by imaging modalities. We summarized and compared the imaging modalities for evaluating acute therapeutic effects after NIR-PIT, and aimed to provide a better understanding of advantages and disadvantages of each modality for evaluation in clinical applications. Fluorescence imaging and fluorescence lifetime, with high resolution, remains high accumulation of fluorescence dyes in the normal organs. High resolution and noninvasiveness are the major advantages of magnetic resonance imaging, while ¹⁸F-fluorodeoxyglucose positron emission tomography provides information about the glucose metabolism. Optical coherence tomography provided more information about the blood vessels. Thus, all of the imaging modalities play an important role in evaluating acute therapeutic effects after NIR-PIT. Clinicians should choose suitable modality according to specific purpose and conditions in clinical application.     

Primary photosynthetic reactions in isolated chloroplasts fixed by glutaric aldehyde]

The primary photosynthetic reactions in isolated pea chloroplasts with the structures fixed by increasing concentrations of glutaric aldehyde were studied. It was shown that under chloroplast fixation by 5--25 mM of glutaric aldehyde, a significant inhibition of processes responsible for energy transformation in biological membranes was observed. The highest sensitivity was observed for the phosphorylation reactions, photo-induced changes in absorption at 520 nm, photo-induced quenching of atebrin fluorescence and slow component of delayed light emission. The photo-induced proton uptake was found to be less sensitive to fixation by glutaric aldehyde. It was also shown that on chloroplast fixation the extent of the steady-state P700 oxidation and the lifetime of the photosystem I and II chlorophyll fluorescence are both increased, a fact is indicating of loss in the effectiveness of light energy transfer from the antenna molecules to the reaction centres. Presumably the conformational changes play an essential role at the initial steps of light energy transformation.     

Hypericin photo-induced apoptosis involves the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and activation of caspase-8

Hypericin (HYP) is a photosensitizing pigment from Hypericum perforatum that displays cytotoxic effects in neoplastic cell lines. Therefore, HYP is presently under consideration as a new anticancer drug in photodynamic therapy. Here, we investigated the mechanism of action of HYP photo-induced apoptosis of Jurkat cells compared to the cytostatic drug paclitaxel (PXL). Both photoactivated HYP and PXL similarly increased the activity of caspase-8 and caspase-3, and drug-induced apoptosis of Jurkat cells was completely blocked by inhibitors of caspase-8 (Z-IETD-FMK) and caspase-3 (Z-DEVD-FMK). The involvement of death receptors was analyzed using neutralizing monoclonal antibodies against Fas (SM1/23), FasL (NOK-2) and TNF-R1 (MAB225), and a polyclonal rabbit anti-human TNF-related apoptosis-inducing ligand (TRAIL) antiserum. TRAIL antibody blocked TRAIL-induced and HYP photo-induced, but not PXL-induced apoptosis of Jurkat cells. In contrast, PXL-induced, but not HYP-induced apoptosis was blocked by the SM1/23 and NOK-2 antibodies. Anti-TNF-R1 antibody had no effect. These findings suggest that HYP photo-induced apoptosis of Jurkat cells is mediated in part by the TRAIL/TRAIL-receptor system and subsequent activation of upstream caspases.     

The two histidine axial ligands of the primary electron donor chlorophylls (P700) in photosystem I are similarly perturbed upon P700+ formation

The extent of delocalization of the positive charge in the oxidized dimer of chlorophyll (Chl) constituting P700, the primary electron donor of photosystem I (PSI), has been investigated by analyzing the perturbation upon P700(+) formation of infrared (IR) vibrational modes of the two His axial ligands of the two P700 Chl molecules. Fourier transform IR (FTIR) difference spectra of the photooxidation of P700 in PSI core complexes isolated from Synechocystis sp. PCC 6803 isotopically labeled either globally with (15)N or more specifically with (13)C on all the His residues reveal isotopic shifts of a differential signal at 1102/1108 cm(-)(1). This signal is assigned to a downshift upon P700(+) formation of the predominantly C(5)-Ntau imidazole stretching mode of His residue(s). The amplitude of this signal is reduced by approximately half in FTIR spectra of Synechocystis mutants in which His PsaB 651, the axial ligand to one of the two Chl molecules in P700, is replaced by Cys, Gln, or Leu. These observations provide further evidence that the positive charge in P700(+) is essentially delocalized over the two Chl molecules, in agreement with a previous FTIR study in which the frequency of the vibrational modes of the 9-keto and 10a-ester C=O groups of the two Chl's in P700, P700(+), and (3)P700 were firmly established for the first time [Breton, J., et al. (1999) Biochemistry 38, 11585-11592]. Only limited perturbations of the amplitude and frequency of the 9-keto and 10a-ester C=O bands of the P700 Chl are elicited by the mutations. On the basis of comparable mutational studies of the primary electron donor in purple bacteria, these perturbations are attributed to small molecular rearrangements of the Chl macrocycle and substituents caused by the repositioning of the P700 dimer in the new protein cavity generated by the mutations. It is proposed that the perturbation of the FTIR spectra upon mutation of a His axial ligand of the P700 Chl recently reported in Chlamydomonas reinhardtii [Hastings, G., et al. (2001) Biochemistry 40, 12943-12949] can be explained by the same effect without the need for a new assignment of the C=O bands of P700. The distribution of charge/spin in P700(+) and (3)P700 determined by FTIR spectroscopy is discussed in relation with the contrasting interpretations derived from recent magnetic resonance experiments.     

Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules

Three major modes of cancer therapy (surgery, radiation and chemotherapy) are the mainstay of modern oncologic therapy. To minimize the side effects of these therapies, molecular-targeted cancer therapies, including armed antibody therapy, have been developed with limited success. In this study, we have developed a new type of molecular-targeted cancer therapy, photoimmunotherapy (PIT), that uses a target-specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IR700, conjugated to monoclonal antibodies (mAbs) targeting epidermal growth factor receptors. Cell death was induced immediately after irradiating mAb-IR700-bound target cells with NIR light. We observed in vivo tumor shrinkage after irradiation with NIR light in target cells expressing the epidermal growth factor receptor. The mAb-IR700 conjugates were most effective when bound to the cell membrane and produced no phototoxicity when not bound, suggesting a different mechanism for PIT as compared to conventional photodynamic therapies. Target-selective PIT enables treatment of cancer based on mAb binding to the cell membrane.     

Short oligonucleotide prodrug having 5-fluoro and 5-iodouracil inhibits the proliferation of cancer cells in a photo-responsive manner

Photo-induced C1′ hydrogen abstraction of 5-fluoro-2′-deoxyuridine was adopted as the key reaction for releasing 5-fluorouracil (5-FU) anticancer drug from oligonucleotide strands. After photoirradiation following 5-FU release, anticancer activity was expected. We demonstrated that oligonucleotide tetramer, d(A^FU^IUA), can release 5-FU under physiological conditions in a photo-responsive manner thorough photo-induced C1′ hydrogen abstraction, and that the 5-FU released from d(A^FU^IUA) having a phosphorothioate backbone clearly suppresses the proliferation of HeLa cells in a photo-responsive manner.     

Dual-targeting multifunctional hyaluronic acid ligand-capped gold nanorods with enhanced endo-lysosomal escape ability for synergetic photodynamic–photothermal therapy of cancer

Au nanorods (AuNRs) have attracted considerable interest as drug delivery systems because of their enhanced cell internalization and stronger drug-loading ability. In addition, the incorporation of photodynamic therapy (PDT) and photothermal therapy (PTT) into one nanosystem presents great promise to defect multiple drawbacks in cancer therapy. Herein, we fabricated a multifunctional and dual-targeting nanoplatform based on hyaluronic acid-grafted-(mPEG/triethylenetetramine-conjugated-lipoic acid/tetra(4-carboxyphenyl)porphyrin/folic acid) polymer ligand capped AuNRs (AuNRs@HA-g-(mPEG/Teta-co-(LA/TCPP/FA)) for combined photodynamic–photothermal therapy of cancer. The prepared nanoparticles displayed high TCPP loading capacity and excellent stability in different biological media. Furthermore, AuNRs@HA-g-(mPEG/Teta-co-(LA/TCPP/FA)) not only could produce a localized hyperthermia to conduct PTT, but also generate cytotoxic singlet oxygen (¹O₂) to perform PDT under laser irradiation. Confocal imaging results disclosed that this nanoparticle endowing the specific function of polymeric ligand could enhance cellular uptake, accelerate endo/lysosomal escape, as well as produce higher reactive oxygen species. Importantly, this combination therapy strategy could also induce higher anticancer potential than PDT or PTT only against MCF-7 tumor cells in vitro. Therefore, this work presented an AuNRs-based therapeutic nanoplatform with great potential in dual-targeting and photo-induced combination therapy of cancer.     

Dichroism of chlorophyll aI absorption change at 700 nm using chloroplastsoriented in a magnetic field.

Using spinach chloroplasts oriented in a magnetic field we have measured the dichroism of the photo-induced absorption change associated with chlorophyll a_I at 700 nm. The greatest absorption change was detected when the electric vector of the measuring beam was polarized parallel to the plane of the photosynthetic membranes. The large dichroic ratio (2.3 ± .1) measured at 700 nm indicates that the transition oscillator involved at this wavelength is nearly parallel to the plane of the photosynthetic membrane. This result is consistent with an earlier study by Junge and Eckhof.     

Study of the acceptor portion of photosystem I according to the temperature relationships of P700 photoconversions

The functioning of the acceptor part of photosystem I was studied by temperature dependence of time course of light induced absorbtion changes at 700 nm of digitonin chloroplast fragments, enriched by photosystem I. Partial irreversibility of P700 photooxidation at low temperatures and appearance of two components (rapid and slow) in the time course of P700+ dark reduction reflect the contribution of different acceptors in electron transport. Thermoinactivation of fragments incubation at acid pH or treatment by glutaraldehyde cause complete inhibition of irreversible P700 photooxidation and slow dark reduction of P700+ at -170 degrees. The slow component of P700+ reduction and irreversible photooxidation of P700 are ascribed to contribution of secondary ferredoxin acceptors. The accurence of rapid component of P700+ dark reduction in light induced signal of treated fragments indicate that this component is due to recombination of reduced primary acceptor and P700+. Because only one electron transport takes at -170 degrees, the occurence of rapid and slow components in dark decay kinetics of P700+ suggests, that secondary acceptors of some reaction centers are incapable to reduction at -170 degrees. The shape of temperature dependence curve of the slow P700+ reduction component is interpreted as an indication of the tunneling electron transport.     

Time-resolved cryo-electron microscopic study of the dissociation of actomyosin induced by photolysis of photolabile nucleotides

The rapid release of a substrate or other ligand from photolabile precursors in a thin layer suspension of biological specimens followed by rapid freezing provides a method of trapping and visualizing short-lived states in a dynamic system. We demonstrate here the first successful application of this method to study the interaction of actin filaments with myosin subfragment 1 (S1) after release of nucleotides. The results obtained suggest that structural changes in actin filaments occur as a result of interaction with S1.     

Adenine nucleotide binding sites on beef heart F1-ATPase. Specificity of cooperative interactions between catalytic sites

Cooperative interactions between nucleotide binding sites on beef heart mitochondrial F1-ATPase have been studied by measuring substrate-promoted release of 5'adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) from a single high affinity site. The site is initially loaded by incubating F1 with an equimolar amount of the nonhydrolyzable ATP analog. When unbound [3H]AMP-PNP is removed and the complex diluted to a concentration below the Kd, release of ligand shows an apparent absolute requirement for medium ADP. Release is biphasic with the extent of release during the initial rapid phase dependent on the concentration of medium ADP. Although phosphate alone has no effect, it enhances the rapid phase of ADP-promoted release over 2-fold with a half-maximal effect at 60 micrometers P1. The binding of efrapeptin (A23871) to the F1.AMP-PNP complex completely prevents ADP-promoted dissociation. Although AMP-PNP release also occurs in the presence of medium ATP, the F1.AMP-PNP complex does not dissociate if an ATP-regenerating system of sufficient capacity to prevent accumulation of medium ADP is added. Consistent with an inability of nucleoside triphosphate to promote release is the failure of medium, nonradioactive AMP-PNP to affect retention of the 3H-labeled ligand. The stability of F1.AMP-PNP complex in the absence of medium nucleotide and the highly specific ability of ADP plus P1 to promote rapid release of the ATP analog are interpreted as support for an ATP synthesis mechanism that requires substrate binding at one catalytic site for product release from an adjacent interacting site.     

Binding of recombinant rat liver fatty acid-binding protein to small anionic phospholipid vesicles results in ligand release: a model for interfacial binding and fatty acid targeting

A number of intracellular proteins bind to negatively charged phospholipid membranes, and this interfacial binding results in a conformational change that modulates the activity of the protein. Using a fluorescent fatty acid analogue, 11-[5-(dimethylamino)naphthalenesulfonyl]undecanoic acid (DAUDA), it is possible to demonstrate the release of this ligand from recombinant rat liver FABP in the presence of phospholipid vesicles that contain a significant proportion of anionic phospholipids. The ligand release that is observed with anionic phospholipids is sensitive to the ionic strength of the assay conditions and the anionic charge density of the phospholipid at the interface, indicating that nonspecific electrostatic interactions play an important role in the process. The stoichiometric relationship between anionic phospholipid and liver FABP suggests that the liver FABP coats the surface of the phospholipid vesicle. The most likely explanation for ligand release is that interaction of FABP with an anionic membrane interface induces a rapid conformational change, resulting in a reduced affinity of DAUDA for the protein. The nature of this interaction involves both electrostatic and nonpolar interactions as maximal release of liver FABP from phospholipid vesicles with recovery of ligand binding cannot be achieved with high salt and requires the presence of a nonionic detergent. The precise interfacial mechanism that results in the rapid release of ligand from L-FABP remains to be determined, but studies with two mutants, F3W and F18W, suggest the possible involvement of the amino-terminal region of the protein in the process. The conformational change linked to interfacial binding of this protein could provide a mechanism for fatty acid targeting within the cell.     

Photo-induced processes in vitamin B6 compounds

In this work, we applied multi-wavelength stopped-flow spectroscopy (MSFS) to study the chemical equilibria between tautomeric or hydrated forms of various vitamin B6 compounds and the Schiff base formed by epsilon-aminocaproic acid (= 6-aminohexanoic acid) with pyridoxal 5'-phosphate at 25 degrees and variable pH. Since some of these compounds are photosensitive, we analyzed the possible occurrence of any secondary photo-induced processes under the conditions of irradiation in the MSFS equipment (continuous irradiation with light from a 75-W Xe lamp spanning the wavelength range of 200-700 nm). To determine the tautomeric composition of these compounds, the electronic absorption spectra were analyzed by means of log-normal curves. Continuous irradiation of pyridoxamine and pyridoxal 5'-phosphate over the wavelength range of 200-700 nm displaces the chemical equilibrium between the tautomeric or hydrated forms of these compounds. However, the Schiff base of epsilon-aminocaproic acid with pyridoxal 5'-phosphate is insensitive to the radiation used. The photo-induced processes detected in pyridoxamine and pyridoxal 5'-phosphate should be taken into account in examining vitamers by MSFS. In fact, these additional processes should be considered in studying the mechanism of action of vitamin B6-dependent enzymes by the MSFS technique, whenever some free vitamer may be present in solution.     

Development of a liposomal delivery system for temperature-triggered release of a tumor targeting agent, Ln(III)-DOTA-phenylboronate

Liposomes, capable of temperature-triggered content release at the site of interest, can be of great importance for imaging and therapy of tumors. The delivery of imaging agents or therapeutics can be improved by application of liposomes with a gel-to-liquid phase-transition temperature suitable for mild hyperthermia (41-43 °C), and by prolonging their circulation time by incorporation of lipids containing polyethyleneglycol moieties. Still, the rapid wash out of the delivered material from the tumor tissue is a major obstacle for both imaging and therapy. In this study, we developed an optimized temperature sensitive liposomal system to be used with mild hyperthermia: highly stable at physiological temperature and with a sharp transition of the bilayer at 41.5 °C, with subsequent rapid release of entrapped compounds such as calcein or tumor cell-targeting contrast agents. Intravital microscopy on calcein/rhodamine containing liposomes was applied to demonstrate the applicability of this system in vivo. The calcein loaded liposomes were injected iv into nude mice with a human BLM melanoma tumor implanted in a dorsal skin-fold window chamber. Arrival of the liposomes at the tumor site and content release after temperature increase were monitored. The results demonstrated not only accumulation of the liposomes at the tumor site, but also a massive release of calcein after increase of the temperature to 41 °C. The versatility of the thermosensitive liposomes was further demonstrated by encapsulation of a tumor cell-targeting DOTA-phenylboronate conjugate and its release at elevated temperatures. The DOTA ligand in this system is able to chelate a variety of metals suitable for both diagnostic and therapeutic applications, whereas the phenylboronate function is able to target specifically to tumor cells through a covalent binding with sialic acid moieties over-expressed on their surface upon heat-triggered release from the liposomal carrier.     

A model for ligand binding to hexacoordinate hemoglobins

Hexacoordinate hemoglobins are heme proteins capable of reversible intramolecular coordination of the ligand binding site by an amino acid side chain from within the heme pocket. Examples of these proteins are found in many living organisms ranging from prokaryotes to humans. The nonsymbiotic hemoglobins (nsHbs) are a class of hexacoordinate heme proteins present in all plants. The nsHb from rice (rHb1) has been used as a model system to develop methods for determining rate constants characterizing binding and dissociation of the His residue responsible for hexacoordination. Measurement of these reactions exploits laser flash photolysis to initiate the reaction from the unligated, pentacoordinate form of the heme protein. A model for ligand binding is presented that incorporates the reaction following rapid mixing with the reaction starting from the pentacoordinate hemoglobin (Hb). This model is based on results indicating that ligand binding to hexacoordinate Hbs is not a simple combination of competing first order (hexacoordination) and second order (exogenous ligand binding) reactions. Ligand binding following rapid mixing is a multiphasic reaction displaying time courses ranging from milliseconds to minutes. The new model incorporates a "closed", slow reacting form of the protein that is not at rapid equilibrium with the reactive conformation. It is also demonstrated that formation of the closed protein species is not dependent on hexacoordination.     

The mechanism of GTP hydrolysis by Ras probed by Fourier transform infrared spectroscopy

Time-resolved Fourier transform infrared spectroscopy (FTIR) in combination with photo-induced release of (18)O-labeled caged nucleotide has been employed to address mechanistic issues of GTP hydrolysis by Ras protein. Infrared spectroscopy of Ras complexes with nitrophenylethyl (NPE)-[alpha-(18)O(2)]GTP, NPE-[beta-(18)O(4)]GTP, or NPE-[gamma-(18)O(3)]GTP upon photolysis or during hydrolysis afforded a substantially improved mode assignment of phosphoryl group absorptions. Photolysis spectra of hydroxyphenylacyl-GTP and hydroxyphenylacyl-GDP bound to Ras and several mutants, Ras(Gly(12))-Mn(2+), Ras(Pro(12)), Ras(Ala(12)), and Ras(Val(12)), were obtained and yielded valuable information about structures of GTP or GDP bound to Ras mutants. IR spectra revealed stronger binding of GDP beta-PO(3)(2-) moiety by Ras mutants with higher activity, suggesting that the transition state is largely GDP-like. Analysis of the photolysis and hydrolysis FTIR spectra of the [beta-nonbridge-(18)O(2), alphabeta-bridge-(18)O]GTP isotopomer allowed us to probe for positional isotope exchange. Such a reaction might signal the existence of metaphosphate as a discrete intermediate, a key species for a dissociative mechanism. No positional isotope exchange was observed. Overall, our results support a concerted mechanism, but the transition state seems to have a considerable amount of dissociative character. This work demonstrates that time-resolved FTIR is highly suitable for monitoring positional isotope exchange and advantageous in many aspects over previously used methods, such as (31)P NMR and mass spectrometry.     

Dynamics of excitation in the photosystem I of cyanobacteria: transfer in the antenna, capture by the reaction site, and dissipation

The structure of a complex of photosystem I (PSI) of cyanobacteria and the mechanisms of the functioning of the antenna and PSI reaction site were described. The complex of PSI in thylakoids of cyanobacteia is organized as a trimer whose antenna is enriched in long-wave chlorophylls. The energy absorbed by these chlorophyls migrates to P700, inducing its oxidation. Long-wave chlorophyls are also involved in the dissipation of excessive energy; both the cation radical of P700 and the triplet of P700 effectively quench the fluorescence of long-wave chlorophyll of PSI. The energy exchange between the antennas of monomers in the trimer of PSI stimulates the dissipation of electron excitation energy, protecting the complex against photodestruction. The kinetics of energy migration in the antenna and charge separation in the reaction site of PSI trimers was studied using subpicosecond spectroscopy. Long-wave chlorophylls of PSI do not substantially affect the energy migration in the heterogeneous antenna of PSI but slow down the capture of energy of P700. The separation of changes in the reaction site of PSI is the most rapid among the known reaction sites.     

RuII(NO+)]3+-core complexes with 4-methyl-pyrimidine and ethyl-isonicotinate: synthesis, X-ray structure, spectroscopy, and computational and NO-release studies upon UVA irradiation

The reaction of RuCl(3)(NO).H(2)O with 4-methylpyrimidine (MePYM) and ethylisonicotinate (EINT), in absolute ethanol at 40-55 degrees C afforded crystalline trans-[RuCl(3)(NO)L(2)] complexes. Structural studies via X-ray diffraction, and spectroscopic methods (NMR, IR, UV-visible (UV-Vis)) revealed that the molecular structures have the two Ls in trans positions (axial) and the chloride anions and the NO(+) cation as equatorial ligands; pyrimidine...pyrimidine pairing pattern via two weak C-H...N interactions occur. The molecular structures for the EINT derivative was inferred from spectroscopy and computations. Under irradiation at 366 nm several solutions of the title compounds deliver NO via first order processes. Visible light (420-700 nm) does not produce significant NO release from CH(2)Cl(2) and CH(3)CN solutions within 24h.