Diacylglycerol Kinase Is Stimulated by Arachidonic Acid in Neural Membranes.

The effect of arachidonic acid (AA) on the activity of diacylglycerol (DG) kinase in neural membranes was investigated. When rat brain cortical membranes were incubated with 0.5 mM dipalmitin and [gamma-P-32]ATP, formation of phosphatidic acid (PA) was observed. It was linear up to 5 min, and the initial rate was similar to 1.0 nmol/min/mg of protein. The DG kinase activity was stimulated twofold by 0.25 mM AA. The stimulation was apparent at the earliest time point measured (1 min) and with the lowest concentration of AA tested (62.5 mu M). The stimulation was proportional to the concentration of AA up to 250 mu M. AA was the most potent stimulator of DG kinase, and linolenic acid showed similar to 40% stimulation. Oleic acid showed no effect, whereas linoleic and the saturated fatty acids tested were inhibitory. AA stimulation of DG kinase was observed only with membranes of cerebrum, cerebellum, and myelin and not with brain cytosol or liver membranes. AA also stimulated the formation of PA in the absence of added dipalmitin (endogenous activity) with membranes prepared from whole brain. DG kinase of neural membranes was extracted with 2 M NaCl, which on dialysis yielded a precipitate. Both the precipitate and the supernatant showed DG kinase activity, but only the enzyme in the precipitate was stimulated by AA at concentrations as low as 25 mu M. It is suggested that AA, through its effect on DG kinase, regulates the level of DG in neural membranes, which in turn regulates protein kinase C activity.

Biotin-conjugated tumour-targeting photocytotoxic iron(III) complexes

Iron(III) complexes FeL(B)] (1-4) of a tetradentate phenolate-based ligand (H3L) and biotin-conjugated dipyridophenazine bases (B), viz. 7-aminodipyrido 3,2-a: 2',3'-c]-phenazine (dppza in 1), (N-dipyrido3,2-a: 2',3'-c]-phenazino) amidobiotin (dppzNB in 2), dipyrido 3,2-a: 2',3'-c]-phenazine-11-carboxylic acid (dppzc in 3) and 2-((2-biotinamido) ethyl) amidodipyrido 3,2-a: 2',3'-c]-phenazine (dppzCB in 4) are prepared, characterized and their interaction with streptavidin and DNA and their photocytotoxicity and cellular uptake in various cells studied. The high-spin iron(III) complexes display Fe(III)/Fe(II) redox couple near -0.7V versus saturated calomel electrode in dimethyl sulfoxide-0.1M tetrabutylammonium perchlorate. The complexes show non-specific interaction with DNA as determined from the binding studies. Complexes with appended biotin moiety show similar binding to streptavidin as that of free biotin, suggesting biotin conjugation to dppz does not cause any loss in its binding affinity to streptavidin. The photocytotoxicity of the complexes is tested in HepG2, HeLa and HEK293 cell lines. Complex 2 shows higher photocytotoxicity in HepG2 cells than in HeLa or HEK293, forming reactive oxygen species. This effect is attributed to the presence of overexpressed sodium-dependent multi-vitamin transporters in HepG2 cells. Microscopic studies in HepG2 cells show internalization of the biotin complexes 2 and 4 essentially occurring by receptor-mediated endocytosis, which is similar to that of native biotin and biotin fluorescein isothiocyanate conjugate.

Ferrocenyl-L-amino acid copper(II) complexes showing remarkable photo-induced anticancer activity in visible light

Ferrocene-conjugated copper(II) complexes Cu(Fc-aa)(aip)](ClO4) (1-3) and (Cu(Fc-aa)(pyip)](ClO4) (4-6) of L-amino acid reduced Schiff bases (Fc-aa), 2-(9-anthryl)-1H-imidazo4,5-f]1,10]phenanthroline (aip) and 2-(1-pyrenyl)-1H-imidazo4,5-f] 1,10]phenanthroline (pyip), where Fc-aa is ferrocenylmethyl-L-tyrosine (Fc-Tyr in 1, 4), ferrocenylmethyl-L-tryptophan (Fc-Trp in 2, 5) and ferrocenylmethyl-L-methionine (Fc-Met in 3, 6), were prepared and characterized, and their photocytotoxicity was studied (Fc = ferrocenyl moiety). Phenyl analogues, viz. (Cu(Ph-Met)(aip)](ClO4) (7) and (Cu(Ph-Met)(pyip)](ClO4) (8), were prepared and used as control compounds. The bis-imidazophenanthroline copper(II) complexes, viz. (Cu(aip)(2)(NO3)](NO3) (9) and Cu(pyip)(2)(NO3)](NO3) (10), were also prepared and used as controls. Complexes 1-6 having a redox inactive cooper(II) center showed the Fc(+)-Fc redox couple at similar to 0.5 V vs. SCE in DMF-0.1 mol (Bu4N)-N-n](ClO4). The copper(II)-based d-d band was observed near 600 nm in DMF-Tris-HCl buffer (1 :1 v/v). The ferrocenyl complexes showed low dark toxicity, but remarkably high photocytotoxicity in human cervical HeLa and human breast adenocarcinoma MCF-7 cancer cells giving an excellent photo-dynamic effect while their phenyl analogues were inactive. The photo-exposure caused significant morphological changes in the cancer cells when compared to the non-irradiated ones. The photophysical processes were rationalized from the theoretical studies. Fluorescence microscopic images showed 3 and 6 localizing predominantly in the endoplasmic reticulum (ER) of the cancer cells, thus minimizing any undesirable effects involving nuclear DNA.

Progress towards the total synthesis of a spermine-conjugated dynemicin analog

Progress towards the synthesis of the spermine-conjugated Dynemicin analog 4 is described. The synthetic route starts with the Michael addition of menthyl acetoacetate to trans-ethyl crotonate followed by a Dieckman condensation to form the cyclohexanedione 14 which, through a series of chemical reactions, is transformed into the quinone imine 6. Key features in the route include the Suzuki coupling reaction of the aryl boronic acid 11 and the enol triflate 12, thermal deprotection/internal amidation of the biaryl 19, cis addition of the (Z)-enediyne 33 to the quinoline 25, intramolecular acetylide addition to a carbonyl within the ketone 29, and an addition/elimination of the cyanophthalide to the quinone imine 6 to form the anthraquinone 36 utilizing the Kraus and Sugimoto methodology.

Development of a Cationic Mucic Acid Polymer-Based Nanoparticle siRNA Delivery System

Cancer chemotherapy has advanced from highly toxic drugs to more targeted treatments in the last 70 years. Chapter 1 opens with an introduction to targeted therapy for cancer. The benefits of using a nanoparticle to deliver therapeutics are discussed. We move on to siRNA in particular, and why it would be advantageous as a therapy. Specific to siRNA delivery are some challenges, such as nuclease degradation, quick clearance from circulation, needing to enter cells, and getting to the cytosol. We propose the development of a nanoparticle delivery system to tackle these challenges so that siRNA can be effective.

Chapter 2 of this thesis discusses the synthesis and analysis of a cationic mucic acid polymer (cMAP) which condenses siRNA to form a nanoparticle. Various methods to add polyethylene glycol (PEG) for stabilizing the nanoparticle in physiologic solutions, including using a boronic acid binding to diols on mucic acid, forming a copolymer of cMAP with PEG, and creating a triblock with mPEG on both ends of cMAP. The goal of these various pegylation strategies was to increase the circulation time of the siRNA nanoparticle in the bloodstream to allow more of the nanoparticle to reach tumor tissue by the enhanced permeation and retention effect. We found that the triblock mPEG-cMAP-PEGm polymer condensed siRNA to form very stable 30-40 nm particles that circulated for the longest time – almost 10% of the formulation remained in the bloodstream of mice 1 h after intravenous injection.

Chapter 3 explores the use of an antibody as a targeting agent for nanoparticles. Some antibodies of the IgG1 subtype are able to recruit natural killer cells that effect antibody dependent cellular cytotoxicity (ADCC) to kill the targeted cell to which the antibody is bound. There is evidence that the ADCC effect remains in antibody-drug conjugates, so we wanted to know whether the ADCC effect is preserved when the antibody is bound to a nanoparticle, which is a much larger and complex entity. We utilized antibodies against epidermal growth factor receptor with similar binding and pharmacokinetics, cetuximab and panitumumab, which differ in that cetuximab is an IgG1 and panitumumab is an IgG2 (which does not cause ADCC). Although a natural killer cell culture model showed that gold nanoparticles with a full antibody targeting agent can elicit target cell lysis, we found that this effect was not preserved in vivo. Whether this is due to the antibody not being accessible to immune cells or whether the natural killer cells are inactivated in a tumor xenograft remains unknown. It is possible that using a full antibody still has value if there are immune functions which are altered in a complex in vivo environment that are intact in an in vitro system, so the value of using a full antibody as a targeting agent versus using an antibody fragment or a protein such as transferrin is still open to further exploration.

In chapter 4, nanoparticle targeting and endosomal escape are further discussed with respect to the cMAP nanoparticle system. A diboronic acid entity, which gives an order of magnitude greater binding (than boronic acid) to cMAP due to the vicinal diols in mucic acid, was synthesized, attached to 5kD or 10kD PEG, and conjugated to either transferrin or cetuximab. A histidine was incorporated into the triblock polymer between cMAP and the PEG blocks to allow for siRNA endosomal escape. Nanoparticle size remained 30-40 nm with a slightly negative ca. -3 mV zeta potential with the triblock polymer containing histidine and when targeting agents were added. Greater mRNA knockdown was seen with the endosomal escape mechanism than without. The nanoparticle formulations were able to knock down the targeted mRNA in vitro. Mixed effects suggesting function were seen in vivo.

Chapter 5 summarizes the project and provides an outlook on siRNA delivery as well as targeted combination therapies for the future of personalized medicine in cancer treatment.

The comparative analysis of the energies of conjugated triple base triplets and their applications

We built 64 sets of 3D models of DNA triplex base triplets (TBT) and minimized their energies. The TBTs were divided into 32 pairs of conjugated ones on the basis of their sequence characteristic, and the energies of each pair of them were compared and analyzed, the results showed: (i) The duplex DNA of which any strand contains at least a couple of A or T, has a preference for selecting the oligodeoxyribonucleic acid (ODN) strand containing abundant T to form TBT. (ii) The duplex DNA of which any strand contains at least a couple of G or C has a preference for selecting ODN containing abundant G to form symmetric antiparallel TBT, but selecting ODN containing abundant C to form asymmetric parallel TBT. (iii) The duplex DNA of which any strand contains only one of A, T, G or C has a preference for selecting ODN containing abundant pyrimidines (T or C) to form antiparallel TBT. Additionally, two examples of TBTs applications, in designing ODN to form triplex with duplex were presented. The energy calculation result revealed that 15-TCG is the best ligand of the HIV PPT duplex. The comparative analysis of energies of the conjugated TBTs provides directive significance for designing ODN strand that is easy to form triplex in theory. (C) 2002 Elsevier Science B.V. All rights reserved.

Flourescent Switch Constructed Based on Hemin-Sensitive Anionic Conjugated Polymer and Its Applications in DNA-Related Sensors

Here, a fluorescent switch is constructed combining hemin, hemin aptamer, and a newly synthesized anionic conjugated polymer (ACP), poly(9,9-bis(6'-phosphate-hexyl) fluorenealt-1,4-phenylene) sodium salt (PFHPNa/PFP). In the "off-state", the fluorescence of PFP is sensitively quenched by hemin, with a high K-sv value of similar to 10(7). While in the "on-state", the formation of the aptamer/hemin complex recovers the fluorescence intensity. The fluorescent switch is sensitive and selective to hemin. To testify the universality and practicality of the fluorescent switch, a series of label-free DNA-related sensing platforms are developed, containing three DNA sensing strategies and one ATP recognition strategy. The fluorescent switch developed is simple, sensitive, and universal, which extends applications of the anionic conjugated polymers.

Enhanced charge collection in polymer photovoltaic cells by using an ethanol-soluble conjugated polyfluorene as cathode buffer layer

We report enhanced polymer photovoltaic (PV) cells by utilizing ethanol-soluble conjugated poly (9, 9-bis (6'-diethoxylphosphorylhexyl) fluorene) (PF-EP) as a buffer layer between the active layer consisting of poly(3-hexylthiophene)/[6, 6]-phenyl C61-butyric acid methyl ester blend and the Al cathode. Compared to the control PV cell with Al cathode, the introduction of PF-EP effectively increases the shunt resistance and improves the photo-generated charge collection since the slightly thicker semi-conducting PF-EP layer may restrain the penetration of Al atoms into the active layer that may result in increased leakage current and quench photo-generated excitons. The power conversion efficiency is increased ca. 8% compared to the post-annealed cell with Al cathode.

Dye-Sensitized Solar Cells Based on Organic Sensitizers with Different Conjugated Linkers: Furan, Bifuran, Thiophene, Bithiophene, Selenophene, and Biselenophene

Six organic dyes with different conjugated linkers such as furan, bifuran, thiophene, bithiophene, selenophene, and biselenophene have been prepared in combination with the dihexyloxy-substituted triphenylamine donor and the cyanoacrylic acid acceptor. In conjunction with an acetonitrile-based electrolyte and a solvent-free ionic liquid electrolyte, these dyes exhibit 6.88-7.77% and 6.39-7.00% efficiencies, respectively. We have demonstrated that furan and selenophene can be employed as building blocks of sensitizers in stable solar cells for the first time. We have also studied the influence of heteroatoms on photocurrents and photovoltages with the aid of quantum calculations and transient photoelectrical decay measurements. Temperature-dependent electrical impedance experiments have shown that a relatively low external quantum efficiency of the dye with biselenophene linker is not related to the charge collection yield in the case of an acetonitrile electrolyte.

Fluorescent conjugated polymer-stabilized gold nanoparticles for sensitive and selective detection of cysteine

We report a new fluorescent detection method for cysteine based on one-step prepared fluorescent conjugated polymer-stabilized gold nanoparticles. The as-prepared fluorescent conjugated polymer-stabilized gold nanoparticles fluoresce weakly due to the fluorescence resonance energy transfer between the fluorophore and the gold nanoparticles. Upon the addition of cysteine, a thiol-containing amino acid, the fluorescence of the colloidal solution increases significantly, indicating that cysteine can modulate the energy transfer between fluorophore and gold. This phenomenon then allows for sensitive detection of cysteine with a limit of detection (LOD) of 25 nM. The linear range of determination of cysteine is from 5 x 10(-8) to 4 x 10(-6) M. None of the other amino acids found in proteins interferes with the determination. Moreover, due to the excellent protecting ability of the fluorescent conjugated polymers, the synthesis of metal nanoparticles and modifying with fluorophores can be accomplished within one step, which makes our method much simpler than conventional methods. We also expect that it will be possible to detect other biologically important analytes based on the fluorescent conjugated polymer-stabilized metal nanoparticles.

Nucleic Acid-Induced Aggregation and Pyrene Excimer Formation

Nucleic acid was found to induce the aggregation of the positively charged pyrene probe (compound 1); as a result, strong pyrene excimer emission was observed. The intensity of the excimer emission was dependent on the concentration of the pyrene probe and the oligonucleotide length, sequence, and concentration. These results suggest a new strategy for label-free nucleic acid-based biosensing applications.

Synthesis of novel nitrogen- and sulfur-containing conjugated polymers used as hole-transporting materials for organic light-emitting diodes

Two series of highly soluble novel nitrogen- and sulfur-containing conjugated polymers were synthesized via an acid-induced self-polycondensation of functional monomers with methyl sulfinyl and aromatic groups. The well-defined structures of synthesized polymers were confirmed by their NMR and IR spectra. The highest occupied molecular orbital energy values for these materials, estimated by cyclic voltammetry, showed a broad range of values from about 5.0 to 5.2 eV used as hole-transport layers (HTL) in two-layer light-emitting diodes ITO/HTL/Alq(3)/Mg:Ag [ITO = indium tin oxide, and Alq(3) = tris(8-quinolinato) aluminum]. The typical turn-on voltage of these diodes was about 4-5 V. The maximum brightness of the device was about 3440 cd/m(2) at 20 V. The maximum efficiency was estimated to be 0.15 1m/W at 10 V.

Design, synthesis and characterization of novel nitrogen-and sulfur-containing polymers with well-defined conjugated length

A series of novel nitrogen- and sulfur-containing conjugated polymers with well-defined conjugation length have been synthesized via an acid-induced self-polycondensation of functional monomers with methylsulfinyl groups. Synthesized polymers exhibit good solubility in common solvents, such as CHCl3, THF, DMF, DMSO, and NMP. With increased numbers of aminophenyl groups, these polymers have shown similar electrical properties to polyaniline (PAn), and these are demonstrated by UV-vis spectroscopy and cyclic voltammetry (CV) measurements on the polymers. The conductivity of preliminarily protonic-doped poly[phenylene sulfide-alt-tetrakis(aniline)] (PPSTEA) is up to 10(-1) S cm(-1).

Nitrogen- and sulfur-containing conjugated polymers as the hole-transporting materials

A series of novel nitrogen- and sulfur-containing conjugated polymers have been synthesized via an acid-induced self polycondensation of functional monomers with methyl sulfinyl group. They exhibit good solubility in common solvents, such as CHCl3, THF, DMF, DMSO, et al; and thus show excellent film-forming properties. They are used as hole-transport layer (HTL) in two-layer light-emitting diodes (ITO/polymer/Alq3/Mg:Ag). The typical turn-on voltage of these diodes is about 4 similar to 5V. The maximum brightness is about 3440cd/m(2) at 20 V. The maximum efficiency is estimated to be 0.15 Im/W at 10V.