Biodistribution of a carborane-containing porphyrin as a targeting agent for Boron Neutron Capture Therapy of oral cancer in the hamster cheek pouch

Boron Neutron Capture Therapy (BNCT) is a bimodal cancer treatment based on the selective accumulation of 10B in tumors and concurrent irradiation with thermalized neutrons. The short-range, high-LET radiation produced by the capture of neutrons by 10B could potentially control tumor while sparing normal tissue if the boron compound targets tumor selectively within the treatment volume. In previous studies, we proposed and validated the hamster cheek pouch model of oral cancer for BNCT studies, proved that absolute and relative uptake of the clinically employed boron compound boronophenylalanine (BPA) would be potentially therapeutic in this model and provided evidence of the efficacy of in vivo BPA-mediated BNCT to control hamster oral mucosa tumors with virtually no damage to normal tissue. We herein present the biodistribution and pharmacokinetics of a lipophilic, carborane-containing tetraphenylporphyrin (CuTCPH) in the hamster oral cancer model. CuTCPH is a novel, non-toxic compound that may be advantageous in terms of selective and absolute delivery of boron to tumor tissues. For potentially effective BNCT, tumor boron concentrations from a new agent should be greater than 30 ppm and tumor/blood and tumor/normal tissue boron concentration ratios should be greater than 5/1 without causing significant toxicity. We administered CuTCPH intraperitoneally (i.p.) as a single dose of 32 microg/g body weight (b.w.) (10 microg B/g b.w.) or as four doses of 32 microg/g b.w. over 2 days. Blood (Bl) and tissues were sampled at 3, 6, 12, 24, 48, and 72 h in the single-dose protocol and at 1-4 days after the last injection in the multidose protocol. The tissues sampled were tumor (T), precancerous tissue surrounding tumor, normal pouch (N), skin, tongue, cheek and palate mucosa, liver, spleen, parotid gland and brain. The maximum mean B ratios for the single-dose protocol were T/N: 9.2/1 (12h) and T/Bl: 18.1/1 (72 h). The B value peaked to 20.7+/-18.5 ppm in tumor at 24h. The multidose protocol maximum mean ratios were T/N: 11.9/1 (3 days) and T/Bl: 235/1 (4 days). Absolute boron concentration in tumor reached a maximum value of 116 ppm and a mean value of 71.5+/-48.3 ppm at 3 days. The fact that absolute and relative B values markedly exceeded the BNCT therapeutic threshold with no apparent toxicity may confer on this compound a therapeutic advantage. CuTCPH-mediated BNCT would be potentially useful for the treatment of oral cancer in an experimental model.     

Enhanced electrochemical and electrocatalytic activity of a new supramolecular manganese-porphyrin species containing four bis(bipyridine)(aqua)ruthenium(II) complexes

A novel supramolecular species [MnTPyP{Ru(bipy)₂H₂O}₄](PF₆)₉ containing four cis-bis(bipyridine)(aqua)ruthenium(II) complexes attached to the peripheral pyridyl groups of meso-tetra(4-pyridyl)porphyrin manganese(III) (MnTPyP) has been synthesized, and employed as the precursor of a rather promising multinuclear oxo-transfer catalyst. This complex forms molecular films by dip coating. Such films are soluble in water, but can be stabilized by exchanging the PF^?₆ anions by [Fe(CN)₆]^4? or [Zn–TPPS]^4?. They exhibit remarkable electrochemical and electrocatalytic activity, as demonstrated in the oxidation of nitrite ions. In this case, experimental evidence supports the involvement of electrochemically generated [ORu^IV]²⁺ groups in the oxidation reaction, reflecting the characteristic reactivity of high-valence metal–oxo complexes.     

Spectral and electrochemical studies of axial ligand binding reactions of carbonylruthenium(II) meso-tetramesitylporphyrin

The spectral and electrochemical properties of carbonylruthenium(II) meso-tetramesitylporphyrin, Ru(CO)(TMP) in the presence of imidazole, N-methylimidazole and hydroxide anion as axial ligands were investigated. Spectrophotometric titration of Ru(CO)(TMP) of the nitrogeneous bases caused the absorption spectrum to shift to longer wavelengths. Larger shifts in wavelength were observed in the titration using tetrabutylammonium hydroxide. The formation constants for these ligands coordinated to the ruthenium center were calculated. The effect of axial ligand ligation caused the decrease of vibrational frequency of CO as detected from FT-IR spectroscopy. The CO stretching frequency (ν_CO) of Ru(CO)TMP in CH₂Cl₂ is 1940 cm^?1, which is lowered to 1936 and 1913 cm^?1 upon coordination of nitrogenous bases and hydroxide anion, respectively. Cyclic voltammetry of Ru(CO)(TMP) in CH₂Cl₂ showed an irreversible reduction wave at ?1.63 V and two reversible oxidations at E_1/2 = 0.78 and 1.27 V vs. Ag|AgCl, respectively. Addition of imidazole and hydroxide into Ru(CO)(TMP) solution caused shifts in the redox potential and accordingly, the binding constants of the ligands to the one- and two-electron oxidized ruthenium porphyrins were estimated and compared. Spectroelectrochemical methods were used to characterize the above electron-transfer reactions.     

Protective effects of a magnesium magnetic isotope (Mg25)‐exchanging nanoparticle (25MgPMC16) on mitochondrial functional disorders in esmolol‐induced cardiac arrest in rats

1 In cardiac surgery, agents are needed to produce temporary cardiac arrest (cardioplegia). One of these agents is esmolol (ESM) which is a short‐acting selective beta‐1 adrenergic receptor antagonist and its overdose causes diastolic ventricular arrest. 2 The ²⁵MgPMC₁₆ (porphyrin adducts of cyclohexil fullerene‐C60) is known as a nanoparticle which has a cardioprotective effect when the heart is subjected to stressful conditions. 3 In this study, we aimed to confirm the deleterious effects of ESM overdose on cardiac mitochondria and identify any protective effects of ²⁵MgPMC₁₆ in male Wistar rats. Esmolol 100 mg kg^?1 (LD50 = 71 mg kg^?1) was injected intravenously (i.v.) into tail vein to induce cardiac arrest. This dose was obtained from an ESM dose–response curve which induces at least 80% arrest in rats. 4 ²⁵MgPMC₁₆ at three different doses (45, 90 and 224 mg kg^?1) was injected i.v. as pretreatment, eight hours before ESM injection. ²⁵MgCl₂ or ²⁴MgPMC₁₆ were used as controls. Following cardiac arrest, the heart was removed and the mitochondria extracted. Mitochondrial viability and the adenosine 5′‐diphosphate sodium salt hydrate/Adenosine 5′‐triphosphate disodium salt hydrate (ADP/ATP) ratio were measured as biomarkers of mitochondrial function. 5 Results indicate that ²⁵MgPMC₁₆ caused a significant increase in mitochondrial viability and decrease in ADP/ATP ratio. No significant changes were seen with ²⁴MgPMC₁₆ or ²⁵MgCl₂. It is concluded that cardiac arrest induced by ESM overdose leads to a significant decrease in mitochondrial viability and their ATP levels, whereas pretreatment by ²⁵MgPMC₁₆ can protect mitochondria by increasing ATP level through liberation of Mg into cells and the improvement of hypoxia.     

Efficient Polymer Solar Cells Based on New Random Copolymers with Porphyrin‐Incorporated Side Chains

Two new wide bandgap block copolymers (PL1 and PL2) with porphyrin‐incorporated side chains are designed and used as electron donors for solution‐processed bulk heterojunction polymer solar cells. The photophysical, electrochemical, and photovoltaic properties, charge transport mobility and film morphology of these two block copolymers are investigated. Detailed investigations reveal that the different alkyl groups and electron‐withdrawing substituents on the porphyrin pendant units have significant influence on the polymer solubility, absorption energy level, band gap, and charge separation in the bulk‐heterojunction thin films, and thus the overall photovoltaic performances. Organic photovoltaic devices derived from these copolymers and ([6,6]‐phenyl‐C₇₁‐butyric acid methyl ester) (PC₇₁BM) acceptor show the best power conversion efficiencies of 5.83% and 7.14%, respectively. These results show that the inclusion of a certain proportion of side chain porphyrin group as a pendant in the traditional donor‐acceptor (D‐A) type polymer can broaden the molecular absorption range and become a full‐color absorbing molecule. The size of the porphyrin pendant also has an obvious effect on the properties of the molecule.     

Myeloid-derived suppressor cells are increased in frequency but not maximally suppressive in peripheral blood of Type 1 Diabetes Mellitus patients

Type 1 Diabetes Mellitus (T1D) results from the destruction of insulin-producing beta cells in the pancreas by autoreactive T cells. Myeloid derived suppressor cells (MDSCs) are a recently identified immune cell subset that down-regulate T cells. Whether defects in MDSC numbers or function may contribute to T1D pathogenesis is not known. We report here that MDSCs are unexpectedly enriched in peripheral blood of both mice and patients with autoimmune diabetes. Peripheral blood MDSCs from T1D patients suppressed T cell proliferation in a contact-dependent manner; however, suppressive function could be enhanced with in vitro cytokine induction. These findings suggest that native T1D MDSCs are not maximally suppressive and that strategies to promote MDSC suppressive function may be effective in preventing or treating T1D.     

Reactive oxygen species involved cancer cellular specific 5-aminolevulinic acid uptake in gastric epithelial cells

Photodynamic therapy and photodynamic diagnosis using 5-aminolevulinic acid (ALA) are clinically useful for cancer treatments. Cancer cells have been reported that 5-aminolevulinic acid is incorporated via peptide transporter 1, which is one of the membrane transport proteins, and has been reported to be significantly expressed in various gastrointestinal cancer cells such as Caco-2. However, the mechanism of this protein expression has not been elucidated. Concentration of reactive oxygen species (ROS) is higher in cancer cells in comparison with that of normal cells. We have previously reported that ROS derived from mitochondria is likely related to invasions and proliferations of cancer cells. Since 5-aminolevulinic acid is the most important precursor of heme which is necessary protein for cellular proliferations, mitochondrial ROS (mitROS) may be also related to peptide transporter 1 expressions. In this study, we used a rat gastric mucosal cell line RGM1 and its cancer-like mutated cell line RGK1, and we clarified the ALA uptake mechanism and its relations between mitROS and peptide transporter 1 expression in RGK1. We also used our self-established stable clone of cell which over-expresses manganese superoxide dismutase, a mitROS scavenger. We studied differences of the photodynamic therapy effects in these cells after ALA administrations to clear the influence of mitROS.     

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl₂, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

The identity of a metal center often defines enzymatic activity, and swapping the native metal for an alternative one or introducing a new metal center has profound effects. More generally, the chemical utility of natural cofactors has inspired decades of study into synthetic analogs with distinct properties, and researchers have subsequently sought straightforward ways to put these novel cofactors back into proteins (1). Substituted metalloenzymes constitute one of the simplest cases. Changing the identity of the metal ion in metalloproteins has enabled powerful spectroscopic and functional studies of these proteins (2–10) in addition to new biocatalytic activities (11–20). However, most methods for producing such proteins with new-to-nature cofactors are limited by the inability to produce the novel protein–cofactor complex in vivo.Hemoproteins, in particular, have been studied through metal substitution because of their important biological functions and utility as biocatalysts. Heme is a ubiquitous and versatile cofactor in biology, and heme-dependent proteins serve essential gas sensing functions (21), metabolize an array of xenobiotic molecules (22), and perform synthetically useful oxygen activation and radical-based chemistry (23). Metal-substituted hemoproteins have enabled key spectroscopic studies of hemoprotein function and the development of biocatalysts with novel reactivity. For example, electron paramagnetic resonance (EPR) studies on cobalt-substituted sperm whale myoglobin (CoMb) enabled detailed characterization of the paramagnetic CoMbO₂ complex (3, 4, 24, 25). In analogous oxygen-binding studies in CoMb and cobalt-substituted hemoglobin (5, 6, 26), resonance Raman was used to identify the O–O stretching mode because cobalt-substituted proteins exhibit enhancement of this vibrational mode compared to the native iron proteins.Metal substitution has a profound effect on catalytic activity of hemoproteins, enabling numerous synthetic applications. Substitution of the native iron for cobalt in several hemoproteins, including a thermostable cytochrome c variant, enabled the reduction of water to H₂ under aerobic, aqueous conditions (27–29). Reconstitution of apoprotein with selected metalloporphyrins has been used to generate metal-substituted myoglobin and cytochrome P450s variants. These enzymes were effective as biocatalysts for C–H activation and carbene insertion reactions (11–14). In a tour de force of directed evolution, which required purification and cofactor reconstitution of each individual variant, Hartwig and coworkers generated a cytochrome P450 variant that utilizes a nonnative Ir(Me)mesoporphyrin cofactor to perform desirable C–H activation chemistry (14). These activities may not be unique to the Ir-substituted protein, as synthetic cobalt porphyrin complexes have been shown to mediate a variety of Co(III)-aminyl and -alkyl radical transformations, including C–H activation (30–32). Indeed, a number of cobalt porphyrin carbene complexes display significant carbon-centered radical character (33–35), whereas the corresponding Fe-porphyrin complexes are closed shell species (36, 37), indicating that cobalt porphyrins may possess distinct, complementary modes of reactivity (38–40).Inspired by these applications, researchers have sought strategies for generating metal-substituted hemoproteins. For many metalloproteins, metal substitution is carried out by removal of the native metal with a chelator and replacement with an alternate metal of similar coordination preference. This method is inapplicable to hemoproteins, as porphyrins do not readily exchange metal ions. Consequently, diverse methods have been employed to make metal-substituted hemoproteins (41–46). Early on, copper, cobalt, nickel, and manganese-substituted horseradish peroxidase (HRP) were prepared by a multistep process that subjected protein to strong acid and organic solvents (41, 42). Variations of this method have been used repeatedly (24, 43, 47–49). However, this method is applicable only to a narrow range of hemoproteins that tolerate the harsh treatment. With the advent of overexpression methods, significant improvement of metalloporphyrin-substituted protein yield was achieved by direct expression of the apoprotein and reconstitution with the desired metalloporphyrin in lysate prior to purification (50). Although this approach has many virtues, direct expression of apoprotein is ineffective for many hemoproteins, again limiting the utility of this method.As an alternative to the above in vitro approaches, researchers have pursued systems for direct in vivo expression of metal substituted hemoproteins. Two specialty strains of Escherichia coli (E. coli) were engineered to incorporate metalloporphyrin analogs from the growth medium into hemoproteins during protein expression. The engineered RP523 strain cannot biosynthesize heme and bears an uncharacterized heme permeability phenotype. Together, these two features enable this strain to assimilate and incorporate various metalloporphyrins into overexpressed hemoproteins with no background heme incorporation (44, 51–53). However, heme auxotrophy makes RP523 cells exceedingly sensitive to O₂, and, in many situations, RP523 cultures must be grown anaerobically. An alternative BL21(DE3)-based engineered strain harbors a plasmid bearing the heme transporter ChuA, which facilitates import of exogenous heme analogs (45). Production of metalloporphyrin-substituted protein with this ChuA-containing strain relies on growth in iron-limited minimal media, thereby diminishing heme biosynthesis. This method was used successfully to express metal-substituted versions of the heme domain of cytochrome P450 BM3 (45) and several myoglobin variants (11, 12). Because these cells biosynthesize a small quantity of their own heme, they are far more robust than the RP523 cells. Unfortunately, this advantage comes at the cost of increased heme contamination in the product protein (2 to 5%) (45).A set of intriguing papers reported the production of cobalt-substituted human cystathionine β-synthase (CoCBS) that relies on the de novo biosynthesis of CoPPIX from CoCl₂ and δ-aminolevulinic acid (δALA), a biosynthetic precursor to heme (46, 54). This method yielded significant amounts of CoCBS—albeit with modest heme contamination (7.4%)—sufficient for spectroscopic and functional characterization of the CoPPIX-substituted protein (8, 46). As cobalt is known to be toxic to E. coli, the researchers passaged the CBS expression strain through cobalt-containing minimal media for 12 d, enabling the cells to adapt to high concentrations of cobalt prior to protein expression. It is plausible that this serial passaging alters the E. coli cells, enabling the biosynthesis of CoPPIX and in vivo production of metal-substituted protein. The adaptation process is slow (>10 d), and it is unknown how genomic instability under these mutagenic conditions affects the reproducibility of this passaging approach.The possibility of facile CoPPIX production is particularly attractive for future biocatalysis efforts. As described above, synthetic cobalt porphyrins have been shown to perform a range of radical-mediated reactions. The ability to produce a CoPPIX center in vivo may enable engineering these unusual reactivities via directed evolution in addition to spectroscopic applications. We therefore set out to explore the unusual phenotype of CoPPIX production by E. coli and to ascertain whether it was possible to efficiently biosynthesize cobalt-containing hemoproteins in vivo from a single “generalist” cell line. Our goal was to achieve an efficient and facile method of cobalt-substituted hemoprotein production with minimal contamination of the native cofactor. Herein, we report the surprising discovery that native E. coli BL21(DE3) can biosynthesize a new-to-nature CoPPIX cofactor (Fig. 1). We use this insight to produce cobalt-substituted hemoproteins in vivo without requirement for complex expression methods or specialized strains.Open in a separate windowFig. 1.Chemical structures of iron protoporphyrin IX (FePPIX or heme b), cobalt protoporphyrin IX (CoPPIX), and free base protoporphyrin IX (H₂PPIX).