Design of Bifunctional Dendritic 5-Aminolevulinic Acid and Hydroxypyridinone Conjugates for Photodynamic Therapy.

Iron chelators have recently attracted interest in the field of photodynamic therapy (PDT) owing to their role in enhancement of intracellular protoporphyrin IX (PpIX) generation induced by 5-aminolevulinic acid (ALA) via the biosynthetic heme cycle. Although ALA is widely used in PDT, cellular uptake of ALA is limited by its hydrophilicity. In order to improve ALA delivery and enhance the PpIX production, several dendrimers incorporating both ALA and 3-hydroxy-4-pyridinone (HPO) were synthesized. The ability of the dendrimers to enter cells and be metabolized to the PpIX photosensitizer was studied in several human cancer cell lines. The dendrimers were found to be significantly more efficient than ALA alone in PpIX production. The higher intracellular PpIX levels showed a clear correlation with enhanced cellular phototoxicity following light exposure. Dendritic derivatives are therefore capable of efficiently delivering both ALA and HPO, which act synergistically to amplify in vitro PpIX levels and enhance PDT efficacy.

Resonance Raman Spectra of Five-Coordinate Heme-Nitrosyl Cytochromes c': Effect of the Proximal Heme-NO Environment.

Five-coordinate heme nitrosyl complexes (5cNO) underpin biological heme-NO signal transduction. Bacterial cytochromes c' are some of the few structurally characterized 5cNO proteins, exhibiting a distal to proximal 5cNO transition of relevance to NO sensing. Establishing how 5cNO coordination (distal vs proximal) depends on the heme environment is important for understanding this process. Recent 5cNO crystal structures of Alcaligenes xylosoxidans cytochrome c' (AXCP) and Shewanella frigidimarina cytochrome c' (SFCP) show a basic residue (Arg124 and Lys126, respectively) near the proximal NO binding sites. Using resonance Raman (RR) spectroscopy, we show that structurally characterized 5cNO complexes of AXCP variants and SFCP exhibit a range of ν(NO) (1651-1671 cm(-1)) and ν(FeNO) (519-536 cm(-1)) vibrational frequencies, depending on the nature of the proximal heme pocket and the sample temperature. While the AXCP Arg124 residue appears to have little impact on 5cNO vibrations, the ν(NO) and ν(FeNO) frequencies of the R124K variant are consistent with (electrostatically) enhanced Fe(II) → (NO)π* backbonding. Notably, RR frequencies for SFCP and R124A AXCP are significantly displaced from the backbonding trendline, which in light of recent crystallographic data and density functional theory modeling may reflect changes in the Fe-N-O angle and/or extent of σ-donation from the NO(π*) to the Fe(II) (dz(2)) orbital. For R124A AXCP, correlation of vibrational and crystallographic data is complicated by distal and proximal 5cNO populations. Overall, this study highlights the complex structure-vibrational relationships of 5cNO proteins that allow RR spectra to distinguish 5cNO coordination in certain electrostatic and steric environments.

Conformational control of the binding of diatomic gases to cytochrome c'.

The cytochromes c' (CYTcp) are found in denitrifying, methanotrophic and photosynthetic bacteria. These proteins are able to form stable adducts with CO and NO but not with O2. The binding of NO to CYTcp currently provides the best structural model for the NO activation mechanism of soluble guanylate cyclase. Ligand binding in CYTcps has been shown to be highly dependent on residues in both the proximal and distal heme pockets. Group 1 CYTcps typically have a phenylalanine residue positioned close to the distal face of heme, while for group 2, this residue is typically leucine. We have structurally, spectroscopically and kinetically characterised the CYTcp from Shewanella frigidimarina (SFCP), a protein that has a distal phenylalanine residue and a lysine in the proximal pocket in place of the more common arginine. Each monomer of the SFCP dimer folds as a 4-alpha-helical bundle in a similar manner to CYTcps previously characterised. SFCP exhibits biphasic binding kinetics for both NO and CO as a result of the high level of steric hindrance from the aromatic side chain of residue Phe 16. The binding of distal ligands is thus controlled by the conformation of the phenylalanine ring. Only a proximal 5-coordinate NO adduct, confirmed by structural data, is observed with no detectable hexacoordinate distal NO adduct.

Cytochrome bd confers nitric oxide resistance to Escherichia coli.

The aerobic respiratory chain of Escherichia coli has two terminal quinol oxidases: cytochrome bo and cytochrome bd. Cytochrome bd was thought to function solely to facilitate micro-aerobic respiration. However, it has recently been shown to be overexpressed under conditions of nitric oxide (NO) stress; we show here that cytochrome bd is crucial for protecting E. coli cells from NO-induced growth inhibition by virtue of its fast NO dissociation rate.

Macromolecular delivery of 5-aminolaevulinic acid for photodynamic therapy using dendrimer conjugates.

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (5-ALA) has been widely used in photodynamic therapy. However, cellular uptake of 5-ALA is limited by its hydrophilicity, and improved means of delivery are therefore being sought. Highly branched polymeric drug carriers known as dendrimers present a promising new approach to drug delivery because they have a well-defined structure capable of incorporating a high drug payload. In this work, a dendrimer conjugate was investigated, which incorporated 18 aminolaevulinic acid residues attached via ester linkages to a multipodent aromatic core. The ability of the dendrimer to deliver and release 5-ALA intracellularly for metabolism to the photosensitizer, protoporphyrin IX, was studied in the transformed PAM 212 murine keratinocyte and A431 human epidermoid carcinoma cell lines. Up to an optimum concentration of 0.1 mmol/L, the dendrimer was significantly more efficient compared with 5-ALA for porphyrin synthesis. The intracellular porphyrin fluorescence levels showed good correlation with cellular phototoxicity following light exposure, together with minimal dark toxicity. Cellular uptake of the dendrimer occurs through endocytic routes predominantly via a macropinocytosis pathway. In conclusion, macromolecular dendritic derivatives are capable of delivering 5-ALA efficiently to cells for sustained porphyrin synthesis.

Hydroxypyridinone and 5-Aminolaevulinic Acid Conjugates for Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising treatment strategy for malignant and nonmalignant lesions. 5-Aminolaevulinic acid (ALA) is used as a precursor of the photosensitizer, protoporphyrin IX (PpIX), in dermatology and urology. However, the effectiveness of ALA-PDT is limited by the relatively poor bioavailability of ALA and rapid conversion of PpIX to haem. The main goal of this study was to prepare and investigate a library of single conjugates designed to coadminister the bioactive agents ALA and hydroxypyridinone (HPO) iron chelators. A significant increase in intracellular PpIX levels was observed in all cell lines tested when compared to the administration of ALA alone. The higher PpIX levels observed using the conjugates correlated well with the observed phototoxicity following exposure of cells to light. Passive diffusion appears to be the main mechanism for the majority of ALA-HPOs investigated. This study demonstrates that ALA-HPOs significantly enhance phototherapeutic metabolite formation and phototoxicity.

Investigation of a novel dendritic derivative of 5-aminolaevulinic acid for photodynamic therapy.

Photodynamic therapy is a treatment for malignant and certain non-malignant lesions that involves administration of a photosensitising drug. The use of 5-aminolaevulinic acid-induced porphyrins has become one of the most active fields of photodynamic therapy research. Since the efficacy of the treatment is somewhat limited by the hydrophilic nature of 5-aminolaevulinic acid, chemical modifications such as esterification with aliphatic alcohols have been made to induce higher porphyrin production. In an attempt to improve delivery of 5-aminolaevulinic acid to tissue, we have investigated the use of dendritic derivatives capable of bearing several drug molecules. The aim of this work was to evaluate in vivo and in vitro the efficacy of the first generation dendron, aminomethane tris-methyl 5-aminolaevulinic acid (containing three 5-aminolaevulinic acid residues) in terms of porphyrin synthesis. In LM3 cells, the dendron induced similar porphyrin levels compared to equimolar concentrations of 5-aminolaevulinic acid. Although the dendron is taken up with comparable efficiency to 5-aminolaevulinic acid, we found that there is only partial intracellular liberation of 5-aminolaevulinic acid residues. Both systemic and topical administration of the dendron to tumour-bearing mice induced higher porphyrin levels than the widely investigated hexyl ester derivative in most tissues studied, although it was not possible to surpass the levels induced by 5-aminolaevulinic acid. In conclusion, aminomethane tris-methyl 5-aminolaevulinic acid is capable of being taken up by cells efficiently, and liberating the active residues, although in vivo it was not possible to improve upon the efficacy of 5-aminolevulinic acid. Studies of accessibility and regulation of the esterases are needed to improve the design of these dendritic derivatives.

Enhanced porphyrin accumulation using dendritic derivatives of 5-aminolaevulinic acid for photodynamic therapy: an in vitro study.

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid has been widely used in photodynamic therapy for a range of malignant and certain non-malignant lesions. However, cellular uptake of 5-aminolaevulinic acid is limited by its hydrophilic nature and improved means of delivery are therefore being sought. Highly branched polymeric drug carriers known as dendrimers are a promising new approach to drug delivery. The aim of this study was to investigate the efficacy of dendrimers conjugated with 5-aminolaevulinic acid for porphyrin production in the transformed PAM 212 keratinocyte cell line and skin explants. Each dendritic derivative incorporated three 5-aminolaevulinic acid residues which were conjugated as esters via methyl or propyl linkers to a central tertiary carbon whose remaining terminal bore an amino, aminobenzyloxycarbonyl or nitro group. In the cell line, all compounds were more efficient at low concentrations compared to equimolar 5-aminolaevulinic acid for porphyrin production, with the most efficient incorporating the longer propyl linker. This compound was also the most lipophilic according to partition coefficient measurements. The intracellular porphyrin fluorescence levels showed good correlation with cellular phototoxicity following light exposure for all the compounds, together with minimal dark toxicity. Our findings indicate that the key factors influencing the efficacy of the dendritic derivatives are lipophilicity and steric hindrance within the dendritic structure which could restrict access to intracellular esterases for liberation of 5-aminolaevulinic acid. These findings should be taken into account in the design of larger dendrimers of 5-aminolaevulinic acid.

Mechanisms of 5-aminolevulinic acid ester uptake in mammalian cells.

The porphyrin precursor 5-aminolevulinic acid (ALA) is being widely used in photodynamic therapy of cancer. Improvement in ALA delivery has been sought through the use of ALA derivatives, in particular the esterification of ALA with aliphatic alcohols, which in certain cases can improve cellular penetration and selectivity. ALA uptake systems appear to be distinctive for each cell type. The LM3 mammary adenocarcinoma cell line takes ALA up by BETA transporters. In this work, we investigated ALA derivative transport systems through the inhibition of radiolabelled ALA uptake in the LM3 cells. We also performed inhibition studies of gamma-aminobutyric acid (GABA) uptake. The more lipohilic ALA derivatives hexyl-ALA and undecanoyl-ALA inhibit ALA uptake, whereas methyl-ALA, R, S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester and the dendron aminomethane tris methyl 5-ALA does not inhibit ALA uptake. A similar pattern was found for GABA, except that the dendron inhibited GABA uptake. However, hexyl-ALA and undecanoyl-ALA are not taken up by BETA transporters, but by simple diffusion, although they still inhibit ALA uptake by binding to the cell membrane. These results show that different modifications to the ALA molecule lead to different uptake mechanisms. Whereas ALA is taken up by BETA transporters, none of the ALA derivatives shares the same mechanism. Knowledge of the mechanisms of ALA derivatives entry into the cells is essential to understand and improve ALA-mediated PDT and to the design of new ALA derivatives that may be taken up at a higher rate than ALA.

Dissimilatory iron(III) reduction by Rhodobacter capsulatus.

The photosynthetic proteobacterium Rhodobacter capsulatus was shown to be capable of dissimilatory Fe(III) reduction. Activity was expressed during anaerobic phototrophic and microaerobic growth with malate as the carbon source, but not during equivalent aerobic growth. A variety of Fe(III) complexes were demonstrated to act as substrates for intact cells and membrane fractions of strain N22DNAR+ using a ferrozine assay for Fe(II) formation. Rates of reduction appeared to be influenced by the reduction potentials of the Fe(III) complexes. However, Fe(III) complexed by citrate, which is readily reduced by Shewanella putrefaciens, was a poor substrate for dissimilation by R. capsulatus. The Fe(III)-reducing activity of R. capsulatus was located solely in the membrane fraction. The reduction of Fe(III) complexes by intact cells was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), suggesting the involvement of ubiquinol: cytochrome c oxidoreductases in the electron transport chain. Lack of sensitivity to myxothiazol plus data from mutant strains implies that the cytochrome bc 1 complex and cytochrome c 2 are not obligatory for dissimilation of Fe(III)(maltol)3. Alternative pathways of electron transfer to Fe(III) must hence operate in R. capsulatus. Using strain N22DNAR+, the reduction rate of Fe(III) complexed by nitrilotriacetic acid (NTA) was elevated compared to that of Fe(III)(maltol)3, and moreover was sensitive to myxothiazol. However, these differences were not observed in the absence of the electron donor malate. The governing factor for the reduction rate of Fe(III)(maltol)3 thus appears to be the limited Fe(III)-reducing activity, whilst the reduction rate of Fe(III) complexed by NTA is controlled by the flux of electrons through the respiratory chain. The use of mutant strains confirmed that the role of the cytochrome bc 1 complex in Fe(III) reduction becomes apparent only with the superior substrate. The energy-conserving nature of Fe(III) reduction by R. capsulatus was demonstrated by electrochromic measurements, with the endogenous carotenoid pigments being employed as indicators of membrane potential generation in intact cells. Using Fe(III)EDTA as electron acceptor, periods of membrane potential generation were directly proportional to the quantity of complex added, and were extended in the presence of HQNO. Fe(III)-dependent carotenoid bandshifts were abolished by addition of the protonophoric uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone.

Sustained and efficient porphyrin generation in vivo using dendrimer conjugates of 5-ALA for photodynamic therapy.

The use of endogenous protoporphyrin IX (PpIX) after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). However the efficacy of ALA-PDT is sub-optimal for thicker tumours and improved ALA delivery and therapeutic response are required. We have investigated the conjugation of ALA to a second-generation dxcendrimer for enhancing porphyrin synthesis in vitro and in vivo in a murine tumour model using systemic i.p. administration. In vitro, the dendrimer was more efficient than ALA for porphyrin synthesis at low concentrations in good correlation with higher cellular ALA dendrimer accumulation. In vivo, the porphyrin kinetics from ALA exhibited an early peak between 3 and 4 h in most tissues, whereas the dendrimer induced sustained porphyrin production for over 24 h and basal values were not reached until 48 h after administration. Integrated porphyrin accumulation from the dendrimer and ALA, at equivalent molar ratios, was comparable showing that the majority of ALA residues were liberated from the dendrimer. The porphyrin kinetics appear to be governed by the rate of enzymatic cleavage of ALA from the dendrimer, which is consistent with in vitro results. ALA dendrimers may be useful for metronomic PDT, and multiple low-dose ALA-PDT treatments.

Characterization of transcriptional regulation of Shewanella frigidimarina Fe(III)-induced flavocytochrome c reveals a novel iron-responsive gene regulation system.

The bacterium Shewanella frigidimarina can grow anaerobically by utilizing Fe(III) as a respiratory electron acceptor. This results in the synthesis of a number of periplasmic c-type cytochromes, which are absent when the organism is grown in the absence of added Fe(III). One cytochrome, IfcA, is synthesized when Fe(III) is present as the sole respiratory electron acceptor or when it is present in combination with oxygen, fumarate, or nitrate. The ifcA gene was thus selected for a study of iron-responsive gene regulation of respiratory proteins in S. frigidimarina. The monocistronic ifcA gene clusters with two other monocistronic genes, ifcO, encoding a putative outer membrane porin, and ifcR, encoding a putative transcriptional regulator of the LysR superfamily. Analysis of transcription of all three genes under a range of growth conditions in the wild type and an ifcR insertion mutant and analysis of a strain that constitutively expresses ifcR revealed that iron regulation is exerted at the level of ifcR transcription. In the presence of Fe(III) IfcR is synthesized and acts positively to regulate expression of ifcO and ifcA. Control of Fe(III) respiration by this novel regulatory system differs markedly from Fur-mediated regulation of iron assimilation, in which Fur serves as an Fe(II)-activated repressor.

Characterization of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA: expression in Escherichia coli confers the ability to reduce soluble Fe(III) chelates.

Shewanella oneidensis MR-1 has the metabolic capacity to grow anaerobically using Fe(III) as a terminal electron acceptor. Growth under these conditions results in the de novo synthesis of a number of periplasmic c-type cytochromes, many of which are multiheme in nature and are thought to be involved in the Fe(III) respiratory process. To begin a biochemical study of these complex cytochromes, the mtrA gene that encodes an approximate 32-kDa periplasmic decaheme cytochrome has been heterologously expressed in Escherichia coli. Co-expression of mtrA with a plasmid that contains cytochrome c maturation genes leads to the assembly of a correctly targeted holoprotein, which covalently binds ten hemes. The recombinant MtrA protein has been characterized by magnetic circular dichroism, which shows that all ten hemes have bis-histidine axial ligation. EPR spectroscopy detected only eight of these hemes, all of which are low spin and provides evidence for a spin-coupled pair of hemes in the oxidized state. Redox titrations of MtrA have been carried out with optical- and EPR-monitored methods, and the hemes are shown to reduce over the potential range -100 to -400 mV. In intact cells of E. coli, MtrA is shown to obtain electrons from the host electron transport chain and pass these onto host oxidoreductases or a range of soluble Fe(III) species. This demonstrates the promiscuous nature of this decaheme cytochrome and its potential to serve as a soluble Fe(III) reductase in intact cells.