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1.  Synthesis and Characterization of Mono-, Di-, and Tri-Polyethylene Glycol Chlorin e6 Conjugates for the Photokilling of Human Ovarian Cancer Cells 
The Journal of organic chemistry  2012;77(23):10.1021/jo301889s.
Pegylated chlorin e6 photosensitizers were synthesized with triethylene glycol attached at the ester bond(s) for a 1:1 conjugate at the 173-position, a 2:1 conjugate at the 152- and 173-positions, and a 3:1 conjugate at the 131-, 152-, and 173-positions. These chlorin sensitizers were studied for hydrolytic stability, solubility, as well as ovarian OVCAR-5 cancer cell uptake, localization, and phototoxicity. Increasing numbers of the PEG groups in the mono-, di-, and tri-PEG chlorin conjugates increased the water solubility and sensitivity to hydrolysis and uptake into the ovarian cancer cells. The PEG chlorin conjugates accumulated in the cytoplasm and mitrochondria, but not in lysosomes. Higher phototoxicity was roughly correlated with higher numbers of PEG groups, with the tri-PEG chlorin conjugate showing the best overall ovarian cancer cell photokilling of the series. Singlet oxygen lifetimes, solvent deuteration, and the effects of additives azide ion and D-mannitol were examined to help clarify the photokilling mechanisms. A Type-II (singlet oxygen) photosensitized mechanism is suggested for the di- and tri-PEG chlorin conjugates, however, a more complicated process based in part on a Type-I (radicals or radical ions) mechanism is suggested for the parent chlorin e6 and the mono-PEG chlorin conjugate.
doi:10.1021/jo301889s
PMCID: PMC3815657  PMID: 23126407
2.  Syntheses and Photodynamic Activity of Pegylated Cationic Zn(II)-Phthalocyanines in HEp2 Cells 
Theranostics  2012;2(9):850-870.
Di-cationic Zn(II)-phthalocyanines (ZnPcs) are promising photosensitizers for the photodynamic therapy (PDT) of cancers and for photoinactivation of viruses and bacteria. Pegylation of photosensitizers in general enhances their water-solubility and tumor cell accumulation. A series of pegylated di-cationic ZnPcs were synthesized from conjugation of a low molecular weight PEG group to a pre-formed Pc macrocycle, or by mixed condensation involving a pegylated phthalonitrile. All pegylated ZnPcs were highly soluble in polar organic solvents but were insoluble in water; they have intense Q absorptions centered at 680 nm and fluorescence quantum yields of ca. 0.2 in DMF. The non-pegylated di-cationic ZnPc 6a formed large aggregates, which were visualized by atomic force microscopy. The cytotoxicity, cellular uptake and subcellular distribution of all cationic ZnPcs were investigated in human carcinoma HEp2 cells. The most phototoxic compounds were found to be the α-substituted Pcs. Among these, Pcs 4a and 16a were the most effective (IC50 ca. 10 μM at 1.5 J/cm2), in part due to the presence of a PEG group and the two positive charges in close proximity (separated by an ethylene group) in these macrocycles. The β-substituted ZcPcs 6b and 4b accumulated the most within HEp2 cells but had low photocytoxicity (IC50 > 100 μM at 1.5 J/cm2), possibly as a result of their lower electron density of the ring and more extended conformations compared with the α-substituted Pcs. The results show that the charge distribution about the Pc macrocycle and the intracellular localization of the cationic ZnPcs mainly determine their photodynamic activity.
doi:10.7150/thno.4547
PMCID: PMC3475216  PMID: 23082098
phthalocyanine; PDT; pegylation; cationic photosensitizer
3.  Somatostatin Analogues for Receptor Targeted Photodynamic Therapy 
PLoS ONE  2014;9(8):e104448.
Photodynamic therapy (PDT) is an established treatment modality, used mainly for anticancer therapy that relies on the interaction of photosensitizer, light and oxygen. For the treatment of pathologies in certain anatomical sites, improved targeting of the photosensitizer is necessary to prevent damage to healthy tissue. We report on a novel dual approach of targeted PDT (vascular and cellular targeting) utilizing the expression of neuropeptide somatostatin receptor (sst2) on tumor and neovascular-endothelial cells. We synthesized two conjugates containing the somatostatin analogue [Tyr3]-octreotate and Chlorin e6 (Ce6): Ce6-K3-[Tyr3]-octreotate (1) and Ce6-[Tyr3]-octreotate-K3-[Tyr3]-octreotate (2). Investigation of the uptake and photodynamic activity of conjugates in-vitro in human erythroleukemic K562 cells showed that conjugation of [Tyr3]-octreotate with Ce6 in conjugate 1 enhances uptake (by a factor 2) in cells over-expressing sst2 compared to wild-type cells. Co-treatment with excess free Octreotide abrogated the phototoxicity of conjugate 1 indicative of a specific sst2-mediated effect. In contrast conjugate 2 showed no receptor-mediated effect due to its high hydrophobicity. When compared with un-conjugated Ce6, the PDT activity of conjugate 1 was lower. However, it showed higher photostability which may compensate for its lower phototoxicity. Intra-vital fluorescence pharmacokinetic studies of conjugate 1 in rat skin-fold observation chambers transplanted with sst2+ AR42J acinar pancreas tumors showed significantly different uptake profiles compared to free Ce6. Co-treatment with free Octreotide significantly reduced conjugate uptake in tumor tissue (by a factor 4) as well as in the chamber neo-vasculature. These results show that conjugate 1 might have potential as an in-vivo sst2 targeting photosensitizer conjugate.
doi:10.1371/journal.pone.0104448
PMCID: PMC4128677  PMID: 25111655
4.  Receptor-Targeting Phthalocyanine Photosensitizer for Improving Antitumor Photocytotoxicity 
PLoS ONE  2012;7(5):e37051.
Photodynamic therapy (PDT) is a promising therapeutic modality which uses a photosensitizer to capture visible light resulting in phototoxicity in the irradiated region. PDT has been used in a number of pathological indications, including tumor. A key desirable feature of the photosensitizer is the high phototoxicity on tumor cells but not on normal cells. In this study, we conjugate a gonadotropin-releasing hormone (GnRH) to a photosensitizer, Zinc phthalocyanine (ZnPc), in order to enhance its specificity to breast cancer, which over-expresses GnRH receptor. ZnPc has unique advantages over other photosensitizers, but is difficult to derivatize and purify as a single isomer. We previously developed a straight-forward way to synthesize mono-substituted β-carboxy-phthalocyanine zinc (ZnPc-COOH). Photophysical and photochemical parameters of this ZnPc-GnRH conjugate including fluorescence quantum yield (Фf), fluorescence decay time (τs) and singlet oxygen quantum yield (ФΔ) were evaluated and found comparable with that of ZnPc, indicating that addition of a GnRH peptide does not significantly alter the generation of singlet oxygen from ZnPc. Cellular uptakes and phototoxicities of this conjugate were tested and found significantly enhanced on human breast cancer cell lines overexpressing GnRH receptors (MDA-MB-231 and MCF-7 cells) compared to cells with low levels of GnRH receptors, such as human embryonic lung fibroblast (HELF) and human liver carcinoma (HepG2) cells. In addition, the cellular uptake of this conjugate toward MCF-7 cells were found clearly alleviated by a GnRH receptor blocker Cetrorelix, suggesting that the cellular uptake of this conjugate was GnRH receptor-mediated. Put together, these findings revealed that coupling ZnPc with GnRH analogue was an effective way to improve the selectivity of ZnPc towards tumors with over-expressed GnRH receptors.
doi:10.1371/journal.pone.0037051
PMCID: PMC3365043  PMID: 22693566
5.  Photodynamic Inactivation of Bacteria Using Polyethylenimine–Chlorin(e6) Conjugates: Effect of Polymer Molecular Weight, Substitution Ratio of Chlorin(e6) and pH 
Lasers in surgery and medicine  2011;43(4):313-323.
Background and Objectives
Antimicrobial photodynamic therapy (APDT) is a novel technique to treat local infections. Previously we reported that the attachment of chlorin(e6) to polyethylenimine (PEI) polymers to form PEI-ce6 conjugates is an effective way to improve ce6 PDT activity against bacteria. The aim of this work was to explore how the polymer molecular weight, substitution ratio (SR) of ce6 and pH value affect the PDT efficacy.
Study Design/Materials and Methods
We have synthesized PEI-ce6(10) (MW = 60,000, SR = 1) and PEI-ce6(11) (MW = 60,000, SR = 5) and compared these with the previous PEI-ce6(9) (MW = 10,000, SR = 1). We tested the PDT efficacy of these three conjugates against Gram-negative E. coli and Gram-positive bacteria (S. aureus and E. fecalis) at three different pH values (5.0, 7.4, 10.0) that may affect the charge on both the bacterial cells and on the conjugate (that has both basic and acidic groups).
Results
PEI-ce6(9) and PEI-ce6(10) were the most effective against these tested bacteria. The PDT effect of all three conjugates depended on pH values. The effective order was pH = 10.0 > pH = 7.4 > pH = 5.0 on E. coli. For S. aureus and E. fecalis the order was pH = 5.0 > pH = 10.0 > pH = 7.4. PEI-ce6(11) PDT activity was worse than PEI-ce6(10) activity which is probably connected to the fact that ce6 molecules are self-quenched within the PEI-ce6(11) molecule. Ce6 quenching within the PEI-ce6 molecules was proved by analyzing fluorescence spectra of PEI-ce6 conjugates at different pH values. There were no differences in bacterial uptake between different pH values in three PEI-ce6 conjugates.
Conclusion
We assume high pH (rather than low pH as was hypothesized) disaggregates the conjugates, so the higher pH was more effective than the lower pH against E. coli. But for Gram-positive bacteria, low pH was more effective possibly due to more overall positive charge on the conjugate. Lasers Surg. Med. 43:313–323, 2011.
doi:10.1002/lsm.21056
PMCID: PMC3449165  PMID: 21500226
antimicrobial photodynamic therapy; polyethylenimine–chlorine(e6) conjugate; pH value; polymer molecular weight; substitution ratio; static and dynamic quenching
6.  Synthesis of ApoSense compound [18F]2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid ([18F]NST732) by nucleophilic ring-opening of an aziridine precursor 
Nuclear Medicine and Biology  2012;39(5):687-696.
Introduction
The small molecule 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid (NST732) is a member of the ApoSense® family of compounds, capable of selective targeting, binding and accumulation within cells undergoing apoptotic cell death. It has application in molecular imaging and blood clotting particularly for monitoring anti-apoptotic drug treatments. We are investigating a fluorine-18 radiolabeled analog of this compound for positron emission tomography studies.
Methods
We prepared the tosylate precursor methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(tosyloxymethyl)butanoate (4) to synthesize fluorine-18 labeled NST732. Fluorination reaction of the tosylate precursor in 1:1 acetonitrile, dimethylsulfoxide with tetrabutyl ammonium fluoride (TBAF) proceeds through an aziridine intermediate (4A) to afford two regioisomers 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-fluorobutanoate (5) and methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoate (6). Acid hydrolysis of the fluoromethylbutanoate (6) isomer produced NST732. As the fluorination reaction of the tosylate precursor proceeds through an aziridine intermediate (4A) and the fluorination conceivably could be done directly on the aziridine, we have separately prepared an aziridine precursor (4A). Fluorine-18 labeling of the aziridine precursor (4A) was performed with [18F]tetrabutyl ammonium fluoride to afford the same two regioisomers (5 and 6). The [18F]2-((5-dimethylamino)naphthalene-1-sulfonamido)methyl)-2-fluorobutanoic acid (NST732) was then obtained by the hydrolysis of corresponding [18F]-labeled ester (6) with 6N hydrochloric acid.
Results
Two regioisomers obtained from the fluorination reaction of aziridine were easily separated by HPLC. The total radiochemical yield was 15 ± 3% (uncorrected, n = 18) from the aziridine precursor, in a 70 min synthesis time with a radiochemical purity > 99%.
Conclusion
Fluorine-18 labeled aposense coumpound [18F]NST732 is prepared in moderate yield by direct fluorination of an aziridine precursor.
doi:10.1016/j.nucmedbio.2011.12.008
PMCID: PMC3357448  PMID: 22336374
7.  Substrate affinity of photosensitizers derived from chlorophyll-a: The ABCG2 transporter affects the phototoxic response of side population stem cell-like cancer cells to photodynamic therapy 
Molecular pharmaceutics  2010;7(5):1789-1804.
Photosensitizers (PS) synthesized with the aim of optimizing photodynamic therapy (PDT) of tumors do not always fulfill their potential when tested in vitro and in vivo in different tumor models. The ATP-dependent transporter ABCG2 a multi-drug resistant pump expressed at variable levels in cancerous cells, can bind and efflux a wide range of structurally different classes of compounds including several PS used pre-clinically and clinically such as porphyrins and chlorins. ABCG2 may lower intracellular levels of substrate PS below the threshold for cell death in tumors treated by PDT, leaving resistant cells to re-populate the tumor. To determine some of the structural factors that affect substrate affinity of PS for ABCG2, we used an ABCG2 expressing cell line (HEK 293 482R) and its non-expressing counterpart, and tyrosine kinase ABCG2 inhibitors in a simple flow cytometric assay to identify PS effluxed by the ABCG2 pump. We tested a series of conjugates of substrate PS with different groups attached at different positions on the tetrapyrrole macrocycle to examine whether a change in affinity for the pump occurred and whether such changes depended on the position or the structure/type of the attached group. PS without substitutions including pyropheophorbides and purpurinimides were generally substrates for ABCG2, but carbohydrate groups conjugated at positions 8, 12, 13 and 17 but not at position 3 abrogated ABCG2 affinity regardless of structure or linking moiety. At position 3, affinity was retained with the addition of iodobenzene, alkyl chains and monosaccharides, but not with disaccharides. This suggests that structural characteristics at position 3 may offer important contributions to requirements for binding to ABCG2. We examined several tumor cell lines for ABCG2 activity, and found that although some cell lines had negligible ABCG2 activity in bulk, they contained a small ABCG2-expressing side population (SP) thought to contain cells which are responsible for initiating tumor regrowth. We examined the relevance of the SP to PDT resistance with ABCG2 substrates in vitro and in vivo in the murine mammary tumor 4T1. We show for the first time in vivo that the substrate PS HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a) but not the non-substrate PS HPPH-Gal (a galactose conjugate of HPPH) selectively preserved the SP which was primarily responsible for regrowth in vitro. The SP could be targeted by addition of imatinib mesylate, a tyrosine kinase inhibitor which inhibits the ATPase activity of ABCG2, and prevents efflux of substrates. A PDT resistant SP may be responsible for recurrences observed both pre-clinically and clinically. To prevent ABCG2 mediated resistance, choosing non-substrate PS or administering an ABCG2 inhibitor alongside a substrate PS might be advantageous when treating ABCG2 expressing tumors with PDT.
doi:10.1021/mp100154j
PMCID: PMC3017217  PMID: 20684544
ABCG2; cancer stem cells; HPPH; photodynamic therapy; photosensitizer; resistance; side population; tetrapyrrole; tyrosine kinase inhibitor
8.  Identification and Quantification of Metallo–Chlorophyll Complexes in Bright Green Table Olives by High-Performance Liquid Chromatrography–Mass Spectrometry Quadrupole/Time-of-Flight 
Journal of agricultural and food chemistry  2011;59(20):10.1021/jf201643s.
Five different samples of table olives, two regular Spanish table olives and three “bright green table olives”, have been analyzed by HPLC–MS/MS to determine their pigment profile. Typical pigment profiles of almost all table olives show primarily chlorophyll derivatives lacking metals (e.g., pheophytin a/b and 152-Me-phytol-chlorin e6). Bright green table olives have a unique profile including metallo–chlorophyll complexes (Cu-152-Me-phytol-chlorin e6 with 26–48% and Cu-pheophytin a with 3–18%) as their major pigments. New tentative structures have been identified by MS such as 152-Me-phytol-rhodin g7, 152-Me-phytol-chlorin e6, 152-Me-phytol-isochlorin e4, Cu-152-Me-phytol-rhodin g7, Cu-152-Me-phytol-chlorin e6, and Cu-152-Me-phytol-isochlorin e4, and new MS/MS fragmentation patterns are reported for Cu-152-Me-phytol-rhodin g7, Cu-152-Me-phytol-chlorin e6, Cu-pheophytin b, Cu-pheophytin a, Cu-pyropheophytin b, and Cu-pyropheophytin a. The presence of metallo–chlorophyll derivatives is responsible for the intense color of bright green table olives, but these metallo–chlorophyll complexes may be regarded as a “green staining” defect that is unacceptable to consumers.
doi:10.1021/jf201643s
PMCID: PMC3849830  PMID: 21905735
high-perfomance liquid chromatography; mass spectrometry; table olives; Cu-pheophytin; metallo–chlorophylls; green staining alteration; regreening
9.  Biodistribution of charged 17.1A photoimmunoconjugates in a murine model of hepatic metastasis of colorectal cancer 
British Journal of Cancer  2000;83(11):1544-1551.
Optimizing photodynamic therapy involves attempting to increase both the absolute tumour content of photosensitizer and the selectivity between tumour and surrounding normal tissue. One reason why photodynamic therapy has not been considered suitable for treatment of metastatic tumours in the liver, is the poor selectivity of conventional photosensitizers for tumour compared to normal liver. This report details an alternative approach to increasing this selectivity by the use of antibody-targeted photosensitizers (or photoimmunoconjugates) to target intrahepatic tumours caused by human colorectal cancer cells in the nude mouse, and explores the role of molecular charge on the tumour-targeting efficiency of macromolecules. The murine monoclonal antibody 17.1A (which recognizes an antigen expressed on HT 29 cells) was used to prepare site-specific photoimmunoconjugates with the photosensitizer chlorine6. The conjugates had either a predominant cationic or anionic charge and were injected i.v. into tumour-bearing mice. Biodistribution 3 or 24 h later was measured by extraction of tissue samples and quantitation of chlorine6 content by fluorescence spectroscopy. The photoimmunoconjugates were compared to the polylysine conjugates in an attempt to define the effect of molecular charge as well as antibody targeting. The anionic 17.1A conjugate delivered more than twice as much photosensitizer to the tumour at 3 h than other species (5 times more than the cationic 17.1A conjugate) and had a tumour:normal liver ratio of 2.5. Tumour-to-liver ratios were greater than one for most compounds at 3 h but declined at 24 h. Tumour-to-skin ratios were high (> 38) for all conjugates but not for free chlorine6. Cationic species had a high uptake in the lungs compared to anionic species. The photoimmunoconjugates show an advantage over literature reports of other photosensitizers, which can result in tumour:normal liver ratios of less than 1. © 2000 Cancer Research Campaign http://www.bjcancer.com
doi:10.1054/bjoc.2000.1486
PMCID: PMC2363424  PMID: 11076666
photodynamic therapy; photoimmunotherapy; monoclonal antibody; photosensitizer; polylysine; intraperitoneal PDT
10.  Analysis of the Conformational Behavior and Stability of the SAP and TSAP Isomers of Lanthanide(III) NB-DOTA-Type Chelates 
Inorganic chemistry  2011;50(17):7966-7979.
Controlling the water exchange kinetics of macrocyclic Gd3+ chelates, a key parameter in the design of improved magnetic resonance imaging (MRI) contrast media, may be facilitated by selecting the coordination geometry of the chelate. The water exchange kinetics of the mono- capped twisted square antiprism (TSAP) being much closer to optimal than those of the mono capped square antiprism (SAP) render the TSAP isomer more desirable for high relaxivity applications. Two systems have been developed that allow for selection of the TSAP coordination geometry in 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-type Gd3+ chelates, both based upon the macrocycle nitrobenzyl cyclen. In this paper we report investigations into the stability and formation of these chelates. Particular focus is given to the production of two regioisomeric chelates during the chelation reaction. These regioisomers are distinguished by having the nitrobenzyl substituent either on a corner or on the side of the macrocyclic ring. The origin of these two regioisomers appears to stem from a conformation of the ligand in solution in which it is hypothesized that pendant arms lie both above and below the plane of the macrocycle. The conformational changes that then result during the formation of the intermediate H2GdL+ chelate give rise to differing positions of the nitrobenzyl substituent depending upon from which face of the macrocycle the Ln3+ approaches the ligand.
doi:10.1021/ic2012843
PMCID: PMC3204396  PMID: 21819053
11.  Radioiodinated O6-Benzylguanine Derivatives Containing an Azido Function 
Nuclear medicine and biology  2010;38(1):77-92.
Introduction
Drug resistance to alkylator chemotherapy has been primarily attributed to the DNA repair protein alkylguanine-DNA alkyltransferase (AGT); thus, personalizing chemotherapy could be facilitated if tumor AGT content could be quantified prior to administering chemotherapy. We have been investigating the use of radiolabeled O6-benzylguanine (BG) analogues to label and quantify AGT in vivo. BG derivatives containing an azido function were sought to potentially enhance the targeting of these analogues to AGT, which is primarily present in the cell nucleus, either by conjugating them to nuclear localization sequence (NLS) peptides or by pretargeting via bioorthogonal approaches.
Methods
Two O6-(3-iodobenzyl)guanine (IBG) derivatives containing an azido moiety—O6-(4-azidohexyloxymethyl-3-iodobenzyl)guanine (AHOMIBG) and O6-(4-azido-3-iodobenzyl)guanine (AIBG)—and their tin precursors were synthesized in multiple steps and the tin precursors were converted to radioiodinated AHOMIBG and AIBG, respectively. Both unlabeled and radioiodinated AHOMIBG analogues were conjugated to alkyne-derivatized NLS peptide heptynoyl-PK3RKV. The ability of these radioiodinated compounds to bind to AGT was determined by a trichloroacetic acid precipitation assays and gel electrophoresis/phosphor imaging. Labeling of an AGT-AIBG conjugate via Staudinger ligation using the 131I-labeled phosphine ligand, 2-(diphenylphosphino)phenyl 4-[131I]iodobenzoate, also was investigated.
Results
[131I]AHOMIBG was synthesized in two steps from its tin precursor in 52.2 ± 7.5% (n = 5) radiochemical yield and conjugated to the NLS peptide via click reaction in 50.7 ± 4.9% (n = 6) yield. The protected tin precursor of AIBG was radioiodinated in an average radiochemical yield of 69.6 ± 4.5% (n = 7); deprotection of the intermediate gave [131I]AIBG in 17.8 ± 4.2% (n = 9) yield. While both [131I]AHOMIBG and its NLS conjugate bound to AGT pure protein, their potency as a substrate for AGT was substantially lower than that of [125I]IBG. Uptake of [131I]AHOMIBG-NLS conjugate in DAOY medulloblastoma cells was up to 8-fold higher than that of [125I]IBG; however, the uptake was not changed when the cellular AGT content was first depleted with BG treatment. [131I]AIBG was almost equipotent as [125I]IBG with respect to binding to pure AGT; however, attempts to radiolabel AGT by treatment with unlabeled AIBG followed by Staudinger ligation using the radiolabeled phosphine ligand, 2-(diphenylphosphino)phenyl 4-[131I]iodobenzoate were not successful.
Conclusion
Although AHOMIBG, and AIBG were synthesized successfully in both unlabeled and radioiodinated forms, the radioiodinated compounds failed to label AGT either after NLS peptide conjugation or via Staundiger ligation. Currently, other bioorthogonal approaches are being evaluated for labeling AGT by pretargeting.
doi:10.1016/j.nucmedbio.2010.07.006
PMCID: PMC3052924  PMID: 21220131
Alkylguanine-DNA alkyltransferase (AGT); O6-benzylguanine; Nuclear localization sequence peptide; Bioorthogonal conjugation; click chemistry; Staudinger ligation
12.  Toward A General Synthesis of Chlorins 
Recently we described a new synthesis of C,D-ring symmetric chlorins 11, involving 2+2 condensation of bis-formyl-dihydrodipyrrins 9 with symmetrically substituted dipyrromethane diacids 10 (Method I). However, while versatile in many aspects, Method I was unsuited to the broader goal of synthesizing fully non-symmetric chlorins of general structure 15, which requires regioselective control over the reacting centers in the A,B- and C,D-ring components. In this paper we describe four new 2+2 strategies that accomplish this differentiation (Methods II-V). Of these, Method V, which combines operational simplicity with moderate to high product yields, proved to be the most effective route, exploiting reactivity differences between the two formyl groups of A,B-rings 9 to impart excellent regioselectivity. Methods II-IV are also useful alternatives to Method V, although in some cases the appropriately functionalized precursors are less readily available. All four approaches generate single regioisomers of diversely substituted chlorins, and in every case the 2+2 condensation is accomplished in a simple, one-flask procedure without need for additives such as oxidizing agents or metals. Taken together, these methodologies provide expanded access to an array of chlorins for SAR studies that may advance the effectiveness of PDT and other applications.
doi:10.1021/ja0780075
PMCID: PMC2443860  PMID: 18166060
13.  Advance in Photosensitizers and Light Delivery for Photodynamic Therapy 
Clinical Endoscopy  2013;46(1):7-23.
The brief history of photodynamic therapy (PDT) research has been focused on photosensitizers (PSs) and light delivery was introduced recently. The appropriate PSs were developed from the first generation PS Photofrin (QLT) to the second (chlorins or bacteriochlorins derivatives) and third (conjugated PSs on carrier) generations PSs to overcome undesired disadvantages, and to increase selective tumor accumulation and excellent targeting. For the synthesis of new chlorin PSs chlorophyll a is isolated from natural plants or algae, and converted to methyl pheophorbide a (MPa) as an important starting material for further synthesis. MPa has various active functional groups easily modified for the preparation of different kinds of PSs, such as methyl pyropheophorbide a, purpurin-18, purpurinimide, and chlorin e6 derivatives. Combination therapy, such as chemotherapy and photothermal therapy with PDT, is shortly described here. Advanced light delivery system is shown to establish successful clinical applications of PDT. Phtodynamic efficiency of the PSs with light delivery was investigated in vitro and/or in vivo.
doi:10.5946/ce.2013.46.1.7
PMCID: PMC3572355  PMID: 23423543
Photochemotherapy; Photosensitizing agents; Chlorophyll and chlorins; Photothermal therapy; Light delivery
14.  Targeted Antimicrobial Photochemotherapy 
Antimicrobial Agents and Chemotherapy  1998;42(10):2595-2601.
This study explores a new approach for antimicrobial therapy with light activation of targeted poly-l-lysine (pL)–chlorin e6 (ce6) conjugates. The goal was to test the hypothesis that these conjugates between pL and ce6 would efficiently target photodestruction towards gram-positive (Actinomyces viscosus) and gram-negative (Porphyromonas gingivalis) oral species while sparing an oral epithelial cell line (HCPC-1). Conjugates of ce6 with pL (average molecular weight, 2,000) having a positive, neutral, or negative charge were prepared. Illumination with red light (λmax = 671 nm) from a diode array produced a dose-dependent loss of CFU from the bacteria, under conditions that did not affect the viability of the epithelial cells. For P. gingivalis, the cationic conjugate produced 99% killing, while the neutral conjugate killed 91% and the anionic conjugate killed 76% after 1 min of incubation and exposure to red light for 10 min. For A. viscosus, the cationic conjugate produced >99.99% killing while HCPC-1 cells remained intact. The importance of the positive charge was shown by the effectiveness of ce6-monoethylenediamine monoamide (a monocationic derivative of ce6) in killing both bacteria. The clinically employed benzoporphyrin derivative under the same conditions killed epithelial cells while leaving P. gingivalis relatively unharmed. A mixture of ce6 with pL did not show phototoxicity comparable with that of the cationic conjugate. These results were explained by the selective uptake of the conjugates by bacteria (20- to 100-fold) compared to that by mammalian cells, while free ce6 showed much less selectivity for bacteria (5- to 20-fold). The data suggest that the cationic pL-ce6 conjugate may have an application for the photodynamic therapy of periodontal disease.
PMCID: PMC105903  PMID: 9756761
15.  In vivo fluorescence imaging of the transport of charged chlorine6 conjugates in a rat orthotopic prostate tumour 
British Journal of Cancer  1999;81(2):261-268.
Polymeric drug conjugates are used in cancer therapy and, varying their molecular size and charge, will affect their in vivo transport and extravasation in tumours. Partitioning between tumour vasculature and tumour tissue will be of particular significance in the case of photosensitizer conjugates used in photodynamic therapy, where this partitioning can lead to different therapeutic effects. Poly-l-lysine chlorine6 conjugates (derived from polymers of averageMr 5000 and 25 000) were prepared both in a cationic state and by poly-succinylation in an anionic state. A fluorescence scanning laser microscope was used to follow the pharmacokinetics of these conjugates in vivo in an orthotopic rat prostate cancer model obtained with MatLyLu cells. Fluorescence was excited with the 454–528 nm group of lines of an argon laser and a 570 nm long pass filter used to isolate the emission. Results showed that the conjugates initially bound to the walls of the vasculature, before extravasating into the tissue, and eventually increasing in fluorescence. The anionic conjugates produced tissue fluorescence faster than the cationic ones, and surprisingly, the largerMr conjugates produced tissue fluorescence faster than the smaller ones with the same charge. These results are consistent with differences in aggregation state between conjugates. © 1999 Cancer Research Campaign
doi:10.1038/sj.bjc.6690686
PMCID: PMC2362866  PMID: 10496351
photosensitizer; pharmacokinetics; photodynamic therapy; tumour vasculature; polymer conjugate
16.  Comparative Tumor Imaging and PDT Efficacy of HPPH Conjugated in the Mono- and Di-Forms to Various Polymethine Cyanine Dyes: Part - 2 
Theranostics  2013;3(9):703-718.
Previous reports from our laboratory have shown that a bifunctional agent obtained by conjugating a photosensitizer (HPPH) to a cyanine dye (CD) can be used for fluorescence image-guided treatment of tumor by photodynamic therapy (PDT). However, the resulting HPPH-CD conjugate showed a significant difference between the tumor-imaging and therapeutic doses. It was demonstrated that the singlet oxygen (1O2*, a key cytotoxic agent in PDT) produced by the conjugate upon excitation of the HPPH moiety was partially quenched by the CD-moiety; this resulted in a reduced PDT response when compared to HPPH-PDT under similar treatment parameters. To improve the therapeutic potential of the conjugate, we synthesized a series of dual functional agents in which one or two HPPH moieties were separately conjugated to three different dyes (Cypate, modified IR820 or modified IR783). The newly synthesized conjugates were compared with our lead compound HPPH-CD in terms of photophysical properties, in vitro and in vivo PDT efficacy, tumor uptake and imaging potential. Among the analogs investigated, the conjugate, in which two HPPH moieties were linked to the modified IR820 produced enhanced tumor uptake and tumor contrast in both Colon 26 (a murine Colon carcinoma) and U87 (a human glioblastoma) cell lines. The long-term PDT efficacy (cure) of this conjugate in BALB/c mice, bearing Colon 26 tumors was also enhanced; however, its efficacy in Nude mice bearing U87 tumors was slightly reduced. It was also found that in all the conjugates the singlet oxygen generation and, consequently, PDT efficacy were compromised by a competing pathway, whereby an electronic excitation of HPPH, the energy donor, is deactivated through an electronic excitation energy transfer (Forster Resonance Energy Transfer, FRET) to the CD fluorophore, the energy acceptor, resulting in overall reduction of the singlet oxygen production. Conjugates with increased FRET showed reduced singlet oxygen production and PDT efficacy. Among the conjugates investigated, the bifunctional agent in which two HPPH moieties were linked to the benzoindole-based cyanine dye 11 showed superiority over the lead candidate 9 (mono HPPH-cyanine dye).
doi:10.7150/thno.5923
PMCID: PMC3767117  PMID: 24019855
Photodynamic therapy, Fluorophores, Reactive Oxygen species; Imaging. Forster Resonance Energy Transfer.
17.  Photoactive Porphyrin Derivative with Broad-Spectrum Activity against Oral Pathogens In Vitro 
Antimicrobial Agents and Chemotherapy  2000;44(12):3364-3367.
Photodynamic therapy (PDT) has historically been used as a means to treat cancerous tumors but has recently been used to kill bacterial cells through the use of targeted photosensitizers. PDT is a potential adjunct to scaling and root planing in the treatment of periodontal disease. However, the effectiveness of porphyrin derivatives against microorganisms has been limited because some gram-negative bacteria are refractory to photodynamic treatment with these agents. We have designed a porphyrin derivative conjugated to a pentalysine moeity that endows the molecule with activity against gram-positive and gram-negative bacteria. Whereas the porphyrin, chlorin e6, showed in vitro activity against a limited spectrum of bacteria, chlorin e6 conjugated to pentalysine showed in vitro activity against all oral microorganisms tested, including Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. polymorphum, Actinomyces viscosus, and the streptococci. Potent antimicrobial activity (≥5-log-unit reduction in the numbers of CFU per milliliter) was retained in the presence of up to 25% whole sheep blood. The use of potent, selective agents such as this chlorin e6–pentalysine conjugate to more effectively reduce the pathogenic bacteria in the periodontal pocket may be a significant tool for the treatment of periodontal disease.
PMCID: PMC90206  PMID: 11083641
18.  Photodynamic Therapy for Acinetobacter baumannii Burn Infections in Mice▿  
Multidrug-resistant Acinetobacter baumannii infections represent a growing problem, especially in traumatic wounds and burns suffered by military personnel injured in Middle Eastern conflicts. Effective treatment with traditional antibiotics can be extremely difficult, and new antimicrobial approaches are being investigated. One of these alternatives to antimicrobials could be the combination of nontoxic photosensitizers (PSs) and visible light, known as photodynamic therapy (PDT). We report on the establishment of a new mouse model of full-thickness thermal burns infected with a bioluminescent derivative of a clinical Iraqi isolate of A. baumannii and its PDT treatment by topical application of a PS produced by the covalent conjugation of chlorin(e6) to polyethylenimine, followed by illumination of the burn surface with red light. Application of 108 A. baumannii cells to the surface of 10-s burns made on the dorsal surface of shaved female BALB/c mice led to chronic infections that lasted, on average, 22 days and that were characterized by a remarkably stable bacterial bioluminescence. PDT carried out on day 0 soon after application of the bacteria gave over 3 log units of loss of bacterial luminescence in a light exposure-dependent manner, while PDT carried out on day 1 and day 2 gave an approximately 1.7-log reduction. The application of PS dissolved in 10% or 20% dimethyl sulfoxide without light gave only a modest reduction in the bacterial luminescence from mouse burns. Some bacterial regrowth in the treated burn was observed but was generally modest. It was also found that PDT did not lead to the inhibition of wound healing. The data suggest that PDT may be an effective new treatment for multidrug-resistant localized A. baumannii infections.
doi:10.1128/AAC.00027-09
PMCID: PMC2737832  PMID: 19564369
19.  Mono- and tri-cationic porphyrin–monoclonal antibody conjugates: photodynamic activity and mechanism of action 
Immunology  2011;132(2):256-265.
Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin®, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of 18fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin® or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.
doi:10.1111/j.1365-2567.2010.03359.x
PMCID: PMC3045661  PMID: 21039468
bioconjugation; head and neck cancer; monoclonal antibody; photodynamic therapy
20.  Mono- and tri-cationic porphyrin–monoclonal antibody conjugates: photodynamic activity and mechanism of action 
Immunology  2011;132(2):256-265.
Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin®, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of 18fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin® or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.
doi:10.1111/j.1365-2567.2010.03359.x
PMCID: PMC3045661  PMID: 21039468
bioconjugation; head and neck cancer; monoclonal antibody; photodynamic therapy
21.  Site-Directed Photoproteolysis of 8-Oxoguanine DNA Glycosylase 1 (OGG1) by Specific Porphyrin-Protein Probe Conjugates: A Strategy to Improve the Effectiveness of Photodynamic Therapy for Cancer 
The specific light-induced, non-enzymatic photolysis of mOGG1 by porphyrin-conjugated or rose bengal-conjugated streptavidin and porphyrin-conjugated or rose bengal-conjugated first specific or secondary anti-IgG antibodies is reported. The porphyrin chlorin e6 and rose bengal were conjugated to either streptavidin, rabbit anti-mOGG1 primary specific antibody fractions or goat anti-rabbit IgG secondary antibody fractions. Under our experimental conditions, visible light of wavelengths greater than 600 nm induced the non-enzymatic degradation of mOGG1 when this DNA repair enzyme either directly formed a complex with chlorin e6-conjugated anti-mOGG1 primary specific antibodies or indirectly formed complexes with either streptavidin-chlorin e6 conjugates and biotinylated first specific anti-mOGG1 antibodies or first specific anti-mOGG1antibodies and chlorin e6-conjugated anti-rabbit IgG secondary antibodies. Similar results were obtained when rose bengal was used as photosensitizer instead of chlorine e6. The rate of the photochemical reaction of mOGG1 site-directed by all three chlorine e6 antibody complexes was not affected by the presence of the singlet oxygen scavenger sodium azide. Site-directed photoactivatable probes having the capacity to generate reactive oxygen species (ROS) while destroying the DNA repair system in malignant cells and tumors may represent a powerful strategy to boost selectivity, penetration and efficacy of current photodynamic (PDT) therapy methodologies.
doi:10.1016/j.jphotobiol.2006.12.004
PMCID: PMC1868704  PMID: 17251034
antibody; PDT; streptavidin; avidin; chlorin e6; porphyrin; proteolysis; OGG1; light
22.  The Vascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid Improves the Antitumor Efficacy and Shortens Treatment Time Associated with Photochlor-sensitized Photodynamic Therapy In Vivo 
In this report, we examined the antitumor activity of photodynamic therapy (PDT) in combination with 5,6-dimethylxanthenone- 4-acetic acid (DMXAA), a vascular disrupting agent currently undergoing clinical evaluation. BALB/c mice bearing subcutaneous CT-26 colon carcinomas were treated with PDT using the second-generation chlorin-based sensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (Photochlor) with or without DMXAA. Long-term (60-days) treatment outcome, induction of tumor necrosis factor-alpha (TNF-α) and interleukin- 6 (IL-6), vascular damage (microvessel density, MVD) were evaluated as endpoints. In addition, treatment selectivity was evaluated using magnetic resonance imaging (MRI) and the foot response assay. A highly synergistic interaction was observed with the combination of low-dose DMXAA and PDT (48 J cm−2 at 112 mW cm−2) resulting in ~60% long-term cures. The duration of the PDT session for this combination therapy protocol was only 7 min, while the duration of a monotherapy PDT session, selected to yield the equivalent cure rate, was 152 min. MRI showed markedly less peritumoral edema after DMXAA + short-duration PDT compared with long-duration PDT monotherapy. Similarly, DMXAA + PDT caused significantly less phototoxicity to normal mouse foot tissue than PDT alone. Increased induction of cytokines TNF-α and IL-6 (P < 0.001) was observed at 4 h followed by extensive vascular damage, demonstrated by a significant reduction in MVD at 24 h after combination treatment. In conclusion, Photochlorsensitized PDT in combination with DMXAA exhibits superior efficacy and improved selectivity with clinically feasible illumination schemes. Clinical evaluation of this novel combination strategy is currently being planned.
doi:10.1111/j.1751-1097.2008.00395.x
PMCID: PMC2897069  PMID: 18643909
23.  Protease-Stable Polycationic Photosensitizer Conjugates between Polyethyleneimine and Chlorin(e6) for Broad-Spectrum Antimicrobial Photoinactivation 
We previously showed that covalent conjugates between poly-l-lysine and chlorin(e6) were efficient photosensitizers (PS) of both gram-positive and gram-negative bacteria. The polycationic molecular constructs increased binding and penetration of the PS into impermeable gram-negative cells. We have now prepared a novel set of second-generation polycationic conjugates between chlorin(e6) and three molecular forms of polyethyleneimine (PEI): a small linear, a small cross-linked, and a large cross-linked molecule. The conjugates were characterized by high-pressure liquid chromatography and tested for their ability to kill a panel of pathogenic microorganisms, the gram-positive Staphylococcus aureus and Streptococcus pyogenes, the gram-negative Escherichia coli and Pseudomonas aeruginosa, and the yeast Candida albicans, after exposure to low levels of red light. The large cross-linked molecule efficiently killed all organisms, while the linear conjugate killed gram-positive bacteria and C. albicans. The small cross-linked conjugate was the least efficient antimicrobial PS and its remarkably low activity could not be explained by reduced photochemical quantum yield or reduced cellular uptake. In contrast to polylysine conjugates, the PEI conjugates were resistant to degradation by proteases such as trypsin that hydrolyze lysine-lysine peptide bonds, The advantage of protease stability combined with the ready availability of PEI suggests these molecules may be superior to polylysine-PS conjugates for photodynamic therapy of localized infections.
doi:10.1128/AAC.50.4.1402-1410.2006
PMCID: PMC1426948  PMID: 16569858
24.  Photodynamic targeting of human retinoblastoma cells using covalent low-density lipoprotein conjugates. 
British Journal of Cancer  1997;75(1):54-61.
Combination of photosensitizers with carrier molecules has been shown to enhance the efficiency of photodynamic therapy (PDT). Owing to an increased expression of their receptors on some malignant and proliferating cells, low-density lipoproteins (LDLs) are potential endogenous carriers. A photosensitizer, chlorin e6 (Ce6), was covalently bound to LDL via carbodiimide activation. The Ce6-LDL conjugate was evaluated on a fibroblast cell line with defined LDL receptor expression and a retinoblastoma cell line (Y79). Uptake of free Ce6 and Ce6 either covalently bound to or complexed with LDL was measured by spectrofluorimetry. Phototoxicity after irradiation at 660 nm was determined by a mitochondrial activity assay (MTT). Covalent binding to LDL significantly increased the uptake of Ce6 for both cell lines by a factor of 4-5. A Ce6: LDL binding ratio of 50:1 was optimal. A receptor-mediated uptake was demonstrated by saturability and competitive inhibition by free LDL. Binding also occurred at 2 degrees C and was attributed to non-specific associations. Irradiation with 10 J cm-2 of 660 nm light after treatment of cells with Ce6-LDL conjugate reduced the MTT activity by 80%, while free or mixed Ce6 induced a maximum of 10% reduction in the MTT activity following identical treatment conditions. These data suggest that targeting of LDL receptor-bearing cells using covalently bound carriers, such as LDL, might increase the efficiency and selectivity of PDT. Intraocular tumours such as retinoblastomas could be appropriate targets for such an approach owing to the ease of access of light sources and the need for non-invasive approaches in sensitive ocular sites.
PMCID: PMC2222700  PMID: 9000598
25.  Photosensitization and mechanism of cytotoxicity induced by the use of ALA derivatives in photodynamic therapy 
British Journal of Cancer  2001;85(2):279-284.
The use of more lipophilic derivatives of 5-aminolevulinic acid (ALA) is expected to have better diffusing properties, and after conversion into the parent ALA, to reach a higher protoporphyrin IX (PPIX) formation rate, thus improving the efficacy of topical photodynamic therapy (PDT). Here we have analysed the behaviour of 3 ALA derivatives (ALA methyl-ester, hexyl ester and a 2-sided derivative) regarding PPIX formation, efficiency in photosensitizing cells and mechanism of cellular death. The maximum amount of porphyrins synthesized from 0.6 mM ALA was 47 ± 8 ng/105 cells. The same amount was formed by a concentration 60-fold lower of hexyl-ALA and 2-fold higher of methyl-ALA. The 2-sided derivative failed to produce PPIX accumulation. Applying a 0.6 J cm−2 light dose, cell viability decreased to 50%. With the 1.5 J cm−2 light dose, less than 20% of the cells survive, and higher light doses produced nearly total cell killing. Comparing the PPIX production and the induced phototoxicity, the more the amount of porphyrins, the greater the cellular killing, and PPIX formed from either ALA or ALA-esters equally sensitize the cells to photoinactivation. ALA-PDT treated cells exhibited features of apoptosis, independently on the pro-photosensitizer employed. ALA-PDT can be improved with the use of ALA derivatives, reducing the amount of ALA necessary to induce efficient photosensitization. ©2001 Cancer Research Campaign http://www.bjcancer.com
doi:10.1054/bjoc.2001.1875
PMCID: PMC2364046  PMID: 11461090
photodynamic therapy; PDT; aminolevulinic acid; ALA; ALA derivatives; apoptosis

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