Apoptosis of vascular smooth muscle cells induced by photodynamic therapy with protoporphyrin IX

Photodynamic therapy (PDT) had been shown effective in the treatment of intimal hyperplasia, which contributes to restenosis, by eradicating cells in the vessel wall. This study is designed to evaluate the effects of PDT with protoporphyrin IX (PpIX) on the viability of vascular smooth muscle cells (SMCs) and to define the cell-death pathway. Fluorescence microscopy and laser-induced fluorescence spectroscopic detection showed that SMCs selectively uptake PpIX, and the intracellular PpIX concentration increases with the amount of PpIX in the incubation solution. PDT with PpIX impaired cellular viability from 93 ± 3.4% to 36 ± 3.9% when the light intensity increases from 2 to 9 J/cm² and intracellular PpIX concentration increases from 0.5 to 20 μg/ml. Although PDT induced both apoptosis and necrosis, the ratio of apoptotic cells increased with light dosage or intracellular PpIX concentration. The loss of mitochondrial membrane potential coincided with the apoptotic ratio. Our results indicated that the induction of apoptosis of SMCs may be one of the mechanisms by which PDT inhibits restenosis in vivo.     

Metallobacteriochlorophylls as potential dual agents for photodynamic therapy and chemotherapy

A theoretical analysis of bacteriochlorophyll a containing its non-native divalent metal ions: Co, Ni, Cu, Zn, Ru, Rh, Pd, and Pt, has been carried out by means of density functional theory (DFT) calculations. The main stress was put on the derivatives with metals, which already found applications as coordination compounds in anti-tumor therapy (Ru, Pt, Pd, and Rh). The idea was to combine their cytotoxic properties with the known suitability of bacteriochlorophylls macrocycle for photodynamic therapy. The geometries of the studied systems are compared and reveal a number of similarities. The cores of the modified bacteriochlorophylls are flat, and the introduced metal ions lie in plane of the macrocycle, showing its large ability to accommodate metal ions of different sizes. However, four metal–nitrogen bonds, linking the central ions with the macrocycle ligand, are not equivalent. Metals are the strongest attached to nitrogens, which come from the pyrrole, which is fused with isocyclic ring. Based on the known spectroscopic data, the absorption properties of the proposed systems are predicted. Finally, it is found that all studied metal–macrocycle adducts are stable in aqueous media. The only exceptions are Mg-BChla (the finding is reflected by experimental facts) and Zn-BChla. The predicted high stability of Ru-, Rh-, Pt- and Pd-bacteriochlorophylls might turn out beneficial for therapeutic purposes.     

Increase in protoporphyrin IX after 5-aminolevulinic acid based photodynamic therapy is due to local re-synthesis.

Protoporphyrin IX (PpIX) fluorescence that is bleached during aminolevulinic acid (ALA) mediated photodynamic therapy (PDT) increases again in time after treatment. In the present study we investigated if this increase in PpIX fluorescence after illumination is the result of local re-synthesis or of systemic redistribution of PpIX. We studied the spatial distribution of PpIX after PDT with and without cooling using the skin-fold observation chamber model. We were unable to show a correlation between the local PpIX fluorescence increase and the distance from a blood vessel. The spatial distribution of PpIX fluorescence within normal tissue or tumour is not changed in response to the illumination. These observations suggest that there is no diffusion of PpIX into the treated tissue. Cooling the tissue to 12 degrees C, a temperature at which PpIX synthesis is inhibited, inhibited the PpIX fluorescence increase normally observed after illumination. We also found a strong correlation between local PpIX photobleaching during illumination and the fluorescence intensity 1 h after illumination similar to what we have observed in patients treated with ALA-PDT. Therefore we conclude that the increase in PpIX fluorescence after illumination is due to local cellular re-synthesis.     

Water-soluble polymer conjugates of ZnPP for photodynamic tumor therapy

Zn-protoporphyrin (ZnPP) is a promising candidate for cancer therapy. It is known to inhibit heme-oxygenase-1 (HO-1), resulting in suppressed biliverdin/bilirubin production accompanying lowered antioxidative capacity. As a consequence, a significant suppression of tumor growth in vivo was reported. Recent findings also showed that ZnPP efficiently generated reactive singlet oxygen under illumination of visible light. In the present report, we describe the photosensitizing capabilities of water-soluble polymer conjugates of ZnPP as novel compounds for photodynamic therapy against solid tumors. The polymer conjugation made ZnPP water-soluble, thus possible for injection for its aqueous solution. The cellular uptake and photobiological activity of ZnPP derivatives have been tested using a human T-cell leukemia cell line in vitro and demonstrated most potent phototoxic effects of SMA-ZnPP followed by PEG-ZnPP under aerobic conditions.     

Carbaporphyrin ketals as potential agents for a new photodynamic therapy treatment of leishmaniasis

Dimethyl and diethyl carbaporphyrin ketals inhibit the growth of Leishmania tarentolae promastigotes in vitro. The concentration dependency of the inhibitory effect was tested using the MTT assay. The presence of reactive oxygen species, such as singlet oxygen and superoxide, was detected using electron paramagnetic resonance spectroscopy with selected spin traps and confocal microscopy in cultures exposed to these carbaporphyrin ketals. These unique porphyrinoids show promise as potent inhibitors of Leishmania.     

Polyamine conjugates of stigmasterol

Three new polyamine conjugates with stigmasterol [(3β,22E)-stigmasta-5,22-dien-3-ol] were synthesized and subjected to basic antimicrobial and cytotoxic tests. The conjugate derived from spermine, (3β,22E)-stigmasta-5,22-dien-3-yl 4(12-amino-4,9-diaza-dodecylamino)-4-oxobutanoate (5c), displayed considerable antimicrobial activity on Staphylococcus aureus at low concentration (50μgmL(-1)). The cytotoxic activity was tested on cells of human T-lymfoblastic leukemia (IC(50)=35.8±10.3μM (5c) and IC(50)=35.9±5.7μM (5b)) and normal human fibroblasts (IC(50)=38.0±2.8μM (5c) and IC(50)=45.5±1.9μM (5b)). Conjugate 5a displayed no activity in both tests.     

Chlorophyll-a analogues conjugated with aminobenzyl-DTPA as potential bifunctional agents for magnetic resonance imaging and photodynamic therapy

A clinically relevant photosensitizer, 3-devinyl-3-(1-hexyloxyethyl)pyropheophorbide-a (HPPH, a chlorophyll-a derivative), was conjugated with Gd(III)-aminobenzyl-diethylenetriaminepentaacetic acid (DTPA), an experimental magnetic resonance (MR) imaging agent. In vivo reflectance spectroscopy confirmed tumor uptake of HPPH-aminobenzyl-Gd(III)-DTPA conjugate was higher than free HPPH administered intraveneously (iv) to C3H mice with subcutaneously (sc) implanted radiation-induced fibrosarcoma (RIF) tumor cells. In other experiments, Sprague-Dawley (SD) rats with sc implanted Ward Colon Carcinoma cells yielded markedly increased MR signal intensities from tumor regions-of-interest (ROIs) 24 h post-iv injection of HPPH-aminobenzyl-Gd(III)-DTPA conjugate as compared to unconjugated HPPH. In both in vitro (RIF tumor cells) and in vivo (mice bearing RIF tumors and rats bearing Ward Colon tumors) the conjugate produced significant increases in tumor conspicuity at 1.5 T and retained therapeutic efficacy following PDT. Also synthesized were a series of novel bifunctional agents containing two Gd(III) atoms per HPPH molecule that remained tumor-avid and PDT-active and yielded improved MR tumor conspicuity compared to their corresponding mono-Gd(III) analogues. Administered iv at a MR imaging dose of 10 micromol/kg, these conjugates produced severe skin phototoxicity. However, by replacing the hexyl group of the pyropheophorbide-a with a tri(ethylene glycol) monomethyl ether (PEG-methyl ether), these conjugates produced remarkable MR tumor enhancement at 8 h post-iv injection, significant tumoricidal activity (80% of mice were tumor-free on day 90), and reduced skin phototoxicity compared to their corresponding hexyl ether analogues. The poor water-solubility characteristic of these conjugates was resolved by incorporation into a liposomal formulation. This paper presents the synthesis of tumor-avid contrast enhancing agents for MR imaging and thus represents an important milestone toward improving cancer diagnosis and tumor characterization. More importantly, this paper describes a new family of bifunctional agents that combine two modalities into a single cost-effective "see and treat" approach, namely, a single agent that can be used for contrast agent-enhanced MR imaging followed by targeted photodynamic therapy.     

Nanoparticles as vehicles for delivery of photodynamic therapy agents

Photodynamic therapy (PDT) in cancer treatment involves the uptake of a photosensitizer by cancer tissue followed by photoirradiation. The use of nanoparticles as carriers of photosensitizers is a very promising approach because these nanomaterials can satisfy all the requirements for an ideal PDT agent. This review describes and compares the different individual types of nanoparticles that are currently in use for PDT applications. Recent advances in the use of nanoparticles, including inorganic oxide-, metallic-, ceramic-, and biodegradable polymer-based nanomaterials as carriers of photosensitizing agents, are highlighted. We describe the nanoparticles in terms of stability, photocytotoxic efficiency, biodistribution and therapeutic efficiency. Finally, we summarize exciting new results concerning the improvement of the photophysical properties of nanoparticles by means of biphotonic absorption and upconversion.     

Oligonucleotide-peptide conjugates as potential antisense agents.

Oligonucleotide-peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling of pre-synthesized peptides to the 2'-position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support.     

Intracellular photodynamic therapy with photosensitizer-nanoparticle conjugates: cancer therapy using a 'Trojan horse'.

Phthalocyanine-nanoparticle conjugates have been designed and synthesised for the delivery of hydrophobic photosensitizers for photodynamic therapy (PDT) of cancer. The phthalocyanine photosensitizer stabilized gold nanoparticles have an average diameter of 2-4 nm. The synthetic strategy interdigitates a phase transfer reagent between phthalocyanine molecules on the particle surface that solubilises the hydrophobic photosensitizer in polar solvents enabling delivery of the nanoparticle conjugates to cells. The phthalocyanine is present in the monomeric form on the nanoparticle surface, absorbs radiation maximally at 695 nm and catalytically produces the cytotoxic species singlet oxygen with high efficiency. These properties suggest that the phthalocyanine-nanoparticle conjugates are ideally suited for PDT. In a process that can be considered as cancer therapy using a 'Trojan horse', when the nanoparticle conjugates are incubated with HeLa cells (a cervical cancer cell line), they are taken up thus delivering the phthalocyanine photosensitizer directly into the cell interior. Irradiation of the nanoparticle conjugates within the HeLa cells induced substantial cell mortality through the photodynamic production of singlet oxygen. The PDT efficiency of the nanoparticle conjugates, determined using colorimetric assay, was twice that obtained using the free phthalocyanine derivative. Following PDT with the nanoparticle conjugates, morphological changes to the HeLa cellular structure were indicative of cell mortality via apoptosis. Further evidence of apoptosis was provided through the bioluminescent assay detection of caspase 3/7. Our results suggest that gold nanoparticle conjugates are an excellent vehicle for the delivery of surface bound hydrophobic photosensitizers for efficacious photodynamic therapy of cultured tumour cells.     

Prostaglandin E2-bisphosphonate conjugates: potential agents for treatment of osteoporosis.

Conjugates of bisphosphonates (potential bone resorption inhibitors) and prostaglandin E2 (a bone formation enhancer) were prepared and evaluated for their ability to bind to bone and to liberate, enzymatically, free PGE2. The conjugate 3, an amide at C-1 of PGE2 proved to be too stable in vivo while conjugate 6, a thioester, was too labile. Several PGE2, C-15 ester-linked conjugates (18, 23, 24 and 31) were prepared and conjugate 23 was found to bind effectively to bone in vitro and in vivo and to liberate PGE2 at an acceptable rate. A 4-week study in a rat model of osteoporosis showed that 23 was better tolerated and more effective as a bone growth stimulant than daily maximum tolerated doses of free PGE2.     

Folic acid conjugates with photosensitizers for cancer targeting in photodynamic therapy: Synthesis and photophysical properties

Recent researches in photodynamic therapy have focused on novel techniques to enhance tumour targeting of anticancer drugs and photosensitizers. Coupling a photosensitizer with folic acid could allow more effective targeting of folate receptors which are over-expressed on the surface of many tumour cells. In this study, different folic acid–OEG-conjugated photosensitizers were synthesized, characterized and their photophysical properties were evaluated. The introduction of an OEG does not significantly improve the hydrophilicity of the FA–porphyrin. All the FA-targeted photosensitizers present good to very good photophysical properties. The best one appears to be Ce6. Molar extinction coefficient, fluorescence and singlet oxygen quantum yields were determined and were compared to the corresponding photosensitizer alone.     

Dual‐wavelength excitation for fluorescence‐based quantification of zinc protoporphyrin IX and protoporphyrin IX in whole blood

Quantification of erythrocyte zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PPIX), individually or jointly, is useful for the diagnostic evaluation of iron deficiency, iron‐restricted erythropoiesis, lead exposure, and porphyrias. A method for simultaneous quantification of ZnPP and PPIX in unwashed blood samples is described, using dual‐wavelength excitation to effectively eliminate background fluorescence from other blood constituents. In blood samples from 35 subjects, the results of the dual‐wavelength excitation method and a reference high performance liquid chromatography (HPLC) assay were closely correlated both for ZnPP (r_s = 0.943, p < 0.0001; range 37–689 μmol ZnPP/mol heme, 84–1238 nmol/L) and for PPIX (r_s = 0.959, p < 0.0001; range 42–4212 μmol PPIX/mol heme, 93–5394 nmol/L). In addition, for ZnPP, the proposed method is compared with conventional single‐wavelength excitation and with commercial front‐face fluorimetry of washed erythrocytes and whole blood. We hypothesize that dual‐wavelength excitation fluorimetry will provide a new approach to the suppression of background fluorescence in blood and tissue measurements of ZnPP and PPIX. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The potential for gene-targeted radiation therapy of cancers

Targeted cancer therapy is the mantra now chanted by oncologists of all types. Everyone hopes that the rapid expansion in the knowledge of cancer cell genetics, signaling, regulatory factors and other changes that underlie malignant transformation and metastasis will lead to innovative approaches for the treatment of cancers. To date, successful targeted therapies have been derived from pharmaceutical chemistry - designing chemical compounds intended to disrupt a crucial pathway for malignant cells to survive, grow and metastasize. Radiotherapy also has a goal of more-selective targeting of therapeutic radiation effects to only tumor cells. In this review, we describe our efforts to create a form of gene-targeted radiation therapy by using the unique radiation effects of radionuclides that decay by the Auger process attached to oligonucleotide carrier-molecules that are capable of forming triplex DNA structures with target sequences in the genome of the human cancer cell.     

Radioiodine and 211At-labeled guanidinomethyl halobenzoyl octreotate conjugates: potential peptide radiotherapeutics for somatostatin receptor-positive cancers

Derivatives of the somatostatin analogues octreotide and octreotate labeled with radioiosotopes are used in the diagnosis and therapy of somatostatin receptor (SSTR)-positive tumors. A method has been devised to synthesize {N-(4-guanidinomethyl-3-iodobenzoyl)-Phe1-octreotate (GMIBO). Receptor binding assay and scatchard analysis yielded a Kd of 4.83 +/- 0.19 nM for this peptide. Derivatives of this peptide labeled with radioiodine ([*I]GMIBO) and the alpha-particle-emitting radiohalogen 211At N-(3-[211At]astato-4-guanidinomethylbenzoyl)-Phe1-octreotate; [211At]AGMBO} were prepared in a single step from a tin precursor in radiochemical yields of 30-35% and 15-20%, respectively. Paired-label internalization assays performed with the SSTR-positive D341 Med human medulloblastoma cell line demonstrated that [125I]GMIBO and [211At]AGMBO were specifically internalized 20-40% more than Nalpha-(1-deoxy-D-fructosyl)-[131I]I-Tyr3-octreotate ([131I]I-Glu-TOCA), the radioiodinated octreotide derivative previously shown to exhibit maximum internalization in this cell line. Uptake of [131I]GMIBO in D341 Med subcutaneous xenografts in a murine model (8.34 +/- 1.82 versus 8.10 +/- 2.23% ID/g at 1h) and SSTR-expressing normal tissues was comparable to that of [125I]I-Glu-TOCA and was shown to be specific. However, the uptake of [131I]GMIBO also was substantially higher in liver (16.9 +/- 3.15 versus 1.39 +/- 0.45% ID/g at 1 h) and in kidneys (44.33 +/- 6.47 versus 3.44 +/- 0.68% ID/g at 1h) compared to that of [125I]I-Glu-TOCA. These data suggest that these novel peptide conjugates retain their specificity for SSTR both in vitro and in vivo; however, because of their higher accumulation in normal tissues they would be best applied in settings amenable to loco-regional administration such as medulloblastoma neoplastic meningitis.     

Silk fibroin hydrogels for potential applications in photodynamic therapy

In this study, we prepared translucid hydrogels with different concentrations of silk fibroin, extracted from raw silk fibers, and used them as a matrix to incorporate the photosensitizer 5-(4-aminophenyl)-10,15,20-tris-(4-sulphonatophenyl) porphyrin trisodium for application in photodynamic therapy (PDT). The hydrogels obtained were characterized by rheology, spectrophotometry, and scattering techniques to elucidate the factors involved in the formation of the hydrogel, and to characterize the behavior of silk fibroin (SF) after incorporating of the porphyrin to the matrix. The rheology results demonstrated that the SF hydrogels had a shear thinning behavior. In addition, we were able to verify that the structure of the material was able to be recovered over time after shear deformation. The encapsulation of porphyrins in hydrogels leads to the formation of self-assembled peptide nanostructures that prevent porphyrin aggregation, thereby greatly increasing the generation of singlet oxygen. Also, our findings suggest that porphyrin can diffuse out of the hydrogel and permeate the outer skin layers. This evidence suggests that SF hydrogels could be used as porphyrin encapsulation and as a drug carrier for the sustained release of photosensitizers for PDT.     

Aminosquaraines as potential photodynamic agents: Synthesis and evaluation of in vitro cytotoxicity

The synthesis of several aminosquaraine cationic dyes displaying strong absorption within the so-called phototherapeutic window (650–850 nm) is described. Their cytotoxicity, under dark and illuminated conditions, was tested against several human tumor cell lines (breast, lung, cervical and hepatocellular carcinomas) and non-tumor porcine liver primary cells. All compounds showed to inhibit the growth of the tumor cells upon irradiation more than in the absence of light, in more or less extension, clearly exhibiting photodynamic activity. The photosensitizing ability against some cell lines, together with the low toxicity for the non-tumor primary PLP2 cells displayed by some of the compounds synthetized, turns them into potential candidates as photosensitizers for PDT.