Cationic Photosensitizer Research Articles

Luminescence-based assessment of thermodynamic constants for electrostatic binding of non-luminescent dyes and atomic ions to colloidal semiconductor surfaces

Steady-state and time-resolved luminescence experiments were employed to study the electrostatic interaction of cationic photosensitizers and negatively charged colloidal semiconductor particles. Electrostatic adsorption of efficient photosensitizers on semiconductor surfaces is known to quench the photosensitizers’ luminescence. By monitoring the quenching, either directly or competitively, thermodynamic constants for binding of both luminescent and non-luminescent ruthenium and osmium complexes to negatively charged SnO2 particle have been determined. The function of atomic cations, such as Li+, Na+ and K+, in controlling the binding of cationic dyes to colloidal SnO2 has also been studied via competitive luminescence. From the study, semiconductor surface binding constants for these species have also been determined.

Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a cationic hydrophilic porphyrin at various light wavelengths.

Photodynamic treatment by the cationic TMPyP photosensitizer was undertaken on the multiple antibiotic-resistant bacteria Acinetobacter baumannii and Escherichia coli. Total eradication of the bacterial cultures was determined immediately after initiation of illumination when these bacteria were treated with 5, 10, 15, 20-tetra (4-N methylpyridyl)porphine (TMPyP) at a concentration of 29.4 micromol/L and illuminated by blue, green, or red light. Total eradication of both bacteria was obtained also after treatment of bacterial cultures with 3.7 micromol/L TMPyP and illumination with blue light (400-450 nm). On the other hand, an 8- or 16- to 20-fold higher light intensity, respectively, was required for total eradication upon illumination with green (480-550 nm) or red light (600-700 nm). A 407-nm blue light only 7 and 9 joules/cm2, respectively, was needed for total eradication of both bacteria even at a concentration of 3.7 micromol/L TMPyP. X-ray-linked microanalysis demonstrated loss of potassium and a flood of sodium and chloride into the cells, indicating serious damage to the cytoplasmic membrane. Transmission electron microscopy (TEM) revealed structural changes and damage to the membrane of treated E. coli. In A. baumannii-treated cells, mesosomes and black dots that resemble aggregation of polyphosphate polymers could be seen. DNA breakage appeared only after a long period of illumination, when the bacterial cell was no longer viable. It can be concluded that cytoplasmic membrane damage and not DNA breakage is the major cause for bacterial death upon photosensitization.

Cell killing by cationic photosensitizers in the SK-23 and SK-MEL-28 melanoma cell lines

Cell killing by cationic photosensitizers in the SK-23 and SK-MEL-28 melanoma cell lines

Cytotoxicity and Adjuvant Activity of Cationic Photosensitizers in a Multidrug Resistant Cell Line

The toxicities and phototoxicities of methylene blue (MB), toluidine blue O (TBO) and Victoria blue BO (VBBO) in a murine mammary tumour cell line (EMT6-S) and a multidrug resistant (MDR) sub-line (EMT6-R) were measured and their efficacy against the resistant sub-line was compared to that of doxorubicin and cis-platinum. The MDR cell line was considerably more susceptible to VBBO than to the conventional agent doxorubicin. VBBO was also pho-totoxic whereas illumination did not alter the activity of doxorubicin or of cis-platin. Both TBO and MB showed limited light activation (2-fold) in both the sensitive and resistant cell lines.Pre-treatment with VBBO prior to exposure to doxorubicin caused a twofold increase in doxorubicin toxicity in both cell lines. MB and TBO, however, increased doxorubicin toxicity in EMT6-R cells ×2 and ×3 respectively, but had less effect on the sensitive cell line (increase ×l.4 and ×2 respectively). Thus MB and TBO may act on the MDR cell line via a different mechanism to that of VBBO.

Eradication of Acinetobacter baumannii by photosensitized agents in vitro

The photodynamic effects of photosensitizers on Acinetobacter baumannii were studied. These Gram negative bacteria have recently been implicated in various infections, mainly acquired in hospitals. They have outstanding characteristics of multidrug high resistance to antimicrobial agents. The best photodynamic effect was obtained when A. baumannii cultures were treated with light activated deuteroporphyrin (Dp) at a concentration of 34 μmoles 1 1 and polymyxin nonapeptide (PMNP) at a concentration of 200 μmoles 1 −1. At these concentrations the culture in brain heart infusion (BHI) broth was found to be sterile after 1 h of treatment. Some inhibition was also obtained under the same conditions with Cd-texaphyrin (Cd-Tx) in the presence of PMNP. Treatment with various other photosensitizers in the presence of PMNP exhibited only marginal antibacterial activity. The cationic photosensitizer tetra-methylpyridyl porphine (TMPyP) did not exhibit any photodynamic effect on A. baumannii when illuminated during its growth in BHI broth. Bacteria grown in nutrient broth or suspended in saline and treated by TMPyP resulted in a significant photoinactivation by the sensitizer alone even in the absence of PMNP. It was found that a high concentration of the proteins present in BHI or in serum prevent TMPyP from acting as a photosensitizer against A. baumannii. Bovine serum albumin at the same high protein concentration prevents Dp (in the presence of PMNP) to act as a photosensitizer. The anionic photosensitizer tetra-sulfonatophenyl porphine (TPPS 4) did not show any photodynamic effect in high or low protein media. In this study it was found that despite the high resistance of the Acinetobacter baumannii to antibiotics, these bacteria can be significantly photoinactivated by treatment with either Dp + PMNP or TMPyP in low protein content environments. When the protein concentration is high, photoinactivation efficiency depends on the type of protein present in the medium.

Unsymmetrically substituted benzonaphthoporphyrazines: a new class of cationic photosensitizers for the photodynamic therapy of cancer.

Unsymmetrical zinc(II) complexes of benzonaphthoporphyrazines 5a-12a bearing between one and eight pyridyloxy substituents are synthesized by statistical tetramerization of 6-(1,1-dimethylethyl)-2,3-naphthalenedicarbonitrile (1) with 4-(3-pyridyloxy)- or 4,5-bis-(3-pyridyloxy)-1,2-benzenedicarbonitrile (2, 3). Methylation of 5a-12a leads to the catianic pyridyloxybenzonaphthoporphyrazines 5b-12b having between one to eight positive charges. The Q-band transition in the visible spectra exhibits a bathochromic shift from 680 to 760 nm dependent upon the number of annelated naphthalene rings. The singlet oxygen quantum yields of the benzonaphthoporphyrazines determined by the dye-sensitized photooxidation of 1,3-diphenylisobenzofurane is surprisingly high (in the range of zinc phthalocyanine). The photooxidative stabilities of the photosensitizers described quantitatively by first-order kinetics decrease with the number of annelated naphtho groups. A linear correlation between the logarithm of the decomposition rate constant and the position of the highest occupied molecular orbital (HOMO)-level of the photosensitizers is found. Destabilization of the HOMO leads to a decrease of the photostability. Due to their adjustable long wavelength absorption, their intramolecular polarity axis and their different hydrophilic/hydrophobic character, these novel compounds may be suitable photosensitizers for the photodynamic therapy of cancer.

Photoinactivation of bacteria. Use of a cationic water-soluble zinc phthalocyanine to photoinactivate both Gram-negative and Gram-positive bacteria

The photosensitization of microorganisms is potentially useful for sterilization and for the treatment of certain bacterial diseases. Unit now, any broad spectrum approach has been inhibited because, although Gram-positive bacteria can be photoinactivated by a range of photosensitizer, Gram-negative bacteria have not usually been susceptible to photosensitized destruction. In the present work, it has been shown that the Gram-negative bacteria Escheria coli and Pseudomonas aeruginosa, as well as the Gram-positive bacterium Enterococcus seriolicida, can be photoinactivated when illuminated in the presence of a cationic water-soluble zinc pyridinium phthalocyanine (PPC). The degree of photoinactivation is dependent on both the concentration of PPC and the illumination time. In contrast, the three bacteria are not photoinactivated by illumination in the presence of a neutral tetra-diethanolamine phthalocyanine (TDEPC( or negatively charged tetra-sulphonated phthalocyanine (TSPC). Uptake studies have revealed that the lack of activity of TSPC is due to the fact that it has very little affinity for any of the organisms. However, the issue appears to be more complex than simply the gross levels of cellular uptake, since TDEPC and PPC are both taken up by the organisms but only PPC shows activity. This indicates that the localization and subcellular distribution of the phthalocyanines may be a crucial factor in determining their cell killing potential. Further analysis of the uptake data has revealed a cell-bound photosensitizer fraction, which remains tightly associated after several washings, and another weakly bound fraction, which is removed by successive washings. Analysis of the cell killing curves, carried out after successive washings of E. coli exposed to PPC, has revealed that it is the tightly associated fraction that is involved in the photosensitization. Taken together with other data, these results suggest that cationic photosensitizers may have a broader application in the photoinactivation of bacterial cells than the anionic or neutral photosensitizers commonly used in photodynamic therapy.

Effect of density-gradients on the binding of photosensitizing agents to plasma proteins

The binding of photosensitizing agents to low-density lipoprotein is considered an important factor in tumor localization. We examined the affinity of a group of photosensitizers, varying in charge and hydrophobicity, for LDL, under conditions designed to determine whether the high salt concentration involved in conventional KBr gradients affected the results. Density-gradients containing KBr vs D 2O were evaluated; the latter can delineate VLDL and LDL from other plasma components, while the KBr gradient readily resolved VLDL, LDL, HDL and albumin. Distribution of the photosensitizers to plasma fractions was assessed, along with the effect of Cremophor EL, an emulsifier used for formulation of water-insoluble drugs. Both the D 2O and KBr gradients provided similar results with regard to the affinity of anionic, neutral or cationic photosensitizers for LDL. The use of Cremophor EL for drug formulation was associated with an altered electrophoretic lipoprotein profile. In some cases, affinity of CRM-solubilized sensitizers for plasma components varied with the density-gradient employed. The high salt concentration used in density-gradient fractionation had little effect on the affinity of photosensitizing agents to low-density lipoprotein but may introduce artifacts when emulsifiers are used in drug formulation.

Impaired accumulation of a cationic photosensitizing agent by a cell line exhibiting multidrug resistance.

Transport and accumulation of copper benzochlorin iminium salt (CDS1), a cationic photosensitizing agent, were examined using the P388/ADR murine leukemia, which exhibits the MDR (multidrug resistance) phenotype, and the wild-type parent cell line, P388. The recent availability of radioactive CDS1 permitted kinetic studies at drug levels in the submicromolar range. Exclusion of CDS1 by P388/ADR cells could be demonstrated, indicating that this agent is a substrate for the outward transport system associated with MDR. These results have implications with regard to the efficacy of cationic photosensitizers against this common neoplastic phenotype. The CDS1 was readily accumulated by P388 cells and by P388/ADR cells when the outward transport system was inhibited. Under these conditions, CDS1 was tightly bound and could not be washed out even when the outward transport system was reactivated.

Fluorescence formation during photodynamic therapy in the nucleus of cells incubated with cationic and anionic water-soluble photosensitizers

The variations of fluorescence during light exposure of the cationic sensitizers methylene blue (MB) and meso-tetra(4 N-methylpyridyl)porphyrin (T4MPyP) as well as the anionic meso-tetra(4-sulphonatophenyl)porphyrin (TPPS 4) were measured at different intracellular sites using video-intensified microscopy in combination with microspectrofluorometry. Before light exposure the sensitizers were localized in distinct parts of the cytoplasm, especially in fluorescent organelles. During irradiation a drastic fluorescence formation and increase in the cytoplasm and nucleus, which was most pronounced in the nucleoli, could be observed for the cationic sensitizers as well as TPPS 4. In the case of MB the increase in fluorescence was concomitant with a spectral shift in the emission spectra. For TPPS 4 and T4MPyP the formation of a second species with a Soret band shifted towards longer wavelengths was observed and correlated with the fluorescence increase in the nucleoli. Cell deformations also took place.

Photodynamic Therapy in Gynecology

PDT is a technique in which visible light is used in combination with photosensitizing agents to achieve a tumoricidal effect. Hematoporphyrins are the most commonly used photosensitizers in clinical practice. DHE is the active fraction of hematoporphyrin. Intravenously injected DHE is found in highest concentration in the liver followed by the spleen, kidney, tumor, skin, muscle, brain, and lungs. The strongest absorption bands for DHE are in the blue region of the spectrum, and this helps to account for the skin toxicity associated with PDT. Red light, at the wavelength of 630 nm, is usually used clinically because of its greater tissue penetration. Techniques such as photobleaching and use of photosensitizers that have weak absorption bands at the lower wavelengths may reduce cutaneous toxicity in the future. Other approaches, such as the use of monoclonal antibody-linked photosensitizers or cationic photosensitizers that are specifically localized in tumor cells, may also increase the effectiveness of PDT while decreasing toxicity. Light for PDT is usually provided by argon-pumped dye lasers or metal vapor lasers. Diode lasers will be used in the future. The use of fiber-optics and diffusing lenses allows the endoscopic and interstitial use of PDT. The mechanism of action of PDT involves the formation of singlet oxygen, which oxidizes biologic molecules and causes irreversible subcellular damage. The major in vivo effect of PDT is caused by its destruction of tumor vasculature, causing anoxia and necrosis. The use of PDT in gynecology has been limited. Several investigators have reported mixed results in treating lower genital tract intraepithelial and recurrent malignant tumors using a variety of approaches involving PDT. The use of PDT in other similar, though nongynecologic, tumors offers a direction for future research.

Strategies for selective cancer photochemotherapy: antibody-targeted and selective carcinoma cell photolysis.

A principle objective in chemotherapy is the development of modalities capable of selectively destroying malignant cells while sparing normal tissues. One new approach to selective photochemotherapy, antibody-targeted photolysis (ATPL) uses photosensitizers (PS) coupled to monoclonal antibodies (MAbs) which bind to cell surface antigens on malignant cells. Selective destruction of human T leukemia cells (HBP-ALL) was accomplished by coupling the efficient PS chlorin e(6) to an anti-T cell MAb using dextran carriers. Conjugates with chlorin: MAb ratios of 30:1 retained > 85% MA b binding activity, and had a quantum yield for singlet oxygen production of 0.7 +/- 0.1, the same as that of free chlorin e(6). Cell killing was dependent on the doses of both MAb-PS and 630-670 nm light and occurred only in target cell populations which bound the MAb. On the order of 10(10) singlet oxygen molecules were necessary to kill a cell. A second approach to specific photochemotherapy, selective carcinoma cell photolysis (SCCP), relies on preferential accumulation of certain cationic PS by carcinoma cell mitochondria. We have evaluated several classes of cationic dyes, and in the case of N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC) and some of its analogs, have demonstrated highly selective killing of human squamous cell, bladder and colon carcinoma cells in vitro. In isolated mitochondria, EDKC uptake and fluorescence depended on membrane potential, and the dye specifically photosensitized damage to Complex I in the electron transport chain. N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine and some of its analogs accumulated within subcutaneous xenografts of human tumors in nude mice with tumor:skin ratios > 8. Photoirradiation caused significant inhibition of tumor growth, without cutaneous phototoxicity.