Improved glucose detection limit based on phosphorescence from protected metalloporphyrin triplet state.

BACKGROUND: Non-invasive indirect blood glucose monitoring can be realized by detecting low concentrations of glucose (0.05-5 mM) in tears, but sensitive optical indicators are required. The intensity of the phosphorescence of a candidate optical indicator, palladium hematoporphyrin monomethyl ether (Pd-HMME), is increased by oxygen consumption under sealed conditions in the presence of glucose and glucose oxidase. However, the glucose detection limit based on this mechanism is high (800 µM) because the phosphorescence is completely quenched under ambient oxygen conditions and hence a large amount of glucose is required to reduce the oxygen levels such that the phosphorescence signal is detectable. RESULTS: To improve the glucose detection limit of Pd-HMME phosphorescence-based methods, the triplet protector imidazole was introduced, and strong phosphorescence was observed under ambient oxygen conditions. Detectable phosphorescence enhancement occurred at low glucose concentrations (<200 µM). Linear correlation between the phosphorescence intensity and glucose concentration was observed in the range of 30-727 µM (R2 = 99.9 %), and the detection limit was ∼10 µM. The glucose sensor has a fast response time (∼90 s) and excellent selectivity for glucose. SIGNIFICANCE AND NOVELTY: These results indicate the potential of the developed optical indicator for fast, selective, and reliable low-concentration glucose sensing.

[The use of fluorescent microscopy for the detection of blood effusion in putrescent material].

The present article was designed to demonstrate the efficiency of application of fluorescent microscopy for histological studies of preparations of putrescent material suspected to contain effused blood. This method makes it possible to detect haematoporphirin, a product of haemoglobin degradation, the presence of which gives reliable evidence of the presence of effused blood in the study material.

Stereochemistry of hemes and other metalloporphyrins.

Metalloporphyrins, most notably the iron porphyrins, observe clearly defined, internally consistent, structural principles that promise to be fully applicable to the hemes in the several families of the hemoproteins.

Fluorescence of experimental atheromatous plaques with hematoporphyrin derivative.

Fluorescence of hematoporphyrin derivative (HPD) has been used clinically to localize malignant neoplasms because of its selective accumulation in these tissues. We tested the hypothesis that HPD may also be selectively concentrated within atheromatous plaques. 48 h after HPD injection in a variety of species, selective fluorescence of atheromatous plaques of the aorta was seen in each animal (rabbits and Patas monkey) exhibiting such lesions. No fluorescence could be demonstrated in aortic segments free of atheromatous involvement. Since the efficacy of photodynamic destruction of malignant tumors with HPD has been demonstrated in clinical studies, the observations of the present study may have therapeutic implications in atheromatosis.

Active photosensitizers in butter detected by fluorescence spectroscopy and multivariate curve resolution.

In this study, fluorescence excitation and emission matrices and multivariate curve resolution (PARAFAC) were used to detect and characterize active photosensitizers spectrally in butter. Butter samples were packed under high (air) and low oxygen (<0.05%) atmospheres and exposed to violet, green, or red light. Six photosensitizers were found: riboflavin, protoporphyrin, hematoporphyrin, a chlorophyll a-like molecule, and two unidentified tetrapyrrols. By estimation of relative concentrations, we could follow how each sensitizer was photodegraded as function of wavelength, oxygen level, and time. The degradation rate of protoporphyrin, hematoporphyrin, chlorophyll a, and one of the tetrapyrrols correlated well (0.83-0.91) with the formation of sensory measured oxidation. The results suggest that mainly type I photoreactions were responsible for the degradation of photosensitizers in both high and low oxygen atmosphere. Type II photoreactions (generation of singlet oxygen) were involved in the oxidation of butter stored in air. The study shows that PARAFAC modeling of fluorescence landscapes is an excellent tool for studying photooxidation in complex systems.

Biological and biophysical properties of the tumor-localizing component of hematoporphyrin derivative.

Reverse-phase chromatography, aqueous gel exclusion, and nonaqueous gel exclusion were assessed as procedures for preparative fractionation of the tumor-localizing product hematoporphyrin derivative. Porphyrin accumulation, fluorescence, and photodynamic cytotoxicity were monitored using the murine Sarcoma 180 tumor. Aqueous gel exclusion chromatography can provide a hematoporphyrin derivative fraction enriched in the tumor-localizing component. A further enrichment occurs when this procedure is carried out at 55 degrees C, but nonlocalizing porphyrins could not be eliminated. While providing a better separation, reverse-phase chromatography cannot provide a tumor-localizing fraction free from contaminating protoporphyrin. However, this and other contaminants can be eliminated from the tumor-localizing fraction via nonaqueous gel exclusion chromatography. This latter separation provides two tumor-localizing products: a fast-eluting fraction enriched in the major photosensitizing component(s); and a more complex slowly eluting fraction enriched in fluorescence localizers.

Biological studies on the main fractions of hematoporphyrin derivative.

The four main fractions of hematoporphyrin derivative were separated by high-pressure liquid chromatography. Each fraction was studied with respect to photosensitizing capabilities, fluorescence, and tumor tissue uptake in mice bearing EMT-6 tumors. Animals received i.p. injections of 10 mg/kg of each fraction, and 24 h later tumors either were treated with 100 J/cm2 of light (630 nm) to evaluate photosensitizing capabilities, or the animals were sacrificed and tumors removed for fluorescence and fraction uptake determination. The results indicate that the fraction responsible for photosensitization has the highest tumor tissue uptake and retention. Furthermore, this fraction demonstrates the highest overall fluorescence localization in neoplastic tissue. The other poorly photosensitizing fractions have a lower overall fluorescence in vivo due to their poor tumor tissue localization.

Transport and binding of hematoporphyrin derivative and related porphyrins by murine leukemia L1210 cells.

A hematoporphyrin derivative (abbreviated in the literature as "HPD") has been used successfully for phototherapy of tumors in the clinic. The chemical nature of HPD, a complex mixture of porphyrins, is not fully understood. This study was designed to provide an explanation for the superior tumor-localizing ability of HPD. Chromatographic behavior, hydrophobicity, transport, binding, and photosensitizing capacity of different porphyrins were examined and compared. Biological studies were carried out using murine leukemia L1210 cells in vitro. The initial rate of porphyrin uptake was a function of drug hydrophobicity. The most hydrophobic components of HPD were therefore the most potent photosensitizers when irradiation followed a 10-min porphyrin-loading incubation. But these and other hydrophobic porphyrins were readily washed from cells by medium containing serum. Hydrophilic components of HPD were gradually accumulated by L1210 cells via a mode of binding not readily dissociated by washing and appear to be responsible for the preferential affinity of this product for neoplastic cells. A portion of the tightly bound porphyrin could not be dissociated by sodium dodecyl sulfate:polyacrylamide gel electrophoresis but remained bound to a low-molecular weight membrane component.

Components of hematoporphyrin derivatives and their tumor-localizing capacity.

Hematoporphyrin derivative (HPD), a complex mixture of porphyrins derived from hematoporphyrin, has been used for localization and photoradiation therapy of tumors. In this report, we describe characterization of HPD and of its component porphyrins by reverse-phase thin-layer chromatography. Uptake of major HPD components by leukemia L1210 cells in vitro and by the Sarcoma 180 tumor in vivo were also examined. These data suggest that the apparent photosensitization of intact cells mediated by hematoporphyrin was associated with the uptake of more hydrophobic porphyrins present as impurities. In the Sarcoma 180 tumor, the fluorescent porphyrin persisting for two days in vivo after HPD administration was found to migrate with hematoporphyrin in a reverse-phase thin-layer chromatography system. Our results suggest that hematoporphyrin may be unable to cross the cell membrane readily in either direction and that long-persisting fluorescence resulting from exposure of tumor tissues to HPD results from transformation of a membrane-permeable HPD component to hematoporphyrin.

Tumor-localizing components of the porphyrin preparation hematoporphyrin derivative.

Synthetic and analytical approaches were used to characterize the tumor-localizing components of the porphyrin preparation, hematoporphyrin derivative. From studies involving aqueous and nonaqueous gel exclusion and reverse-phase chromatography, we conclude that localization is mediated by hematoporphyrin derivative components which are among the most hydrophobic in the preparation. This apparent hydrophobicity may derive from hydrogen-bonding phenomena, rather than from absence of hydrophilic functional groups.

Photodynamic effect in an experimental bladder tumor treated with intratumor injection of hematoporphyrin derivative.

Photodynamic therapy (PDT) is an experimental treatment modality for malignant tumors. It is based on the principle that a photosensitizer, such as hematoporphyrin derivative (HPD), is retained in higher concentrations in tumors than in surrounding nonmalignant tissues and that photoactivation of the sensitizer can be used to evoke tumor destruction. However, retention of the systemic injection of HPD is not limited to malignant tissues. This lack of specific tumor localization thus reduces the therapeutic ratio of the treatment and causes skin photosensitivity and possible systemic toxicity. Injection of HPD directly into the tumor, on the other hand, has been shown to yield higher levels of the drug in the tumor and lower levels in normal tissues, in comparison with systemic administration. In this study, we examined the photodynamic effect on s.c. implanted mouse bladder tumors subjected to intratumor (i.t.) and i.p. HPD injections. Tumor cell killing, measured by cell survival, was observed in both the it. and i.p. groups and was dependent on fluence and HPD dosage. However, no significant enhancement of cell killing was observed in the i.t. injected tumors, despite the higher porphyrin levels in these tumors. Histological examination of the effect of PDT on the blood vessels indicated that while cell death accompanied severe hemorrhage in the i.p. injected tumors, in the i.t. tumors there was much less hemorrhage and intact blood vessels remained. This observation suggests that with i.t. administration, direct photodynamic action may play a significant role in the tumor cell killing, in contrast to systemic administration, in which destruction of the blood vessels is believed to be the main cause of tumor destruction.

Tumor-localizing and photosensitizing properties of the main components of hematoporphyrin derivative.

Both 3H-labeled and unlabeled hematoporphyrin derivative (HPD) were analyzed by high-pressure liquid chromatography (HPLC) and gel permeation chromatography. Four main components were isolated by HPLC, and two were isolated by gel permeation chromatography. The tumor-localizing ability of each component was tested and compared to that of 67Ga and 3H2O by injection in mice bearing Lewis lung carcinoma. The photosensitizing abilities of the HPLC-separated components in vitro were also tested. Finally, porphyrin extracts of tumors from mice given HPD were analyzed by HPLC. The tumor-localizing ability of the components increased with decreasing polarity. While crude HPD localized in tumor tissue only to the same extent as did 3H2O, Component 7 was almost as effective as was 67Ga in localizing in the tumor. The cellular uptake of HPD components increased with decreasing polarity. In accordance with this, the low-polarity components were the most effective photosensitizers.

Raman microspectroscopy of Hematoporphyrins. Imaging of the noncancerous and the cancerous human breast tissues with photosensitizers.

Raman microspectroscopy combined with fluorescence were used to study the distribution of Hematoporphyrin (Hp) in noncancerous and cancerous breast tissues. The results demonstrate the ability of Raman spectroscopy to distinguish between noncancerous and cancerous human breast tissue and to identify differences in the distribution and photodegradation of Hematoporphyrin, which is a photosensitizer in photodynamic therapy (PDT), photodynamic diagnosis (PDD) and photoimmunotherapy (PIT) of cancer. Presented results show that Hematoporphyrin level in the noncancerous breast tissue is lower compared to the cancerous one. We have proved also that the Raman intensity of lipids and proteins doesn't change dramatically after laser light irradiation, which indicates that the PDT treatment destroys preferably cancer cells, in which the photosensitizer is accumulated. The specific subcellular localization of photosensitizer for breast tissues samples soaked with Hematoporphyrin was not observed.

On the nature of the active component of haematoporphyrin derivative--Part 2.

It has been suggested recently that neither an ester nor an ether linkage was present in the tumour-localising component of Photofrin II (PFII) (a commercial preparation of DHE). We now report that our chemical and spectroscopic studies, establish the presence of an ester linkage with the possibility that an ether linkage may also be involved.

The main photosensitizing components of hematoporphyrin derivative.

Commercial hematoporphyrin (Hp) and the tumor-localizing and photosensitizing agent hematoporphyrin derivative (Hpd) were analysed by means of high pressure lipid chromatography (HPLC). Furthermore, their efficiencies in sensitizing the photoinactivation of human cells in vitro were compared. The comparison showed that the least polar components of Hpd played the major role in this sensitization. In Hpd solutions used for injection in photochemotherapeutic treatment of cancer, these active components seem to be present as aggregates.

Observations on the chemical structure and cytotoxic activity of marycin, a hematoporphyrin derivative.

This paper describes the preparation, chemical structure and cytotoxic activity of marycin, a hematoporphyrin derivative. Marycin has been prepared by condensing hematoporphyrin dimethyl ester in the presence of p-toluenesulfonic acid and reducing the product with lithium aluminum hydride. The product appeared to be pure by thin-layer chromatography (TLC) and high-performance liquid chormatography (HPLC). The product, analyzed by UV-visible absorbance and fluorescence spectra, appears to be related to the parent hematoporphyrin compound. The product was also analyzed by NMR and Mass spectra: a dimeric structure can be assigned to marycin: this appears to have an oxide bridge between C2-chains of two porphyrin units and hydroxyl groups instead of carboxyls. Marycin was screened for cytotoxic activity against ZR-75, MCF-7, HT-29, K-562, human tumor cell lines and the MRC-9 human embryonic cell line. Marycin decreases the growth index, measured in the radiometric assay, as 14CO2 production. The cytotoxic activity was dose-dependent and is attributable to the pure compound, marycin. Marycin is active at low doses but the activity varies with the cell line studied. The compound had low toxicity versus MRC-9 normal cell line. The compound is active without light activation. How marycin acts is a matter of speculation. Marycin is highly liposoluble and would be expected to have high toxicity for tumors.

Localization of potent photosensitizers in human tumor LOX by means of laser scanning microscopy.

By means of laser scanning fluorescence microscopy the intratumoral localization patterns of several photosensitizers in LOX tumors in nude mice were studied. Lipophilic dyes such as P-II (Photofrin II), 3-THPP tetra(3-hydroxyphenyl)porphin, TPPS1 (tetraphenylporphine monosulfonate), TPPS2a (tetraphenylporphine disulfonates with the sulfonate groups on adjacent rings), A1PCS1 (aluminium phthalocyanine monosulfonate) and A1PCS2 (aluminium phthalocyanine disulfonates) localized mainly in tumor cells. The fluorescence intensity of these dyes increased from 4 h to 48 h post-injection and the fluorescence was still observable 120 h post-injection. The more hydrophilic dyes such as TPPS2o (tetraphenylporphine disulfonates with the sulfonates groups on opposite rings), TPPS3 (tetraphenylporphine trisulfoantes), TPPS4 (tetraphenylporphine tetrasulfonates), A1PCS3 (aluminium phthalocyanine trisulfonates) and A1PCS4 (aluminium phthalocyanine tetrasulfonates) localized mainly extracellularly in the tumorous stroma. The fluorescence intensity of these dyes decreased from 4 h to 48 h post-injection. 120 h post-injection no significant fluorescence of these dyes could be seen in the tumors. The data are discussed in relation to what is known about the in vivo photosensitizing efficiency of some of the dyes.

Localization of potent photosensitizers in human tumor LOX by means of laser scanning microscopy.

By means of laser scanning fluorescence microscopy the intratumoral localization patterns of several photosensitizers in LOX tumors in nude mice were studied. Lipophilic dyes such as TPPS1 (tetraphenylporphine monosulfonate), TPPS2a (tetraphenylporphine disulfonates with the sulfonate groups on adjacent rings), AlPCS1 (aluminium phthalocyanine monosulfonate) and AlPCS2 (aluminium phthalocyanine disulfonates) localized mainly in tumor cells. The fluorescence intensity of these dyes increased from 4 h to 48 h postinjection and the fluorescence was still observable 120 h postinjection. The more hydrophilic dyes such as TPPS3 (tetraphenylporphine trisulfonates), TPPS4 (tetraphenylporphine tetrasulfonates), and AlPCS4 (aluminium phthalocyanine tetrasulfonates) localized mainly extracellularly in the tumorous stroma. The fluorescence intensity of these dyes decreased from 4 h to 48 h postinjection. 120 h postinjection no significant fluorescence of these dyes could be seen in the tumors. P-II (Photofrin II), 3-THPP [tetra(3-hydroxyphenyl)porphine], TPPS2o (tetraphenylporphine disulfonates with the sulfonate groups on opposite rings) and AlPCS3 (aluminum phthalocyanine trisulfonates) had a combined localization pattern, i.e. a strongly cytoplasmic membrane-localizing pattern and a weakly intracellular distribution pattern, although some fluorescence could be seen in the tumorous stroma. The data are discussed in relation to what is known about the in vivo photosensitizing efficiency of some of the dyes.

Location of P-II and AlPCS4 in human tumor LOX in vitro and in vivo by means of computer-enhanced video fluorescence microscopy.

The patterns of in vitro intracellular and in vivo intratumoral localization of Photofrin II (P-II) and aluminum phthalocyanine tetrasulfonate (AlPCS4) in human melanoma LOX were studied by means of computer-enhanced video fluorescence microscopy (CEVFM). The hydrophobic drug P-II localized diffusely in the perinuclear fraction of the cytoplasm of the LOX cells cultivated in vitro. Light exposure did not result in any observable change in the localization pattern. The hydrophilic dye AlPCS4 was distributed as granular and grain patterns in the cytoplasm before light exposure, in exactly the same pattern as that of acridine orange incubated in the same cells, which is known to emit red fluorescence from lysosomes, thus indicating that AlPCS4 was also primarily localized in the lysosomes of the LOX cells. After light exposure the distribution of the intracellular AlPCS4 fluorescence was altered and the intensity increased. In vivo, P-II had a combined cellular localization pattern (i.e. a strongly cytoplasmic membrane-localizing pattern and a weakly intracellular distribution pattern) and an extracellular distribution pattern in the tumor tissue, while the AlPCS4 fluorescence was seen mainly in the stroma of the tumor. The total fluorescence intensity of P-II and AlPCS4 in the LOX tumor tissue at different times after injection was quantitatively determined by means of CEVFM.

Targetted phototherapy with sensitizer-monoclonal antibody conjugate and light.

Photodynamic therapy (PDT) was performed in vitro and in vivo using monoclonal antibody conjugated to hematoporphyrin (HP). The antibody (45-2D9) recognized a cell surface glycoprotein on cells derived from NIH 3T3 cells which were transformed with the ras oncogene (45-342). Radionuclide imaging with either In111 or I125 chelated to 45-2D9 or the isotype identical (IgG1) antibody MOPPC-21 revealed selectivity of 45-2D9 for 45-342 flank tumours in nude mice, and minimal targetting for a 45-342 clone which did not express the cell surface glycoprotein. The 45-2D9-HP conjugate resulted in selective killing of the 45-342 line compared with the parent line in vitro. At HP concentrations of 76 micrograms ml-1, the 45-2D9-HP conjugate resulted in significantly more long-term cures of PDT treated flank tumours compared with free HP at the same concentration. 45-2D9 alone had no effect on tumour growth. The antibody-HP conjugate resulted in significantly less local toxicity compared with standard Photofrin II PDT, and also achieved a greater number of long-term cures. This 'photoimmunotherapy' demonstrates the ability to treat established tumours with greater efficacy and decreased morbidity, probably due to specific sensitizer targetting which allows normal surrounding tissue to be spared upon illumination.