两种荧光方法分析血样中锌原卟啉和原卟啉

利用N,N-二甲基甲酰胺同时萃取血样中痕量的锌原卟啉和原卟啉,以三维荧光-交替三线性分解算法和导数恒基体同步荧光法同时分析血样中的锌原卟啉和原卟啉.前者中锌原卟啉和原卟啉分别在0.6～35 μg/L和0.4～27 μg/L范围内呈良好的线性,检出限分别为0.18和 0.12 μg/L;后者中锌原卟啉和原卟啉在0.14～45 μg/L和0.056～28.5 μg/L范围内呈良好的线性,检出限分别为0.12和 0.045 μg/L.实际血样中两种方法的平均回收率分别为(82±9)%和(85±10)%,且两种方法测定20份血样时获得的相关性良好.     

Nanocarbon Materials Modified with the Zinc Complex of Protoporphyrin IX,Recognized Antibiotics in Water Samples

The complex between protoporphyrin IX and zinc was immobilized on nanocarbon paste and on nanodiamond paste to design two stochastic microsensors. The microsensors were used for the recognition and analysis of antibiotics: amoxicillin, ampicillin, and biotin in water samples. Stochastic sensors provided different signatures for the three antibiotics making possible their simultaneous recognition and assay in water samples. Low limits of determination 0.3 pg/mL for amoxicillin and ampicillin, and 0.21 pg/mL for biotin were obtained when nanocarbon paste was used, and 0.03 pg/mL for amoxicillin, 0.30 pg/mL for ampicillin, and 2.14 fg/mL for biotin were obtained when nanodiamond paste was used. Recoveries higher than 99.32 % with RSD lower than 1.00 % were obtained for the assay of the antibiotics in water samples.     

The Temperature Dependence of Protoporphyrin IX Production in Cells and Tissues

The formation of protoporphyrin IX (PpIX) in human skin during topical application of 5-aminolevulinic acid (ALA) was found to be strongly temperature dependent, with an activation energy of about 17 kcal/mol. This temperature dependence is mainly related to porphyrin production and not to ALA penetration into the skin. The penetration of ALA into mouse and human skin was almost temperature independent. The activation energy of PpIX production in mouse skin was practically identical with that in human skin. The activation energy of ALA uptake by cells in vitro was about 10 kcal/mol and that for PpIX production was about 13 kcal/mol. The latter activation energy was within the error limits similar to that for the activity of the enzyme porphobilinogen deaminase, suggesting that this enzyme might represent a rate-limiting step for PpIX production in living tissue.     

Depth Profile of Protoporphyrin IX Fluorescence in an Amelanotic Mouse Melanoma Model

Protoporphyrin IX (PpIX) fluorescence was measured at different depths in a subcutaneous amelanotic melanoma model (LOX) in mice. PpIX was induced by topical application of 5‐aminolevulinic acid (ALA) and two of its derivatives, the methylester (MAL) and hexylester (HAL) onto the normal skin covering the tumor. The PpIX fluorescence intensity on the surface of the tumors was the highest for HAL, followed by ALA and MAL. Using equimolar concentrations (0.5 mmol g^?1), HAL induced nearly twice as much fluorescence as ALA did. The depth profile of PpIX fluorescence was measured at different layers of the tumor, which was carefully sliced and controlled in situ ex vivo. The PpIX fluorescence was mainly localized within the upper 2 mm of the tissue for ALA and within 1 mm for MAL and HAL. There were no significant differences in the shape of the fluorescence excitation spectra, but the long wavelength excitation peak (633 nm) was so weak that these results are unreliable for depth estimation. When considering the low fluorescence intensity (around 5% of the intensity at the tumor surface), the actual penetration depth of HAL was comparable to that of ALA. The fluorescence after topical application of ALA and HAL was significantly above the background level down to a depth of around 6 mm, and there were traces of PpIX fluorescence even at the tumor base (10 mm). The fluorescence after topical application of MAL was detectable down to 1 mm. In the depth of 2–6 mm, the fluorescence was slightly higher for HAL than for ALA. Using the estimated diffusion coefficients for topically applied ALA (0.16 ± 0.03 mm² h^?1), MAL (0.045 ± 0.005 mm² h^?1) and HAL (0.037 ± 0.003 mm² h^?1), the behavior of the drugs after different application times could be estimated in this tumor model.     

5-Aminolevulinic Acid-induced Protoporphyrin IX Levels in Tissue of Human Malignant Brain Tumors

Protoporphyrin IX (PpIX) produced from exogenous, orally administered 5-aminolevulinic acid (ALA) displays high tumor-selective uptake and is being successfully employed for fluorescence-guided resection (FGR) of human malignant gliomas. Furthermore, the phototoxicity of PpIX can be utilized for photodynamic therapy (PDT) of brain tumors, which has been shown previously. Here, the absolute PpIX concentration in human brain tissue was investigated following oral ALA administration (20 mg kg⁻¹ b.w.). An extraction procedure was used to quantify PpIX in macroscopic tissue samples, weighing 0.013–0.214 g, obtained during FGR. The PpIX concentration was significantly higher in vital grade IV tumors (5.8 ± 4.8 μm , mean ± SD, range 0–28.2 μm , n = 8) as compared with grade III tumors (0.2 ± 0.4 μm , mean ± SD, range 0–0.9 μm , n = 4). There was also a large heterogeneity within grade IV tumors with PpIX displaying significantly lower levels in infiltration zones and necrotic regions as compared with vital tumor parts. The average PpIX concentration in vital grade IV tumor parts was in the range previously shown sufficient for PDT-induced tissue damage following irradiation. However, the feasibility of PDT for grade III brain tumors and for grade IV brain tumors displaying mainly necrotic tissue areas without solid tumor parts needs to be further investigated.     

Protoporphyrin IX fluorescence photobleaching during ALA-mediated photodynamic therapy of UVB-induced tumors in hairless mouse skin

Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB-induced tumor tissue in the SKH-HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT-induced damage were examined. Results show that the rate of PpIX photobleaching (i.e., fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB-induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB-induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.     

Complex Formation of Ferric Protoporphyrin IX From the Reaction of Hemin with Ammonia and Small Aliphatic Amines

Complexes of Fe^III protoporphyrin IX (Fe^IIIPPIX) with the amido anion were obtained from the reaction of Fe^IIIPPIX chloride (hemin) with ammonia and small aliphatic amines under solvent free conditions. The reaction of hemin with gaseous ammonia leads to a pentacoordinated complex at the iron site, PPIX–Fe–NH₂, plus NH₄Cl, while at the peripheral propionic acidic groups ammonium carboxylate is formed. The corresponding stoichiometry (1:4 molar ratio of hemin to ammonia) was confirmed by the adsorption isotherm. Analogous reactions and complex formation were observed with EtNH₂ and Et₂NH. These reactions were monitored using X-ray diffraction (XRD), and i.r. and Mössbauer spectroscopies. The isomer shift and quadrupole splitting values of the resulting complexes are in correspondence with the strong -donor character of the amido anion linked to the iron atom. For comparison, the Mössbauer parameters for hemin complexes with arginine and 2-aminoguanidine, which also have pure interaction with the porphyrin iron, were included and discussed.     

Hyperfine Shifts of the 13C-NMR. in Protoporphyrin IX Iron (III) Dicyanide and Deuteroporphyrin IX Iron (III) Dicyanide

The ¹³C-NMR. in Zn(II) (Protoporphyrin IX), Fe(III) (Protoporphrin IX) (CN)₂, Zn(II) (Deuteroporphyrin IX dimethylester), and Fe(III) (Deuteroporphyrin IX) (CN)₂ have been identified, and the ¹³C hyperfine shifts in the iron complexes evaluated. In a partial analysis of these data the parameters Q^H, Q_CCH^H, and Q_C′CH^C, which characterize the isotropic coupling through hyperconjugation between the ¹H and ¹³C nuclei of the porphyrin side chains and the unpaired electron spin density on the aromatic ring carbon atoms, have been re-examined. This paper is part of an investigation of the electronic states in low spin ferric hemes and hemoproteins, and the relations between the electronic structures and the biological roles of these molecules.     

Growth rate and chromosome number of tumor cell lines with different metastatic potential

We investigated whether the metastatic potential of various tumor cell lines was related to chromosome counts or to rate of growth in vitro or in vivo. Clones of known metastatic potential derived from a C3H- fibrosarcoma induced by UV radiation (UV-2237) and from C57BL/6 B16 melanoma were tested for these characteristics. No correlation was found between the growth rate of these clones in monolayer culture or at a subcutaneous site and their ability to produce metastases. The cells from clones of UV-2237 were mainly in the diploid range with only one exception, and the B16 clones were all hyperploid. Thus, there was also no correlation between malignant behavior of the clones and gross changes in chromosome number.     

Detection of Early Stages of Carcinogenesis in Adenomas of Murine Lung by 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence

Abstract— Administration of the heme precursor 5-aminolevulinic acid (ALA) leads to the selective accumulation of the photosensitizer protoporphyrin IX (PpIX) in certain types of normal and abnormal tissues. This phenomenon has been exploited clinically for detection and treatment of a variety of malignant and nonmalignant lesions. The present preclinical study examined the specificity of ALA-induced porphyrin fluorescence in chemically induced murine lung tumors in vivo. During the early stages of tumorigenesis, ALA-induced PpIX fluorescence developed in hyperplastic tissues in the lung and later in early lung tumor foci. In early tumor foci, maximum PpIX fluorescence occurred 2 h after the administration of ALA and returned to background levels after 4 h. There was approximately a 20-fold difference in PpIX fluorescence intensity between tumor foci and the adjacent normal tissue. The specificity of ALA-induced fluorescence for hyperplastic tissues and benign tumors in lung during tumorigenesis suggests a possible use for this fluorochrome in the detection of premalignant alterations in the lung by fluorescence endoscopy. Two non-small cell lung cancer cell lines developed ALA-induced PpIX fluorescence in vitro . These lines exhibited a light-dose-dependent phototoxic response to ALA photodynamic therapy (PDT) in vitro . Because PpIX is a clinically effective photosensitizer for a wide variety of malignancies, these results support the possible use of ALA-induced PpIX PDT for lung cancer.     

Kinetics of Photobleaching of Protoporphyrin IX in the Skin of Nude Mice Exposed to Different Fluence Rates of Red Light

Abstract— The purpose of the present study was to determine the kinetics and the fluence rate dependency of the photo-bleaching of protoporphyrin IX (PpIX) in normal skin of Balb/c nude mice after systemic and topical application of 5-aminolevulinic acid (ALA). ALA was administered systemically (200 mg/kg body weight, i.p.) and topically (20% w/w ALA cream) to the mice. Fluences of up to 40 J/cm² were delivered by a dye laser (636 nm) at fluence rates of 37.5, 75, 150, 300 and 500 mW/cm². The photo-bleaching rate was constant within this range of fluence rates. This result suggests that there is no oxygen effect for PpIX photobleaching in this region for the skin of Balb/c nude mice. During light exposure the fluorescence decay followed neither first- nor second-order kinetics. The decay rate was slightly faster after systemic application than after topical application of ALA, but did not depend on the time (1–8 h) between application and analysis.     

The effects of 5-aminolevulinic acid esters on protoporphyrin IX production in human adenocarcinoma cell lines.

Photodynamic diagnosis (PDD) and photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) is an interesting approach to detect and treat dysplasia and early cancers in the gastrointestinal tract. Because of low lipophilicity resulting in poor penetration across cell membranes, high doses of ALA should be administered in order to reach clinically relevant levels of PPIX. One way of increasing PPIX accumulation is derivatization of ALA into a more lipophilic molecule. In our in vitro study, different esterifications of ALA were investigated to analyze the effects on PPIX accumulation in human adenocarcinoma cell lines. For systematic analysis of cell type-specific PPIX accumulation, three human adenocarcinoma cell lines (SW480, HT29 and CaCo2) and a fibroblast cell line (CCD18) were tested. 3-(4,5-Dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) assays were performed to ensure that the ALA esters showed no cellular dark toxicity. Different concentrations (ranging from 0.012 to 0.6 mmol/L, 3 h) and incubation times (5, 10, 30, 180 min; 0.12 mmol/L) were examined. PPIX accumulation was measured using flow cytometry. ALA esters, especially ALA-hexylester and ALA-benzylester, induced significant higher PPIX levels in adenocarcinoma cell lines when compared with ALA and may be promising candidates for PDT and PDD.     

Protoporphyrin IX fluorescence kinetics in UV-induced tumours and normal skin of hairless mice after topical application of 5-aminolevulinic acid methyl ester

Accumulation of protoporphyrin IX (PpIX) was investigated in normal skin and UV-induced tumours in hairless mice after topical application of a cream containing 2, 8 or 16% of 5-aminolevulinic acid methyl ester (ALA-Me). Higher levels of PpIX were measured in tumours compared to normal skin. The maximal amount of PpIX was reached at 1.5, 3 and 4 h after 2, 8 and 16% ALA-Me application, respectively. Higher tumour to normal skin PpIX fluorescence ratios were measured after application of 8 and 16% ALA-Me than after application of 2%. After irradiation with a broad spectrum of visible light from a slide projector, more than 90% of PpIX was bleached by fluences of 36 and 48 J/cm2, at fluence rates of 10 and 40 mW/cm2 respectively. At these fluences, the PpIX photobleaching rate was significantly higher (P<0.05) in normal mouse skin than in tumours. In addition, for a given fluence, more PpIX was photobleached at the lower fluence rate (10 mW/cm2) than at the higher fluence rate (40 mW/cm2) in normal skin (P<0.001) as well as in tumours (P<0.05) after exposure to 24 J/cm2 of light. In conclusion, the highest tumour to normal skin PpIX ratio was observed 3 h after application of 8% ALA-Me, suggesting that light exposure should be performed at this time in order to achieve an optimal PDT effect in this tumour model.     

Protoporphyrin IX fluorescence kinetics and localization after topical application of ALA pentyl ester and ALA on hairless mouse skin with UVB-induced early skin cancer

In order to improve the efficacy of 5-aminolevulinic acid-based (ALA) photodynamic therapy (PDT), different ALA derivatives are presently being investigated. ALA esters are more lipophilic and therefore may have better skin penetration properties than ALA, possibly resulting in enhanced protoporphyrin IX (PpIX) production. In previous studies it was shown that ALA pentyl ester (ALAPE) does considerably enhance the PpIX production in cells in vitro compared with ALA. We investigated the in vivo PpIX fluorescence kinetics after application of ALA and ALAPE to hairless mice with and without UVB-induced early skin cancer. ALA and ALAPE (20% wt/wt) were applied topically to the mouse skin and after 30 min, the solvent was wiped off and PpIX fluorescence was followed in time with in vivo fluorescence spectroscopy and imaging. At 6 and 12 h after the 30 min application, skin samples of visible lesions and adjacent altered skin (UVB-exposed mouse skin) and normal mouse skin were collected for fluorescence microscopy. From each sample, frozen sections were made and phase contrast images and fluorescence images were recorded. The in vivo fluorescence kinetics showed that ALAPE induced more PpIX in visible lesions and altered skin of the UVB-exposed mouse skin, but not in the normal mouse skin. In the microscopic fluorescence images, higher ALAPE-induced PpIX levels were measured in the stratum corneum, but not in the dysplastic layer of the epidermis. In deeper layers of the skin, PpIX levels were the same after ALA and ALAPE application. In conclusion, ALAPE does induce higher PpIX fluorescence levels in vivo in our early skin cancer model, but these higher PpIX levels are not located in the dysplastic layer of the epidermis.     

Characterization of Endogenous Protoporphyrin IX Induced by δ-Aminolevulinic Acid in Resting and Activated Peripheral Blood Lymphocytes by Four-Color Flow Cytometry

Lymphocytes treated with δ-aminolevulinic acid (ALA) can accumulate the photoactive, fluorescent heme precursor, protoporphyrin IX (PpIX). With visible light illumination, PpIX can be used in photodynamic therapy (ALA-PDT) to kill or functionally alter cells. The aim of this study was to characterize the effects of ALA and ALA-PDT on resting and activated human peripheral blood T lymphocytes. Accumulation of PpIX depends inversely on the rate of its iron-dependent conversion into heme. Activated, replicating lymphocytes have low intracellular iron levels, with corresponding increases in the transferrin receptor (CD71). Thus, we expected activated lymphocytes would preferentially accumulate PpIX. Using four-color flow cytometry, we examined ALA-induced PpIX levels in T-cell subsets of resting and activated human peripheral blood mononuclear cells and the relationship between CD71 and PpIX. Peripheral blood mononuclear cells stimulated by phytohemagglutinin (PHA) were simultaneously phenotyped for PpIX, CD71 and the T-cell markers CD3 and CD4 or CDS. In activated cells treated with 0-6mM ALA for 4 h, PpIX fluorescence was maximal at 1 mM ALA. On a single cell basis, there was a strong correlation between PpIX ac-cumulation and CD71 expression. The ALA-treated, PHA-stimulated, CD71⁺ lymphocytes had an eight-fold greater mean PpIX fluorescence than nonactivated, CD71- cells. Approximately 87% of the CD4* and 85% of the CD8⁺ T cells accumulated PpIX. The PpIX levels of CDS⁺ cells were about 5% greater than CD4⁺ cells. In addition, mixed lymphocyte reaction-stimulated cells treated with ALA accumulated more PpIX than controls. Thus, activated cells preferentially accumulate endogenous PpIX when exogenous ALA is administered. Cytotoxicity studies showed that the majority of the activated cells following ALA-PDT were killed but resting cells were spared. Also, in examining activation markers by flow cytometry the number of cells that were positive for activation markers CD38 or CD71 dramatically decreased after ALA and light treatment in activated populations. The data suggest a role for ALA-PDT as an immunomodulator or photocytotoxic agent targeting activated lymphocytes.     

How to affect number, size, and location of metal particles deposited in conducting polymer layers

This paper presents a review on a series of recent investigations focused on the understanding of the role of various factors for the number, size, and location of the metal particles electrodeposited in conducting polymer (CP) layers. It is demonstrated that the initial oxidation state of the CP layer and its surface and bulk structure play an important role for the location of the metal particles. The use of metal anion complexes instead of the corresponding metal cations presents a helpful tool for affecting the location and number of metal crystals. The involvement of special metal/polymer interactions in the metal electrocrystallization process is another way for influencing the metal deposit. An alternative to the electrodriven deposition is the electroless metal precipitation based on the reducing ability of the CP layers. This approach results in metal particles deposition at the polymer surface and may be effectively controlled through parameters such as CP reduction charge, dipping time, and concentration of the metal-plating solution.     

Biodistribution of Photofrin II® and 5-Aminolevulinic Acid-Induced Protoporphyrin IX in Normal Rat Bladder and Bladder Tumor Models: Implications for Photodynamic Therapy

Photodynamic therapy (PDT) has been considered as a potential therapy for superficial bladder carcinomas. Cutaneous photosensitivity and reduction of bladder capacity are the two well-known complications following systemic administration of the commonly used photosensitizer, Photofrin II® (PII). The objective of the present study was to evaluate whether intravesical. (i.b.) instillation of photosensitizers for PDT of bladder cancer might be a more suitable treatment method. Female Fischer rats were utilized to develop orthotopic and heterotopic bladder tumor models. Rats bearing orthotopic bladder tumors were treated either intravesically or intravenously with graded doses of 5-aminolevulinic acid (ALA) or PII. Normal rats received the same doses of ALA or PII. As well, rats bearing heterotopic tumor were studied for comparison. The biodistribution times (times allowed for tissue uptake and bioconversion following drug administration) were 2, 4 or 6 h. Porphyrin fluorescence intensities within tumor, urothelium, submucosa, bladder muscularis and abdominal muscle were quantitated by confocal laser scanning microscopy. Following intravenous (i.v.) injection of ALA, tumor protoporphyrin IX (PpIX) levels peaked at 4 h and diminished by 6 h. The PpIX ratios of tumor-to-bladder mucosa, submucosa and muscle layers were 3:1, 5:1 and 8:1, respectively, 4 h following 1000 mg/kg ALA injection. After ALA instillation, the optimal biodistribution time appeared to be 4 h. Bladder instillation provided comparable tumor labeling with the i.v. route, but lost selectivity of PpIX accumulation between tumor and normal urothelium. The PpIX ratio of tumor-to-bladder muscularis was 5:1. After i.b. instillation of PII, porphyrin fluorescence was detected only within tumor and urothelium, while porphyrin fluorescence was mainly located in bladder submucosa following i.v. injection. Intravesical administration of ALA or PII might be feasible for PDT of superficial bladder cancers.     

Sensitivity increase in molecular recognition by decrease of the sensing particle size and by increase of the receptor binding site--a case with chemomechanical polymers

For the first time it is shown that diminishing the particle size of a chemomechanical polymer leads to a dramatic sensitivity increase, with a large response triggered e.g. by action of external AMP; as illustrated with carbohydrate model complexes in solution, the necessary high binding affinity can be achieved by providing an excess of recognition units in one of the partners.     

Salmonella-host cell interactions, changes in host cell architecture, and destruction of prostate tumor cells with genetically altered Salmonella.

Increasingly, genetically modified Salmonella are being explored as a novel treatment for cancer because Salmonella preferentially replicate within tumors and destroy cancer cells without causing the septic shock that is typically associated with wild-type S. typhimurium infections. However, the mechanisms by which genetically modified Salmonella strains preferentially invade cancer cells have not yet been addressed in cellular detail. Here we present data that show S. typhimurium strains VNP20009, LT2, and CRC1674 invasion of PC-3M prostate cancer cells. S. typhimurium-infected PC-3M human prostate cancer cells were analyzed with immunofluorescence microscopy and transmission electron microscopy (TEM) at various times after inoculation. We analyzed microfilaments, microtubules, and DNA with fluorescence and immunofluorescence microscopy. 3T3 Phi-Yellow-mitochondria mouse 3T3 cells were used to study the effects of Salmonella infestation on mitochondria distribution in live cells. Our TEM results show gradual destruction of mitochondria within the PC-3M prostate cancer cells with complete loss of cristae at 8 h after inoculation. The fluorescence intensity in YFP-mitochondria-transfected mouse 3T3 cells decreased, which indicates loss of mitochondria structure. Interestingly, the nucleus does not appear affected by Salmonella within 8 h. Our data demonstrate that genetically modified S. typhimurium destroy PC-3M prostate cancer cells, perhaps by preferential destruction of mitochondria.