The effect of dimethylsulfoxide, 1-[2-(decylthio)ethyl]azacyclopentan-2-one and LabrafacCC on porphyrin formation in normal mouse skin during topical application of methyl 5-aminolevulinate: A fluorescence and extraction study

In this work, the effect of 10% of dimethylsulfoxide (DMSO), 1-[2-(decylthio)ethyl]azacyclopentan-2-one (HPE-101) and Labrafac^®CC (a mixture of caprylic and capric acid triglycerides) on porphyrin formation in mouse skin during topical application of methyl 5-aminolevulinate (MAL) was studied. The porphyrin level in mouse skin was determined by measuring directly fluorescence and by extraction method.The porphyrin fluorescence kinetics during continuous application of MAL in creams in concentrations 2, 10 and 20% (wt./wt.) for up to 7 h showed that in this concentration range the kinetics of porphyrin production in the site of application does not depend significantly on the MAL concentration. After 24 h of application of all studied creams the porphyrin fluorescence in the area of treatment was dramatically reduced to be about two-fold higher than the skin autofluorescence, suggesting a significant decrease of the porphyrin concentration in these sites, although in all cases traces of porphyrins were found.It was found by extraction method that a 10% MAL cream with 10% DMSO for 4 h increased the concentration of porphyrin about four-fold compared with 10% MAL cream alone. The presence of 10% HPE-101 or 10% Labrafac^®CC in the 10% MAL cream increased the porphyrin concentration in the area of application about 2.5- and 2-fold, respectively, as compared with MAL cream without enhancers. No statistically significant difference was found between the effects of the creams containing 10% MAL with 10% HPE-101 or 10% Labrafac^®CC. The results obtained after 24 h of mouse skin treatment with the same creams showed a large decrease of porphyrin formation in comparison with results found after 4 h. All porphyrin concentrations measured after this time of MAL creams application were similar. Skin erythema was observed using MAL cream with DMSO and HPE-101, but not with Labrafac^®CC.Our work suggests that the new penetration enhancer Labrafac^®CC in creams with MAL may be used to increase the penetration of MAL through the mouse skin and at the same time increase production of porphyrin in the skin. This may reduce skin erythema, which is induced by DMSO and HPE-101.     

Combination of hand-held probe and microscopy for fluorescence guided surgery in the brain tumor marginal zone

BackgroundVisualization of the tumor is crucial for differentiating malignant tissue from healthy brain during surgery, especially in the tumor marginal zone. The aim of the study was to introduce a fluorescence spectroscopy-based hand-held probe (HHF-probe) for tumor identification in combination with the fluorescence guided resection surgical microscope (FGR-microscope), and evaluate them in terms of diagnostic performance and practical aspects of fluorescence detection.Material and MethodsEighteen operations were performed on 16 patients with suspected high-grade glioma. The HHF-probe and the FGR-microscope were used for detection of protoporphyrin (PpIX) fluorescence induced by 5-aminolevulinic acid (5-ALA) and evaluated against histopathological analysis and visual grading done through the FGR-microscope by the surgeon. A ratio of PpIX fluorescence intensity to the autofluorescence intensity (fluorescence ratio) was used to quantify the spectra detected by the probe.ResultsFluorescence ratio medians (range 0 – 40) measured by the probe were related to the intensity of the fluorescence in the FGR-microscope, categorized as “none” (0.3, n = 131), “weak” (1.6, n = 34) and “strong” (5.4, n = 28). Of 131 “none” points in the FGR-microscope, 88 (67%) exhibited fluorescence with the HHF-probe. For the tumor marginal zone, the area under the receiver operator characteristics (ROC) curve was 0.49 for the FGR-microscope and 0.65 for the HHF-probe.ConclusionsThe probe was integrated in the established routine of tumor resection using the FGR-microscope. The HHF-probe was superior to the FGR-microscope in sensitivity; it detected tumor remnants after debulking under the FGR-microscope. The combination of the HHF-probe and the FGR-microscope was beneficial especially in the tumor marginal zone.     

ALA-induced porphyrin formation and fluorescence in synovitis tissue: In-vitro and in vivo studies

The synovial inflammatory process in rheumatoid arthritis (RA) is accompanied by massive tumor-like proliferation and activation of the connective stroma. These abnormal cells actively invade and destroy the peri-articular bone and cartilage at the margins of joints where synovium and bone are attached. There is still a lack of minimally invasive synovectomy methods, which might be suitable for the smaller joints. Unfortunately, these joints are usually involved in the disease. Photodynamic therapy has been evaluated as a possible treatment modality for RA synovitis. The present study describes the differences of 5-aminolevulinic acid (5-ALA) and 5-ALA ester-induced protoporphyrin IX (PpIX) production in cell cultures obtained from patients with RA, osteoarthritis (OA) and human sarcoma cell line (HS 192.T) and in a collagen-induced arthritis model in rats. The incubation of cells with hexyl aminolevulinate (HAL) induced the same amount of fluorescence as 5-ALA and methyl aminolevulinate (MAL) at about a 100-fold lower concentration. Incubation with HAL-induced accumulation of at least twice as much porphyrins in RA- and HS 192.T-cells than 5-ALA and MAL in OA-cells. Similar levels of porphyrins were accumulated in RA and the malignant cells. In vivo, intra-articular application of 5-ALA induced a significant porphyrin accumulation in synovitis tissue as measured by in situ fluorescence spectroscopy. In contrast to our in vitro results and other reports, we could not detect enhanced fluorescence after application of up to 0.1 mg HAL.     

Fluorescence spectroscopy in the visible range for the assessment of UVB radiation effects in hairless mice skin

Ultraviolet (UV) radiation may induce skin alterations as observed in photoaging. Some recognized modifications are epidermal hyperplasia, amorphous deposition of degraded elastic fibers and reduction in the number of collagen fibers. They alter the tissue biochemical properties that can be interrogated by steady state fluorescence spectroscopy (SSFS). In this study, we monitored the changes in endogenous fluorescence emission from hairless mice skin during a protocol of photoaging using UVB irradiation. To perform the fluorescence spectroscopy, it was used a violet laser (408 nm) to induce the native fluorescence that is emitted in the visible range. Under 408 nm excitation, the emission spectrum showed bands with peaks centered around 510, 633 and 668 nm for irradiated and control groups. A relative increase of the fluorescence at 633 nm emission on the flank was observed with time when compared to the ventral skin at the same animal and the non-irradiated control group. We correlated the emission at 633 nm with protoporphyrin IX (PpIX), and our hypothesis is that the PpIX metabolism in the photoaged and aged skin are different. PpIX fluorescence intensity in the photoaged skin is higher and more heterogeneous than in the aged skin. Notwithstanding, more spectroscopic and biochemistry studies investigating the 510 and 633 nm emission are needed to confirm this hypothesis.     

Photodynamic therapy (PDT) - Initiation of apoptosis via activation of stress-activated p38 MAPK and JNK signal pathway in H460 cell lines

Aims

The purpose of this study was to investigate the photoefficacies of protoporphyrin IX (PpIX) generated by drug precursor 5-aminolevulinic acid (ALA) and its hexyl ester (H-ALA) on two human non-small lung carcinoma cell lines (H460/Bcl-2 and H460/neo).

Main methods

Drug uptake and the photoefficacies of PpIX were measured by flow cytometry and MTT assay; while the mode of cell death and alternation of signal transduction pathways were studied with 4′,6-diamidino-2-phenylindole (DAPI) staining and Western blot analysis, respectively.

Key findings

The flow cytometric analysis of H-ALA (5 μM) uptake revealed optimal fluorescent intensity at 8 h incubation, while ALA (0.5 mM) was still far from saturation. The LD₃₀ of H-ALA-PDT was 30 μM, 2 J/cm², while the LD₃₀ of ALA-PDT was 3 mM, 2 J/cm². The dark toxicities mediated by both pro-drug H-ALA and ALA were negligible. By DAPI staining, apoptotic cell death was observed. In addition, by Western blot analysis, H-ALA- and ALA-mediated PDT initiated apoptotic cell death via the up-regulation and activation of p38 mitogen activated protein kinase (MAPK), the stress-activated c-jun N-terminal kinases (JNK) and ERK.

Significance

These results suggested that H-ALA and ALA mediated PDT displayed similar photocytotoxicities towards the two non-small lung cancer cells. Our present study also demonstrates H-ALA or ALA mediated PDT in H460 cells are closely related to the activation of p38 MAPK and JNK signalling pathway.     

Fluorescence optical imaging and 3T-MRI for detection of synovitis in patients with rheumatoid arthritis in comparison to a composite standard of reference

ObjectivesTo address whether Indocyanine Green (ICG) enhanced fluorescence optical imaging (FOI) is more sensitive than magnetic resonance imaging (MRI) in the detection of synovitis of the wrist and finger joints in rheumatoid arthritis and to analyze the performance of FOI depending on the grade of synovitis.MethodsTwenty patients with highly active rheumatoid arthritis (mean DAS28-ESR 5.25 ± 1.0) and thirteen healthy volunteers underwent clinical examination, FOI and contrast-enhanced 3T-MRI. Joints were rated by three independent readers semiquantitatively (grade 0–3: no, low, moderate and high grade synovitis) and compared to a semiquantitative composite standard of reference (cSOR, grade 0–3) that incorporated clinical parameters, FOI and MRI results.Results2.868 evaluations in 956 joints were performed. FOI had an overall sensitivity of 57.3% and a specificity of 92.1%, whereas MRI had a sensitivity of 89.2% and a specificity of 92.6%. The sensitivity of FOI increased with the degree of synovitis to 65.0% for moderate and severe synovitis (specificity 88.1%) and 76,3% for severe synovitis (specificity 80.5%). The performance of FOI decreased with the degree of synovitis with false negative results predominantly for mild (156/343, 45.5%) and moderate (160/343, 46.6%) synovitis and false positive FOI evaluations predominantly based on weak (grade 1) signals (133/163, 81,6%).ConclusionFOI has a lower sensitivity than 3T-MRI in the detection of synovitis of the hand and finger joints. The diagnostic performance of FOI decreases with the degree of synovitis and with the strength of FOI signals.     

Synthesis,characterisation and in vitro investigation of photodynamic activity of 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride on HeLa cells using low light fluence rate

Photodynamic therapy (PDT) is a treatment that aims to kill cancer cells by reactive oxygen species, mainly singlet oxygen, produced through light activation of a photosensitiser (PS). Amongst photosensitisers that attracted the most attention in the last decade are cationic and amphiphilic molecules based on porphyrin, chlorin and phthalocyanine structures. Our aim was to join this search for more optimal balance of the lipophilic and hydrophilic moieties in a PS. A new amphiphilic porphyrin, 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (5) was synthesised and characterised by ¹H NMR, UV–vis and fluorescence spectroscopy, and by MALDI-TOF/TOF spectrometry. In vitro photodynamic activity of 5 was evaluated on HeLa cell lines and compared to the activity of the hydrophilic 5-(4-acetamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (7). Low fluence rate (2 mW cm⁻²) of red light (643 nm) was used for the activation, and both porphyrins showed a drug dose-response as well as a light dose-response relationship, but the amphiphilic porphyrin was presented with significantly lower IC₅₀ values. The obtained IC₅₀ values for 5 were 1.4 μM at 15 min irradiation time and 0.7 μM when the time of irradiation was 30 min, while for 7 these values were 37 and 6 times higher, respectively. These results confirm the importance of the lipophilic component in a PS and show a potential for 5 to be used as a PS in PDT applications.     

Comparison of semiquantitative fluorescence imaging and PET tracer uptake in mesothelioma models as a monitoring system for growth and therapeutic effects

IntroductionVarious techniques are available for in vivo imaging, and precise understanding of their characteristics is essential for effective use of the imaging results. We established human mesothelioma cell lines expressing red fluorescent protein (RFP) and examined their fluorescence intensity and uptake of positron emission tomography (PET) tracer analogs to compare their characteristics and assess their usefulness in the evaluation of therapeutics.MethodA human mesothelioma cell line was stably transfected to express RFP. Fluorescence, cell number and protein amount were measured during cell growth and treatment with cytotoxic reagents. In in vivo experiments, RFP-expressing cells were injected subcutaneously or into the pleural cavity of nude mice, and fluorescence images were taken with or without pemetrexed treatment. The uptake of [³H]3′-deoxy-3′-fluorothymidine ([³H]FLT) and [¹⁴C]2-fluoro-2-deoxy-d-glucose ([¹⁴C]FDG) under treatment with the above reagents in vitro and in vivo were examined.ResultsStrong correlation was observed between fluorescence intensity and total cell number with or without cytotoxic treatment. The uptake of [³H]FLT and [¹⁴C]FDG decreased rapidly after the initiation of treatment with actinomycin D or cycloheximide. When treated with pemetrexed, the uptake of [³H]FLT temporarily increased. The cells formed subcutaneous and orthotopic tumors, with fluorescence intensity correlating with tumor volume. The correlation was sustained under pemetrexed treatment. The uptake of [³H]FLT in vivo increased significantly early after pemetrexed treatment.ConclusionFluorescence imaging could be used to semiquantitatively monitor tumor size, whereas PET could be used to monitor tumor response to therapeutic treatments, and especially, FLT might be a good marker of the response to anti-folate chemotherapeutics.     

In vitro and in vivo evaluation of a chlorin-based photosensitizer KAE® for cancer treatment

BackgroundPhotodynamic therapy (PDT) has been approved for the clinical treatment of cancers. Photosensitizer (PS) is a crucial element of PDT. In the current study, in vitro and in vivo evaluation of a chlorin-based photosensitizer KAE® was performed.MethodsThe physicochemical characteristics of KAE® were compared with chlorin e6. The intracellular distribution of KAE® in HeLa cells was observed by laser scanning confocal microscopy. Reactive oxygen species (ROS) generation was detected through a 2′, 7-dichlorodihydrofluorescein diacetate probe. The pharmacokinetics of KAE® was studied in mice. The photodynamic activities of KAE® and porphyrin based PSs were compared both in vitro and in vivo. The biosafety of KAE® in mice was evaluated by pathological section observation, blood routine examination and biochemistry assays.ResultsKAE® was readily dissolved in an aqueous solvent in a clinically acceptable concentration and showed a strong absorption at around 660 nm. Most of KAE® was located in the mitochondria of the tumor cells. Compared with hematoporphyrin derivative and 5-aminolevulinic acid, KAE® displayed a higher efficiency in cell killing. Furthermore, it could be completely eliminated from mouse body in 2 days. KAE® had no toxicity to mice under the tested dosage.ConclusionsOur results suggested that KAE® is an effective and safe PS for PDT in cancer therapy and has a promising prospect for clinical application.