Showing papers on "Protoporphyrin IX published in 2020"

ABCG2 influence on the efficiency of photodynamic therapy in glioblastoma cells.

Abstract: Background Photodynamic therapy with 5-aminolevulinic acid (5-ALA PDT) is a promising novel therapeutic approach in the therapy of malignant brain tumors. 5-ALA occurs as a natural precursor of protoporphyrin IX (PpIX), a tumor-selective photosensitizer. The ATP-binding cassette transporter ABCG2 plays a physiologically significant role in porphyrin efflux from living cells. ABCG2 is also associated with stemness properties. Here we investigate the role of ABCG2 on the susceptibility of glioblastoma cells to 5-ALA PDT. Methods Accumulation of PpIX in doxycycline-inducible U251MG glioblastoma cells with or without induction of ABCG2 expression or ABCG2 inhibition by KO143 was analyzed using flow cytometry. In U251MG cells, ABCG2 was inducible by doxycycline after stable transfection with a tet-on expression plasmid. U251MG cells with high expression of ABCG2 were enriched and used for further experiments (sU251MG-V). PDT was performed on monolayer cell cultures by irradiation with laser light at 635 nm. Results Elevated levels of ABCG2 in doxycycline induced sU251MG-V cells led to a diminished accumulation of PpIX and higher light doses were needed to reduce cell viability. By inhibiting the ABCG2 transporter with the efficient and non-toxic ABCG2 inhibitor KO143, PpIX accumulation and PDT efficiency could be strongly enhanced. Conclusion Glioblastoma cells with high ABCG2 expression accumulate less photosensitizer and require higher light doses to be eliminated. Inhibition of ABCG2 during photosensitizer accumulation and irradiation promises to restore full susceptibility of this crucial tumor cell population to photodynamic treatment.

The active site of magnesium chelatase.

Abstract: The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons.

Macroscopic fluorescence-lifetime imaging of NADH and protoporphyrin IX improves the detection and grading of 5-aminolevulinic acid-stained brain tumors.

Abstract: Maximal safe tumor resection remains the key prognostic factor for improved prognosis in brain tumor patients. Despite 5-aminolevulinic acid-based fluorescence guidance the neurosurgeon is, however, not able to visualize most low-grade gliomas (LGG) and infiltration zone of high-grade gliomas (HGG). To overcome the need for a more sensitive visualization, we investigated the potential of macroscopic, wide-field fluorescence lifetime imaging of nicotinamide adenine dinucleotide (NADH) and protoporphyrin IX (PPIX) in selected human brain tumors. For future intraoperative use, the imaging system offered a square field of view of 11 mm at 250 mm free working distance. We performed imaging of tumor tissue ex vivo, including LGG and HGG as well as brain metastases obtained from 21 patients undergoing fluorescence-guided surgery. Half of all samples showed visible fluorescence during surgery, which was associated with significant increase in PPIX fluorescence lifetime. While the PPIX lifetime was significantly different between specific tumor tissue types, the NADH lifetimes did not differ significantly among them. However, mainly necrotic areas exhibited significantly lower NADH lifetimes compared to compact tumor in HGG. Our pilot study indicates that combined fluorescence lifetime imaging of NADH/PPIX represents a sensitive tool to visualize brain tumor tissue not detectable with conventional 5-ALA fluorescence.

pO2-weighted imaging in vivo by delayed fluorescence of intracellular Protoporphyrin IX

Abstract: In this Letter, we report on the imaging of tissue oxygen by means of time-gated wide-field lifetime imaging of the microsecond-scale delayed fluorescence from Protoporphyrin IX (PpIX). PpIX is produced endogenously from a clinically used δ-aminolevulinic acid (5-ALA) by most cells. The lifetime of delayed fluorescence is lengthened by the absence of oxygen to quench its triplet state. Here, the images of the delayed emission from the human skin and the skin of nude mice were captured after topical application of 5-ALA, using a pulsed 635 nm excitation. Macroscopic lifetime-based images were found to be associated with the tissue oxygenation and obtained with a low light dose (∼10mJ/cm2) and “capture-times” of a few seconds.

DNA Strand Break Properties of Protoporphyrin IX by X-Ray Irradiation against Melanoma.

Abstract: Recent reports have suggested that 5-aminolevulinic acid (5-ALA), which is a precursor to protoporphyrin IX (PpIX), leads to selective accumulation of PpIX in tumor cells and acts as a radiation sensitizer in vitro and in vivo in mouse models of melanoma, glioma, and colon cancer. In this study, we investigated the effect of PpIX under X-ray irradiation through ROS generation and DNA damage. ROS generation by the interaction between PpIX and X-ray was evaluated by two kinds of probes, 3′-(p-aminophenyl) fluorescein (APF) for hydroxyl radical (•OH) detection and dihydroethidium (DHE) for superoxide (O2•-). •OH showed an increase, regardless of the dissolved oxygen. Meanwhile, the increase in O2•- was proportional to the dissolved oxygen. Strand breaks (SBs) of DNA molecule were evaluated by gel electrophoresis, and the enhancement of SBs was observed by PpIX treatment. We also studied the effect of PpIX for DNA damage in cells by X-ray irradiation using a B16 melanoma culture. X-ray irradiation induced γH2AX, DNA double-strand breaks (DSBs) in the context of chromatin, and affected cell survival. Since PpIX can enhance ROS generation even in a hypoxic state and induce DNA damage, combined radiotherapy treatment with 5-ALA is expected to improve therapeutic efficacy for radioresistant tumors.

Biodegradable Silica‐Based Nanoparticles with Improved and Safe Delivery of Protoporphyrin IX for the In Vivo Photodynamic Therapy of Breast Cancer

Abstract: Silica‐based nanoplatforms are highly versatile and attractive delivery systems for cancer treatment. These platforms have been used for the effective delivery of pharmacological agents in preclinical settings. Though silicon oxide is found naturally in the human body, a major limitation associated with silica‐based nanoparticles is their slow biodegradability. Therefore, the potential risks related to the longer bioaccumulation of these materials can be significant. In this work, the synthesis and application of a novel silica‐based nanoplatform, polysilsesquioxane nanoparticles (PSilQ NPs) is reported. The developed PSilQ material contains stimuli‐responsive properties, and improves biodegradability for the efficient delivery of a clinically relevant photosensitizer, protoporphyrin IX. Herein, it is demonstrated that the PSilQ nanoplatform is biocompatible and exhibits enhanced biodegradability in an immune‐competent mouse model. In addition, PSilQ NPs show phototherapeutic efficiency for reducing the tumor burden in an orthotopic model of triple‐negative breast cancer. These results may pave the way for the future clinical evaluation of this silica‐based nanoplatform.

Gold Nanoshells Coated 5-Aminolevulinic Liposomes for Photothermal-Photodynamic Antitumor Therapy

Abstract: In this study, a novel antitumor system was designed on the basis of photodynamic therapy and photothermal therapy using gold nanoshells coated 5-aminolevulinic acid (5-ALA) liposomes (GNALs). As a widely used photosensitizer prodrug, 5-aminolevulinic acid (5-ALA)-derived protoporphyrin IX (PpIX) can accumulate to a higher level in tumors than in normal tissues. Protoporphyrin IX (PpIX) initiates a series of cytotoxic reactions after irradiation of target tissue with a measured dose of light with appropriate wavelength, which may be dependent on the generation of reactive oxygen species. Gold nanoshells can strongly absorb NIR lasers at the same time, and can generate hyperthermia to provide photothermic effects. Gold nanoshells coated 5-ALA-Liposomes (GNALs) exhibit a uniform-sized spherical shape of 185.8±0.91 nm, with a zeta potential of 33±1.6 mV. Liposomes with specific sizes ranging from 100 to 200 nm can escape into the tumor interstitial tissue and accumulate preferentially in tumor tissue owing to the EPR effect. In addition, NIR light-excited nanosystems significantly promoted antitumor effects in this study compared to single photodynamic therapy. It was also found that the increased temperature promoted inhibition rate of SKOV3 cells. The novel drug delivery system shows great potential in photothermal-photodynamic antitumor therapy.

An in vitro study of the effect of 5-ALA-mediated photodynamic therapy on oral squamous cell carcinoma.

Abstract: The primary aim of this study was to observe the effect of 5-ALA-mediated photodynamic therapy on oral squamous cell carcinoma in vitro. SCC25 cells were divided into the observation group and the blank control group. Different concentrations of 5-ALA and SCC25 cells were co-incubated for different times, and the concentration of protoporphyrin IX was detected by flow cytometry. SCC25 cells were divided into the 5-ALA group (100 mg/L), the laser irradiation group alone, the 5-ALA plus laser irradiation group, and the blank control group (0 mg/L 5-ALA), and the methyl thiazolyl tetrazolium (MTT) solution method was used (each group was incubated for 4, 8 and 12 h in turn). The cell survival rate was calculated. Using annexin V-fluorescein isothiocyanate/propidium iodide method, the apoptosis of SCC25 cells was detected by flow cytometry. The level of protoporphyrin IX in SCC25 cells increased with increased concentrations of 5-ALA and length of incubation. However, after 12 h, protoporphyrin IX level in SCC25 cells was gradually stabilized, and similar effect was obtained with 100 mg/L or more 5-ALA, indicating that the level of protoporphyrin IX in SCC25 cells was determined by 5-ALA concentration and incubation time. 5-ALA plus laser irradiation exerted an inhibitory effect on the growth of SCC25 cells, which was highly associated with drug dose and incubation time. Compared with the control group, laser irradiation alone or 5-ALA alone had no effect on the apoptosis of SCC25 cells. Different concentrations of 5-ALA combined with laser irradiation showed a remarkable effect of apoptosis, and a higher apoptosis rate was seen with higher drug concentrations. 5-ALA-mediated photodynamic therapy affects the growth of SCC25 cells in vitro, which may provide a new idea for the clinical treatment of oral squamous cell carcinoma.

Potent antiviral effect of protoporphyrin IX and verteporfin on SARS-CoV-2 infection

Abstract: The infection of SARS-CoV-2 has spread to more than 200 countries and regions and the numbers of infected people and deaths worldwide are expected to continue to rise. Current treatment of COVID-19 is limited and mostly supportive. At present, there is no specific therapeutics against SARS-CoV-2. In this study, we discovered that protoporphyrin IX and verteporfin, two FDA-approved drugs for treatment of human diseases, had significant antiviral effect against SARS-CoV-2, with EC50 values for the reduction of viral RNA at nanomolar concentrations. Both drugs completely inhibited the cytopathic effect (CPE) produced by SARS-CoV-2 infection at lower drug concentrations than that of remdesivir. The selection indices of protoporphyrin IX and verteporfin are 952.74 and 368.93, respectively, suggesting wide safety margins. Both drugs were able to prevent SARS-CoV-2 infection as well as suppress established SARS-CoV-2 infection. The compounds share a porphyrin ring structure. Molecular docking indicates that the compounds may interact with viral receptor ACE2 and could block the cell-cell fusion mediated by ACE2 and viral S protein. Our finding suggests that protoporphyrin IX and verteporfin might be potential antivirals against SARS-CoV-2 infection and also sheds new light on the development of a novel class of small compounds against SARS-CoV-2.

Single Nucleotide Mutagenesis of the TaCHLI Gene Suppressed Chlorophyll and Fatty Acid Biosynthesis in Common Wheat Seedlings

Abstract: Wheat (Triticum aestivum L.) is one of the most important crops in the world. Chlorophyll plays a vital role in plant development and crop improvement and further determines the crop productivity to a certain extent. The biosynthesis of chlorophyll remains a complex metabolic process, and fundamental biochemical discoveries have resulted from studies of plant mutants with altered leaf color. In this study, we identified a chlorophyll-deficiency mutant, referred to as chli, from the wheat cultivar Shaannong33 that exhibited an obvious pale-green leaf phenotype at the seedling stage, with significantly decreased accumulation of chlorophyll and its precursors, protoporphyrin IX and Mg-protoporphyrin IX. Interestingly, a higher protoporphyrin IX to Mg-protoporphyrin IX ratio was observed in chli. Lipid biosynthesis in chli leaves and seeds was also affected, with the mutant displaying significantly reduced total lipid content relative to Shaanong33. Genetic analysis indicated that the pale-green leaf phenotype was controlled by a single pair of recessive nuclear genes. Furthermore, sequence alignment revealed a single-nucleotide mutation (G664A) in the gene TraesCS7A01G480700.1, which encodes subunit I of the Mg-chelatase in plants. This single-nucleotide mutation resulted in an amino acid substitution (D221N) in the highly conserved domain of subunit I. As a result, mutant protein Tachli-7A lost the ability to interact with the normal protein TaCHLI-7A, as assessed by yeast two-hybrid assay. Meanwhile, Tachli-7A could not recover the chlorophyll deficiency phenotype of the Arabidopsis thaliana SALK_050029 mutant. Furthermore, we found that in Shaannong33, the protoporphyrin IX to Mg-protoporphyrin IX ratio was growth state-dependent and insensitive to environmental change. Overall, the mutation in Tachli-7A impaired the function of Mg-chelatase and blocked the conversion of protoporphyrin IX to Mg- protoporphyrin IX. Based on our results, the chli mutant represents a potentially useful resource for better understanding chlorophyll and lipid biosynthetic pathways in common wheat.

Evaluation of aminolevulinic acid-mediated protoporphyrin IX fluorescence and enhancement by ABCG2 inhibitors in renal cell carcinoma cells

Abstract: Aminolevulinic acid (ALA) has been approved as an intraoperative molecular imaging probe for protoporphyrin IX (PpIX) fluorescence-guided resection of glioma. Here we explored its potential application for renal cell carcinoma (RCC) that is showing increased incidence in recent years. ALA-mediated PpIX in cell lysates (intracellular) and culture medium was measured in five human RCC cell lines (786-O, 769-P, A-704, Caki-1, Caki-2) and a non-tumor human kidney epithelial cell line HK-2 by spectrofluorometry and flow cytometry. The activity of PpIX bioconversion enzyme ferrochelatase (FECH) and PpIX efflux transporter ABCG2 was determined to correlate with the PpIX level. We found that ALA-PpIX fluorescence was highly variable among RCC cell lines and A-704 was the only RCC cell line exhibiting significantly higher intracellular PpIX than HK-2 cells. Neither the intracellular PpIX level nor the total amount of PpIX (including PpIX in cell lysates and the medium) had significant correlation with the activity of FECH or ABCG2. To enhance the intracellular PpIX, cells were treated with Ko143, a pharmacological inhibitor of ABCG2. Ko143 significantly increased the intracellular PpIX in cell lines with ABCG2 activity, but not in cell lines with little ABCG2 activity. In fact, there was a positive correlation between the ABCG2 activity and Ko143-induced PpIX enhancement across kidney cell lines. To identify clinically relevant ABCG2 inhibitors, small molecule inhibitors targeting various cell signaling pathways, some of which are known to inhibit ABCG2, were evaluated for the enhancement of ALA-PpIX in Caki-2 cells that had the highest ABCG2 activity in the RCC cell panel. Our screening led to the identification of several clinically available inhibitors that significantly increased the intracellular PpIX. Particularly, kinase inhibitor lapatinib exhibited the strongest enhancement effect. These clinical inhibitors can be used for the enhancement of ALA-PpIX fluorescence in tumors with elevated ABCG2 activity.

Fluorescence detection of deep intramucosal cancer excited by green light for photodynamic diagnosis using protoporphyrin IX induced by 5-aminolevulinic acid: an ex vivo study.

Abstract: Significance: The diagnostic depth of photodynamic diagnosis (PDD) for gastric cancer with protoporphyrin IX (PpIX) is limited, which leads to missing intramucosal cancers in screening and surgery. Aim: The reason is that the excitation light, whose wavelength is determined by the highest absorption peak of PpIX (∼405 nm), is strongly attenuated by mucosal tissues. We investigated an excitation wavelength that can extend the diagnostic depth of PpIX fluorescence at the mucosal subsurface. Approach: By calculating the depth-dependent intensity of the excitation light in porcine gastric mucosa for each wavelength, relationships among the wavelength, fluorophore depth, and fluorescence intensity were assessed and fluorescence images of PpIX pellets located at different fluorophore depths were compared experimentally by changing the excitation wavelength. Results: The numerical calculation showed that a 505-nm excitation light provided the highest fluorescence intensities at a fluorophore depth deeper than 1.1 mm. In the fluorescence observation, the fluorescence intensities at fluorophore depths of 0 and 1.0 mm at 405 nm were 5.4 × 103 and 1.0 × 103 arb. units, whereas those at 505 nm were 5.3 × 101 and 1.9 × 102 arb. units, respectively. Conclusion: The experimental results suggest that the diagnosis depth of PDD with PpIX for intramucosal cancer can be extended by 505-nm excitation light.

Probing Hexaminolevulinate Mediated PpIX Fluorescence in Cancer Cell Suspensions in the Presence of Chemical Adjuvants

Abstract: Exogenous administration of hexaminolevulinate (HAL) induces fluorescent protoporphyrin IX (PpIX) accumulation preferentially in cancer cells. However, the PpIX fluorescence intensities between noncancer and cancer cells are highly variable. The contrast between cancer and noncancer cells may be insufficient to reliably discriminate, especially at the single cell level in cancer diagnostics. This study examines the use of the chemical adjuvants dimethylsulphoxide (DMSO) or deferoxamine (DFO) to enhance the HAL induced PpIX accumulation in cancer cells. Our results showed that in some of the incubation conditions tested, the addition of DFO with HAL significantly increased PpIX 21 fluorescence of adherent monolayer cancer cells, but this was never the case for cells in suspension. Permeabilisation with DMSO did not increase PpIX fluorescence. Cell-to-cell interaction may well play an important role in the PpIX accumulation when suspended cells are treated in HAL and adjuvant chemicals.

Antibacterial Activity of Synthetic Cationic Iron Porphyrins.

Abstract: Widespread antibiotic resistance demands new strategies for fighting infections. Porphyrin-based compounds were long ago introduced as photosensitizers for photodynamic therapy, but light-independent antimicrobial activity of such compounds has not been systematically explored. The results of this study demonstrate that synthetic cationic amphiphilic iron N-alkylpyridylporphyrins exert strong bactericidal action at concentrations as low as 5 μM. Iron porphyrin, FeTnHex-2-PyP, which is well tolerated by laboratory animals, efficiently killed Gram-negative and Gram-positive microorganisms. Its bactericidal activity was oxygen-independent and was controlled by the lipophilicity and accumulation of the compound in bacterial cells. Such behavior is in contrast with the anionic gallium protoporphyrin IX, whose efficacy depends on cellular heme uptake systems. Under aerobic conditions, however, the activity of FeTnHex-2-PyP was limited by its destruction due to redox-cycling. Neither iron released from the Fe-porphyrin nor other decomposition products were the cause of the bactericidal activity. FeTnHex-2-PyP was as efficient against antibiotic-sensitive E. coli and S. aureus as against their antibiotic-resistant counterparts. Our data demonstrate that development of amphiphilic, positively charged metalloporphyrins might be a promising approach in the introduction of new weapons against antibiotic-resistant strains.

Polymeric nanoparticles favor the in vitro dermal accumulation of Protoporphyrin IX (PpIX) with optimal biocompatibility and cellular recovery in culture of healthy dermal fibroblasts after Photodynamic Therapy

Abstract: The aim of this study was to investigate (i) the cutaneous accumulation of photosensitizer (PS) protoporphyrin IX (PpIX) encapsulated in nanoparticles (Np) by confocal laser scanning microscopy (CLSM) in animal model skin (ii) the in vitro dark cytotoxicity and phototoxicity of PpIX-Nps in dermal fibroblast cells (L929 fibroblast cells and (iii) the recovery of cell viability after PDT. The CLSM has shown higher PS concentration in the superficial skin; the stratum corneum (SC) for both encapsulated and free PpIX for all times evaluated (4 h, 8 h and 24 h). Although with lower PS accumulation in the deeper layers of the skin, ie; Epidermis + Dermis (EP + D) after 8 h the PS accumulation was significantly higher when encapsulated in Np. In brief, PpIX-Np was not cytotoxic to fibroblast cells in the dark (˜ 100% viable cells; excellent biocompatibility) and in the presence of light 8 h post incubation (phototoxicity) reached CC50 = 7.01 μg / ml (compared to free PpIX, CC50 = 0.84 μg / ml) meaning that PpIX in Nps were phototoxic to cells as expected; however at higher concentrations than free PpIX. Moreover, PpIX-Np has shown 100% of cell recovery 24 h post PDT in the fibroblast cells, indicating photoactivation protection of PpIX by the Nps in the healthy cells.

Protoporphyrin IX and verteporfin prevent SARS-CoV-2 infection in vitro and in a mouse model expressing human ACE2

Abstract: The SARS-CoV-2 infection is spreading rapidly worldwide. Efficacious antiviral therapeutics against SARS-CoV-2 is urgently needed. Here, we discovered that protoporphyrin IX (PpIX) and verteporfin, two FDA-approved drugs, completely inhibited the cytopathic effect produced by SARS-CoV-2 infection at 1.25 μM and 0.31 μM respectively, and their EC50 values of reduction of viral RNA were at nanomolar concentrations. The selectivity indices of PpIX and verteporfin were 952.74 and 368.93, respectively, suggesting broad margin of safety. Importantly, PpIX and verteporfin prevented SARS-CoV-2 infection in mice adenovirally transduced with human ACE2. The compounds, sharing a porphyrin ring structure, were shown to bind viral receptor ACE2 and interfere with the interaction between ACE2 and the receptor-binding domain of viral S protein. Our study suggests that PpIX and verteporfin are potent antiviral agents against SARS-CoV-2 infection and sheds new light on developing novel chemoprophylaxis and chemotherapy against SARS-CoV-2.

5-Aminolevulinic Acid: Pitfalls of Fluorescence-guided Resection for Malignant Gliomas and Application for Malignant Glioma Therapy.

Abstract: 5-Aminolevulinic acid (ALA) has been widely used as an intravital fluorescence marker in the fluorescence-guided resection of malignant gliomas. Although not a photosensitizer itself, 5-ALA is a prodrug that accumulates protoporphyrin IX (PpIX) in the mitochondria of glioma cells; PpIX acts as a photosensitizer. Fluorescence-guided resection for malignant gliomas has some pitfalls. Moreover, 5-ALA is not merely a fluorescence marker but has potential as a mitochondria-targeting drug for malignant glioma therapy. In this article, we review the literature related to 5-ALA, discuss the pitfalls of fluorescence-guided resection using 5-ALA for malignant gliomas, and describe the application of 5-ALA for malignant glioma therapy with personal opinions.

Two photon excited fluorescence and hyper Rayleigh scattering of Protoporphyrin IX

Abstract: The two-photon excited fluorescence (TPEF) cross-section of Protoporphyrin IX (PpIX) in solution is determined at different excitation wavelengths. Maxima observed at 620 and 634 nm are associated with two different forms of PpIX already observed in one-photon excited fluorescence studies. In order to further provide insights into these two forms, first hyperpolarizabilities are also reported for these two forms with corresponding depolarization ratio further demonstrating the existence of specific symmetry differences. The static and the frequency dependent first hyperpolarizability were also calculated using Gaussian 09 and compared to the experimental data. This work shows that PpIX can exhibit two forms that might be targeted in applications like oncology clinical agents.

Simultaneous Determination of Carcinoembryonic Antigen (CEA), Carbohydrate Antigen 19-9 (CA19-9), and Serum Protein p53 in Biological Samples with Protoporphyrin IX (PIX) Used for Recognition by Stochastic Microsensors

Abstract: Three of the most assessed biomarkers in the screening of biological samples for gastric cancer are carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and serum protein p53 (p53). ...