Codelivery of anti-CD47 antibody and chlorin e6 using a dual pH-sensitive nanodrug for photodynamic immunotherapy of osteosarcoma.

Osteosarcoma is a malignant tumor originating from bone tissue that progresses rapidly and has a poor patient prognosis. Immunotherapy has shown great potential in the treatment of osteosarcoma. However, the immunosuppressive microenvironment severely limits the efficacy of osteosarcoma treatment. The dual pH-sensitive nanocarrier has emerged as an effective antitumor drug delivery system that can selectively release drugs into the acidic tumor microenvironment. Here, we prepared a dual pH-sensitive nanocarrier, loaded with the photosensitizer Chlorin e6 (Ce6) and CD47 monoclonal antibodies (aCD47), to deliver synergistic photodynamic and immunotherapy of osteosarcoma. On laser irradiation, Ce6 can generate reactive oxygen species (ROS) to kill cancer cells directly and induces immunogenic tumor cell death (ICD), which further facilitates the dendritic cell maturation induced by blockade of CD47 by aCD47. Moreover, both calreticulin released during ICD and CD47 blockade can accelerate phagocytosis of tumor cells by macrophages, promote antigen presentation, and eventually induce T lymphocyte-mediated antitumor immunity. Overall, the dual pH-sensitive nanodrug loaded with Ce6 and aCD47 showed excellent immune-activating and anti-tumor effects in osteosarcoma, which may lay the theoretical foundation for a novel combination model of osteosarcoma treatment.

Sonodynamic-chemotherapy synergy with chlorin e6-based carrier-free nanoparticles for non-small cell lung cancer.

Sonodynamic therapy (SDT), an emerging cancer treatment with significant potential, offers the advantages of non-invasiveness and deep tissue penetrability. The method involves activating sonosensitizers with ultrasound to generate reactive oxygen species (ROS) capable of eradicating cancer cells, addressing the challenge faced by photodynamic therapy (PDT) where conventional light sources struggle to penetrate deep tissues, impacting treatment efficacy. This study addresses prevalent challenges in numerous nanodiagnostic and therapeutic agents, such as intricate synthesis, poor repeatability, low stability, and high cost, by introducing a streamlined one-step assembly method for nanoparticle preparation. Specifically, the sonosensitizer Chlorin e6 (Ce6) and the chemotherapy drug erlotinib are effortlessly combined and self-assembled under sonication, yielding carrier-free nanoparticles (EC-NPs) for non-small cell lung cancer (NSCLC) treatment. The resulting EC-NPs exhibit optimal drug loading capacity, a simplified preparation process, and robust stability both in vitro and in vivo, owing to their carrier-free characteristics. Under the synergistic treatment of sonodynamic therapy and chemotherapy, EC-NPs induce an excess of reactive oxygen in tumor tissue, prompting apoptosis of cancer cells and reducing their proliferative capacity. Both in vitro and in vivo experiments demonstrate superior therapeutic effects of EC-NPs under ultrasound conditions compared to free Ce6. In summary, our research findings highlight that the innovatively designed carrier-free sonosensitizer EC-NPs present a therapeutic option with commendable efficacy and minimal side effects.

Efficient Assessment of Tumor Vascular Shutdown by Photodynamic Therapy on Orthotopic Pancreatic Cancer Using High-Speed Wide-Field Waterproof Galvanometer Scanner Photoacoustic Microscopy.

To identify the vascular alteration by photodynamic therapy (PDT), the utilization of high-resolution, high-speed, and wide-field photoacoustic microscopy (PAM) has gained enormous interest. The rapid changes in vasculature during PDT treatment and monitoring of tumor tissue activation in the orthotopic pancreatic cancer model have received limited attention in previous studies. Here, a fully two-axes waterproof galvanometer scanner-based photoacoustic microscopy (WGS-PAM) system was developed for in vivo monitoring of dynamic variations in micro blood vessels due to PDT in an orthotopic pancreatic cancer mouse model. The photosensitizer (PS), Chlorin e6 (Ce6), was utilized to activate antitumor reactions in response to the irradiation of a 660 nm light source. Microvasculatures of angiogenesis tissue were visualized on a 40 mm2 area using the WGS-PAM system at 30 min intervals for 3 h after the PDT treatment. The decline in vascular intensity was observed at 24.5% along with a 32.4% reduction of the vascular density at 3 h post-PDT by the analysis of PAM images. The anti-vascularization effect was also identified with fluorescent imaging. Moreover, Ce6-PDT increased apoptotic and necrotic markers while decreasing vascular endothelial growth factor (VEGF) expression in MIA PaCa-2 and BxPC-3 pancreatic cancer cell lines. The approach of the WGS-PAM system shows the potential to investigate PDT effects on the mechanism of angiographic dynamics with high-resolution wide-field imaging modalities.

Targeted Delivery of Catalase and Photosensitizer Ce6 by a Tumor-Specific Aptamer Is Effective against Bladder Cancer In Vivo.

Photodynamic therapy (PDT) is often applied in a clinical setting to treat bladder cancer. However, current photosensitizers report drawbacks such as low efficacy, low selectivity, and numerous side effects, which have limited the clinical values of PDT for bladder cancer. Previously, we developed the first bladder cancer-specific aptamer that can selectively bind to and be internalized by bladder tumor cells versus normal uroepithelium cells. Here, we use an aptamer-based drug delivery system to deliver photosensitizer chlorine e6 (Ce6) into bladder tumor cells. In addition to Ce6, we also incorporate catalase into the drug complex to increase local oxygen levels in the tumor tissue. Compared with free Ce6, an aptamer-guided DNA nanotrain (NT) loaded with Ce6 and catalase (NT-Catalase-Ce6) can specifically recognize bladder cancer cells, produce oxygen locally, induce ROS in tumor cells, and cause mitochondrial apoptosis. In an orthotopic mouse model of bladder cancer, the intravesical instillation of NT-Catalase-Ce6 exhibits faster drug internalization and a longer drug retention time in tumor tissue compared with that in normal urothelium. Moreover, our modified PDT significantly inhibits tumor growth with fewer side effects such as cystitis than free Ce6. This aptamer-based photosensitizer delivery system can therefore improve the selectivity and efficacy and reduce the side effects of PDT treatment in mouse models of bladder cancer, bearing a great translational value for bladder cancer intravesical therapy.

Detection of Arsenic(V) by Fluorescence Sensing Based on Chlorin e6-Copper Ion.

The high toxicity of arsenic (As) can cause irreversible harm to the environment and human health. In this study, the chlorin e6 (Ce6), which emits fluorescence in the infrared region, was introduced as the luminescence center, and the addition of copper ion (Cu2+) and As(V) provoked a regular change in fluorescence at 652 nm, whereas that of As(III) was 665 nm, which was used to optionally detect Cu2+, arsenic (As(III), and As(V)). The limit of detection (LOD) values were 0.212 µM, 0.089 ppm, and 1.375 ppb for Cu2+, As(III), and As(V), respectively. The developed method can be used to determine Cu2+ and arsenic in water and soil with good sensitivity and selectivity. The 1:1 stoichiometry of Ce6 with Cu2+ was obtained from the Job plot that was developed from UV-visible spectra. The binding constants for Cu2+ and As(V) were established to be 1.248 × 105 M-1 and 2.35 × 1012 M-2, respectively, using B-H (Benesi-Hildebrand) plots. Fluorescence lifetimes, B-H plots, FT-IR, and 1H-NMR were used to postulate the mechanism of Cu2+ fluorescence quenching and As(V) fluorescence restoration and the interactions of the two ions with the Ce6 molecule.

Tumor fluorescence and oxygenation monitoring during photodynamic therapy with chlorin e6 photosensitizer.

BACKGROUND: The study is aimed at developing a method for monitoring photodynamic therapy (PDT) of a tumor using chlorin-type photosensitizers (PSs). Lack of monitoring of chlorin e6 (Cе6) photobleaching, hemoglobin oxygenation and blood flow during light exposure can limit the PDT effectiveness. MATERIALS AND METHODS: Phototheranostics includes spectral-fluorescence diagnostics of Ce6 distribution in the NIR range and PDT with simultaneous assessment of hemoglobin oxygenation and tumor blood flow. Fluorescence diagnostics and PDT were performed using the single laser λexc=660 ± 5 nm. RESULTS: Combined spectroscopic PDT monitoring method allowed simultaneous estimation of Ce6 photobleaching, hemoglobin oxygenation and tumor vascular thrombosis during PDT without interrupting the therapeutic light exposure. CONCLUSION: The developed method of tumor phototheranostics using chlorin-type PSs may make it possible to personalize the duration of therapeutic light exposure during PDT.

Ferroptosis boosted oral cancer photodynamic therapy by carrier-free Sorafenib-Ce6 self-assembly nanoparticles.

Oral cancer is a disease with high morbidity and mortality worldwide and greatly impacts the quality of life, especially in patients with advanced stages. Photodynamic therapy (PDT) is one of the most effective clinical treatments for oral cancers. However, most clinically applied photosensitizers have several deficiencies, including oxygen dependence, poor aqueous solubility, and a lack of tumor-targeting ability. Herein, the carrier-free multifunctional Sorafenib (Sor), chlorin e6 (Ce6), and Fe3+ self-assembly co-delivery nanoparticles (Sor-Ce6 NPs) were constructed via combining a ferroptosis inducer Sor and a photosensitizer Ce6 for synergetic therapy. The as-synthesized Sor-Ce6 NPs presented excellent colloidal stability and water dispersity with good in vivo tumor-targeting ability. More significantly, the low dose of Sor-Ce6 NPs had little dark toxicity but produced significantly enhanced ROS and supplied O2 sustainably to increase phototoxicity through ferroptosis pathway. Notably, the Sor-Ce6 NPs showed significantly higher in vitro and in vivo anti-tumor efficacy than the Sor/Ce6 mixture due to the improvement of cellular uptake and the incorporation of foreign Fe ions in the system, which also confer the T1 magnetic resonance-guided imaging ability to the formed Sor-Ce6 NPs. Our study demonstrates a promising self-assembled strategy for overcoming hypoxia-related PDT resistance for oral cancer treatment.

Controllable Assembly of Cu2+ and Chlorin E6 for H2 S-Activatable Recognition of Bacterial Infection and Enhanced Antibacterial Therapy.

Antibacterial photodynamic therapy (APDT) has emerged as one of the intriguing strategies to combat bacterial resistance. However, the antibacterial efficacy of APDT is found to be severely impacted by the hydrogen sulfide (H2 S)-overproduced bacterial infection microenvironment. Herein, a multifunctional APDT platform is developed by assembling Cu2+ and chlorin e6 (Ce6), which exhibits unique H2 S-activatable fluorescence (FL) and antibacterial features. Noteworthily, the assembly conditions are crucial for achievement of Cu-Ce6 nanoassemblies (NAs) with the on-demand responsive properties. The quenched FL and photosensitization of Cu-Ce6 NAs can be selectively activated by the overexpressed H2 S in infected area, enabling specific recognition of bacterial infection and localized antibacterial therapy with minimized side effects. Significantly, amplified oxidative stress is achieved owning to the effective consumption of H2 S by Cu2+ in the NAs, leading to an enhanced APDT. The antibacterial mechanisms including broad-spectrum APDT activity of released Ce6, inherent sterilization effects of produced copper polysulfides and the accompanying disturbance of bacterial sulphide metabolism are further identified. This study may pave a new avenue for the rational design of intelligent APDT platform using minimalist biological building units and thus facilitating the clinical translation of nano-antibacterial agents.

Localized Photodynamic Therapy Using a Chlorin e6-Embedded Silicone-Covered Self-Expandable Metallic Stent as a Palliative Treatment for Malignant Esophageal Strictures.

Localized photodynamic therapy (PDT) uses a polymeric-photosensitizer (PS)-embedded, covered self-expandable metallic stent (SEMS). PDT is minimally invasive and a noteworthy potential alternative for treating esophageal strictures, where surgery is not a viable option. However, preclinical evidence is insufficient, and optimized irradiation energy dose ranges for localized PDT are unclear. Herein, we validated the irradiation energy doses of the SEMS (embedded in a PS using chlorin e6 [Ce6] and covered in silicone) and PDT-induced tissue changes in a rat esophagus. Cytotoxicity and phototoxicity in the Ce6-embedded SEMS piece with laser irradiation were significantly higher than that of the silicone-covered SEMS with or without laser and the Ce6-embedded silicone-covered SEMS without laser groups (all p < 0.001). Moreover, surface morphology, atomic changes, and homogeneous coverage of the Ce6-embedded silicone-covered membrane were confirmed. The ablation range of the porcine liver was proportionally increased with the irradiation dose (all p < 0.001). The ablation region was identified at different irradiation energy doses of 50, 100, 200, and 400 J/cm2. The in vivo study in the rat esophagus comprised a control group and 100, 200, and 400 J/cm2 energy-dose groups. Finally, histology and immunohistochemistry (TUNEL and Ki67) confirmed that the optimized Ce6-embedded silicone-covered SEMS with selected irradiation energy doses (200 and 400 J/cm2) effectively damaged the esophageal tissue without ductal perforation. The polymeric PS-embedded silicone-covered SEMS can be easily placed via a minimally invasive approach and represents a promising new approach for the palliative treatment of malignant esophageal strictures.

Chlorophyllin-Containing Copolymers and Their Responsive Properties.

Copper-chlorophyllin is a water-soluble derivative of chlorophylls and shows low cytotoxicity and antimutagenic properties in cultured cells. It has multiple applications, including its use as a photosensitizer in photothermal therapy because of its green light-activated photothermal performance. In this work, it was copolymerized with a poly(ethylene glycol) methacrylic monomer to yield random copolymers by free radical polymerization, which showed dual temperature- and pH-dependent phase transitions in aqueous solutions. The cloud points of the copolymer solutions were raised by lowering the pH of the aqueous solutions due to the protonation of the carboxylic groups on the chlorophyllin moieties, which decreased the overall hydrophilicity of the polymers. At low pH values, complete protonation of the carboxylic acid groups of the chlorophyllin moieties led to an irreversible aggregation of the copolymers in water. The incorporation of chlorophyllin in the copolymer improved its stability over its single molecular form.

ß-Cyclodextrin Nanophotosensitizers for Redox-Sensitive Delivery of Chlorin e6.

The aim of this study is to prepare redox-sensitive nanophotosensitizers for the targeted delivery of chlorin e6 (Ce6) against cervical cancer. For this purpose, Ce6 was conjugated with ß-cyclodextrin (bCD) via a disulfide bond, creating nanophotosensitizers that were fabricated for the redox-sensitive delivery of Ce6 against cancer cells. bCD was treated with succinic anhydride to synthesize succinylated bCD (bCDsu). After that, cystamine was attached to the carboxylic end of bCDsu (bCDsu-ss), and the amine end group of bCDsu-ss was conjugated with Ce6 (bCDsu-ss-Ce6). The chemical composition of bCDsu-ss-Ce6 was confirmed with 1H and 13C NMR spectra. bCDsu-ss-Ce6 nanophotosensitizers were fabricated by a dialysis procedure. They formed small particles with an average particle size of 152.0 ± 23.2 nm. The Ce6 release rate from the bCDsu-ss-Ce6 nanophotosensitizers was accelerated by the addition of glutathione (GSH), indicating that the bCDsu-ss-Ce6 nanophotosensitizers have a redox-sensitive photosensitizer delivery capacity. The bCDsu-ss-Ce6 nanophotosensitizers have a low intrinsic cytotoxicity against CCD986Sk human skin fibroblast cells as well as Ce6 alone. However, the bCDsu-ss-Ce6 nanophotosensitizers showed an improved Ce6 uptake ratio, higher reactive oxygen species (ROS) production, and phototoxicity compared to those of Ce6 alone. GSH addition resulted in a higher Ce6 uptake ratio, ROS generation, and phototoxicity than Ce6 alone, indicating that the bCDsu-ss-Ce6 nanophotosensitizers have a redox-sensitive biological activity in vitro against HeLa human cervical cancer cells. In a tumor xenograft model using HeLa cells, the bCDsu-ss-Ce6 nanophotosensitizers efficiently accumulated in the tumor rather than in normal organs. In other words, the fluorescence intensity in tumor tissues was significantly higher than that of other organs, while Ce6 alone did not specifically target tumor tissue. These results indicated a higher anticancer activity of bCDsu-ss-Ce6 nanophotosensitizers, as demonstrated by their efficient inhibition of the growth of tumors in an in vivo animal tumor xenograft study.

Targeted treatment of atherosclerosis with protein-polysaccharide nanoemulsion co-loaded with photosensitiser and upconversion nanoparticles.

Macrophages are the most abundant cell group in atherosclerosis (AS) lesions and play a vital role in all stages of AS progression. Recent research has shown that reactive oxygen species (ROS) generation from photodynamic therapy (PDT) induces macrophage autophagy to improve abnormal lipid metabolism and inflammatory environment. Especially in macrophage-derived foam cells, which has become a potential strategy for the treatment of AS. In this study, we prepared the conjugate (DB) of dextran (DEX) and bovine serum albumin (BSA). The DB was used as the emulsifier to prepare nanoemulsion loaded with upconversion nanoparticles (UCNPs) and chlorin e6 (Ce6) (UCNPs-Ce6@DB). The DEX modified on the surface of the nanoemulsion can recognise and bind to the scavenger receptor class A (SR-A) highly expressed on macrophages and promote the uptake of macrophage-derived foam cells in AS plates through SR-A-mediated endocytosis. In addition, UCNPs-Ce6@DB-mediated PDT enhanced ROS generation and induced autophagy in macrophage-derived foam cells, enhanced the expression of ABCA1, a protein closely related to cholesterol efflux, and inhibited the secretion of pro-inflammatory cytokines. Ultimately, UCNPs-Ce6@DB was shown to inhibit plaque formation in mouse models of AS. In conclusion, UCNPs-Ce6@DB offers a promising treatment for AS.

Impact of the lipid phase composition and state on the in vitro digestibility and chlorophyllin bioaccessibility of W1/O/W2 emulsions into whole milk.

Water-in-oil-in-water (W1/O/W2) emulsions offer the potential to deliver hydrophilic bioactive compounds into foods, yet their application remains limited due to their instability. Thus, the impact of lipid phase composition and state on the colloidal stability, in vitro lipid digestibility and chlorophyllin (CHL) bioaccessibility of W1/O/W2 emulsions before and after incorporation into whole milk was studied. Medium-chain triglyceride oil (MCT) was used as a liquid lipid phase and MCT with glyceryl stearate (GS) or pure hydrogenated palm oil (HPO) as gelled lipid phases. The lipid phase composition was crucial to forming W1/O/W2 emulsions. MCT or MCT+GS allowed the successful formation of W1/O/W2 emulsions, being more stable upon gastric conditions those formulated with MCT+GS than pure MCT. In contrast, the use of HPO led to phase separation, which was maintained after the gastric conditions. Regarding their lipid digestibility, W1/O/W2 emulsions formulated with MCT or MCT+GS were fully digested, whereas only 40% of the lipid was digested using HPO. In accordance, the CHL bioaccessibility was higher using MCT or MCT+GS than HPO. When co-digested with whole milk, the colloidal stability and lipid digestibility of the W1/O/W2 emulsions with MCT or MCT+GS were not altered, whereas the W1/O/W2 emulsion-HPO showed enhanced colloidal stability and lipid digestibility (57.71 ± 3.06%), due to the surface-active properties of milk protein. The present study provides useful information to develop stable functional foods enriched with hydrophilic bioactive compounds by using W1/O/W2 emulsions.

Revealing the significance of chlorophyll b in the moss Physcomitrium patens by knocking out two functional chlorophyllide a oxygenase.

The chlorophyllide a oxygenase (CAO) plays a crucial role in the biosynthesis of chlorophyll b (Chl b). In the moss Physcomitrium patens (P. patens), two distinct gene copies, PpCAO1 and PpCAO2, are present. In this study, we investigate the differential expression of these CAOs following light exposure after a period of darkness (24 h) and demonstrate that the accumulation of Chl b is only abolished when both genes are knocked out. In the ppcao1cao2 mutant, most of the antenna proteins associated with both photosystems (PS) I and II are absent. Despite of the existence of LHCSR proteins and zeaxanthin, the mutant exhibits minimal non-photochemical quenching (NPQ) capacity. Nevertheless, the ppcao1cao2 mutant retains a certain level of pseudo-cyclic electron transport to provide photoprotection for PSI. These findings shed light on the dual dependency of Chl b synthesis on two CAOs and highlight the distinct effects of Chl b deprival on PSI and PSII core complexes in P. patens, a model species for bryophytes.

Chlorin-e6 conjugated to the antimicrobial peptide LL-37 loaded nanoemulsion enhances photodynamic therapy against multi-species biofilms related to periodontitis.

In our previous studies, Chlorin-e6 (Ce6) demonstrated a significant reduction of microorganisms' viability against multi-species biofilm related to periodontitis while irradiated with blue light. However, the conjugation of Ce6 and antimicrobial peptides, and the incorporation of this photosensitizer in a nanocarrier, is still poorly explored. We hypothesized that chlorin-e6 conjugated to the antimicrobial peptide LL-37 loaded nanoemulsion could inhibit a multi-species biofilm related to periodontitis during photodynamic therapy (PDT), the pre-treatment with hydrogen peroxide was also tested. The nanoemulsion (NE) incorporated with Ce6 was characterized regarding the physiochemical parameters. Images were obtained by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Later, the Ce6 and LL-37 incorporated in NE was submitted to UV-Vis analysis and Reactive Oxygen Species (ROS) assay. Finally, the combined formulation (Ce6+LL-37 in nanoemulsion) was tested against multi-species biofilm related to periodontitis. The formed nanoformulation was kinetically stable, optically transparent with a relatively small droplet diameter (134.2 unloaded and 146.9 loaded), and weak light scattering. The NE system did not impact the standard UV-VIS spectra of Ce6, and the ROS production was improved while Ce6 was incorporated in the NE. The combination of Ce6 and LL-37 in NE was effective to reduce the viability of all bacteria tested. The treatment with hydrogen peroxide previous to PDT significantly impacted bacterial viability. The current aPDT regimen was the best already tested against periodontal biofilm by our research team. Our results suggest that this combined protocol must be exploited for clinical applications in localized infections such as periodontal disease. - Nanoemulsion demonstrated to be an excellent nanocarrier for photodynamic application. - Chlorin-e6 incorporated in nanoemulsion showed great physicochemical and biophotonic parameters. - The combination of chlorin-e6 and LL-37 peptide in nanoemulsion is effective to eliminate periodontal pathogenic bacteria. - The treatment with hydrogen peroxide previous to PDT significantly impacted bacterial viability.

Modulation of tumour hypoxia by ultrasound-responsive microbubbles to enhance the sono-photodynamic therapy effect on triple-negative breast cancer.

Sono-photodynamic therapy (SPDT) is an oxidative stress-dependant antitumour treatment modality. Due to the hypoxic tumour microenvironment, the antitumour effect of SPDT is limited. In this study, we developed lipid vesicles to transport a photosensitizer (chlorin e6, Ce6) and oxygen into tumours to promote SPDT efficiency on triple-negative breast cancer in vitro and in vivo. The results showed that compared with the same concentration of free Ce6, Lipo-Ce6 produced a higher singlet oxygen level under light irradiation. Cellular Lipo-Ce6 accumulation was 4-fold higher than that of free Ce6. The cytotoxicity on 4T1 cells caused by Lipo-Ce6-SPDT was significantly stronger than that caused by free Ce6-SPDT, and oxygen microbubbles (O2MB) further enhanced the cytotoxicity of Lipo-Ce6-SPDT under hypoxic conditions. Cellular ROS production in the Lipo-Ce6-SPDT+O2MB group was approximately 2.5-fold higher than that in the Lipo-Ce6-SPDT+C3F8MB group. Furthermore, O2MB rapidly relieved 4T1 subcutaneous xenograft hypoxia conditions under ultrasound exposure and significantly improved the antitumour activity of SPDT in vivo. These results indicate that the combination of O2MB and a high-activity liposome photosensitizer can significantly enhance the antitumour efficiency of SPDT for hypoxic tumours.

Evidence of increases of phytol and chlorophyllide by enzymatic dephytylation of chlorophylls in smoothie made from spinach leaves.

Phytol is a diterpene alcohol found abundantly in nature as the phytyl side chain of chlorophylls. Free form of phytol and its metabolites have been attracting attention because they have a potential to improve the lipid and glucose metabolism. On the other hand, phytol is unfavorable for those who suffering from Refsum's disease. However, there is little information on the phytol contents in leafy vegetables rich in chlorophylls. This study indicated that raw spinach leaves contain phytol of 0.4-1.5 mg/100 g fresh weight. Furthermore, crude enzyme extracted from the leaves showed the enzyme activities involved in dephytylation of chlorophyll derivatives and they were high at mild alkaline pH and around 45°C, and lowered at 55°C or above. Under the optimum pH and temperature for such enzymes determined in the model reaction using the crude enzyme, phytol content in the smoothie made from raw spinach leaves increased with an increase of chlorophyllide, another reaction product. Comparison between the increased amounts of phytol and chlorophyllide showed that the enzymatic dephytylation of chlorophylls was critically responsible for the increase of phytol in the smoothie. PRACTICAL APPLICATION: Phytol, which is released by the enzymes related to chlorophyll metabolism in plants, has been investigated because of its potential abilities to improve the lipid metabolism and blood glucose level. In contrast to such health benefits, they are known to be toxic for patients suffering from Refsum's disease. This research for the first time reports the phytol content in raw spinach leaves and that phytol can be increased in the smoothie made from spinach leaves by the action of endogenous enzymes on chlorophyll derivatives under a certain condition. These results help control phytol content in the smoothies.

Porphin e6 complex loaded with gold nanorod mesoporous silica enhances photodynamic therapy in ovarian cancer cells in vitro.

A growing amount of experimental evidence has proven that the application of gold nanorods (AuNRs) in photodynamic therapy (PDT) can significantly enhance its therapeutic efficacy. The aim of this study was to establish a protocol for investigating the effect of gold nanorods loaded with the photosensitizer chlorin e6 (Ce6) on photodynamic therapy in the OVCAR3 human ovarian cancer cell line in vitro and to determine whether the PDT effect was different from that of Ce6 alone. OVCAR3 cells were randomly divided into three groups: the control group, Ce6-PDT group, and AuNRs@SiO2@Ce6-PDT group. Cell viability was measured by MTT assay. The generation of reactive oxygen species (ROS) was measured by a fluorescence microplate reader. Cell apoptosis was detected by flow cytometry. The expression of apoptotic proteins was detected by immunofluorescence and western blotting. The results showed that compared with that of the Ce6-PDT group, the cell viability of the AuNRs@SiO2@Ce6-PDT group was significantly decreased (P < 0.05) in a dose-dependent manner, and ROS production increased significantly (P < 0.05). The flow cytometry results showed that the proportion of apoptotic cells in the AuNRs@SiO2@Ce6-PDT group was significantly higher than that in the Ce6-PDT group (P < 0.05). Immunofluorescence and western blot results showed that the protein expression levels of cleaved caspase-9, cleaved caspase-3, cleaved PARP, and Bax in the AuNRs@SiO2@Ce6-PDT-treated-OVCAR3 cells were higher than those in the Ce6-PDT-treated cells (P < 0.05), and the protein expression levels of caspase-3, caspase-9, PARP, and Bcl-2 were slightly lower than those in the Ce6-PDT group (P < 0.05). In summary, our results show that AuNRs@SiO2@Ce6-PDT has a significantly stronger effect on OVCAR3 cells than the effect of Ce6-PDT alone. The mechanism may be related to the expression of Bcl-2 family and caspase family in the mitochondrial pathway.

Analysis of Chlorophylls/Chlorophyllins in Food Products Using HPLC and HPLC-MS Methods.

Of the different quality parameters of any food commodity or beverage, color is the most important, attractive and choice-affecting sensory factor to consumers and customers. Nowadays, food industries are interested in making the appearance of their food products attractive and interesting in order to appeal to consumers/customers. Natural green colorants have been accepted universally due to their natural appeal as well as their nontoxic nature to consumers. In addition, several food safety issues mean that natural green colorants are preferable to synthetic food colorants, which are mostly unsafe to the consumers but are less costly, more stable, and create more attractive color hues in food processing. Natural colorants are prone to degradation into numerous fragments during food processing, and thereafter, in storage. Although different hyphenated techniques (especially high-performance liquid chromatography (HPLC), LC-MS/HRMS, and LC/MS-MS are extensively used to characterize all these degradants and fragments, some of them are not responsive to any of these techniques, and some substituents in the tetrapyrrole skeleton are insensitive to these characterization tools. Such circumstances warrant an alternative tool to characterize them accurately for risk assessment and legislation purposes. This review summarizes the different degradants of chlorophylls and chlorophyllins under different conditions, their separation and identification using various hyphenated techniques, national legislation regarding them, and the challenges involved in their analysis. Finally, this review proposes that a non-targeted analysis method that combines HPLC and HR-MS assisted by powerful software tools and a large database could be an effective tool to analyze all possible chlorophyll and chlorophyllin-based colorants and degradants in food products in the future.

Predicted Structure of the BciC Enzyme Catalyzing the Removal of the C132-Methoxycarbonyl Group for Biosynthesis of Chlorosomal Chlorophylls: A Mechanism for Dual Catalytic Functions of Hydrolysis and Decarboxylation inside Its Active Site.

Green photosynthetic bacteria, one of the phototrophs, have the largest and most efficient light-harvesting antenna systems, called chlorosomes. The core part of chlorosomes consists of unique bacteriochlorophyll c/d/e molecules. In the biosynthetic pathway of these molecules, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. Two sequential reactions have been proposed for the BciC enzymatic demethoxycarbonylation: the BciC enzyme would catalyze the hydrolysis of the C132-methoxycarbonyl group, and the resulting carboxylic acid would be rapidly decarboxylated to generate pyrochlorophyllide a. In this study, we computationally predicted the three-dimensional structure of the BciC protein. Its active site was proposed based on structural analysis using docking simulation. In vitro enzymatic reaction assays of mutated BciC supported the prediction. The BciC enzymatic hydrolysis would be an aspartic/glutamic acid hydrolase, which involves the amino residues E85 and D180. Furthermore, Y58 and H126 might depend on stabilization and/or recognition with the substrate. Most importantly, H137 would protonate 13-C═O or deprotonate C132-COOH in the hydrolyzed product to promote decarboxylation. In conclusion, the BciC enzyme has the dual functions of hydrolysis and decarboxylation.