Effects of Silencing Heme Biosynthesis Enzymes on 5‐Aminolevulinic Acid‐mediated Protoporphyrin IX Fluorescence and Photodynamic Therapy

Aminolevulinic acid (ALA)‐mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA‐mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA‐mediated PDT. Silencing PBGS or PBGD significantly reduced ALA‐stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA‐stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA‐stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA‐PDT, while increased sensitivity to ALA‐PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA‐mediated PpIX fluorescence and PDT efficacy.     

The p53‐Dependent Expression of Frataxin Controls 5‐Aminolevulinic Acid‐Induced Accumulation of Protoporphyrin IX and Photo‐Damage in Cancerous Cells

Mitochondrial frataxin is involved in various functions such as iron homeostasis, iron–sulfur cluster biogenesis, the protection from oxidative stress and apoptosis and acts as a tumor suppressor protein. We now show that the expression of frataxin is stimulated in a p53‐dependent manner and prove that frataxin is a direct p53 target gene by showing that the p53‐responsive element in the promoter of the mouse frataxin gene is bound by p53. The bacterial expression of human frataxin stimulated maturation of human ferrochelatase, which catalyzes the insertion of iron into protoporphyrin at the last step of heme biosynthesis. Overexpression of frataxin in human cancer A431 and HeLa cells lowered 5‐aminolevulinic acid(ALA)‐induced accumulation of protoporphyrin and induced resistance to ALA‐induced photo‐damage, whereas p53 silencing with siRNA in non tumor HEK293T cells down‐regulated the expression of frataxin and increased the accumulation of protoporphyrin. Thus, the decrease of the expression of frataxin unregulated by p53 in tumor cells enhances ALA‐induced photo‐damage, by down‐regulation of mitochondrial functions.     

Enhancement of Acid‐Catalyzed Esterification by the Addition of Base

General acid‐catalyzed reaction can be enhanced by the addition of base. Self‐catalyzed esterification of benzoic acid and octan‐1‐ol was enhanced by the addition of certain base such as imidazole. The rate of the esterification was accelerated as the concentration of imidazole increased. Trans‐esterification of 4‐nitrophenyl acetate was promoted in chloroform by the mixture of benzoic acid and imidazole, but not by benzoic acid or imidazole alone.     

Acid‐mediated regioselective alkylation of 1,2,3‐triazole

1,2,3‐Triazole reacts with isopropyl alcohol in concentrated sulfuric acid to yield 1‐isopropyl‐1H‐1,2,3‐triazole as the only reaction product.     

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.     

Enhancement of Fatty Acid‐based Polyurethanes Cytocompatibility by Non‐covalent Anchoring of Chondroitin Sulfate

For tissue engineering purpose biopolymer chondroitin sulfate (CS), one of the major components of cartilage and bone extracellular matrix, was immobilized onto the surface of amino‐functionalized polyurethane (PU) films derived from naturally occurring oleic and 10‐undecenoic acids. The amino‐functionalized PUs were prepared by aminolysis with 1,6‐hexamethylenediamine of synthesized PUs containing methyl ester groups. FTIR‐ATR, XPS, SEM, and water contact angle measurements were used to confirm the surface changes at each step of treatment, both in morphologies and chemical composition. Cytotoxicity and cell morphology analysis using osteoblast cell line MG63 showed that PU‐CS films are suitable materials for cell growth, spreading, and differentiation.

     

Neurotransmitter Transporter Family Including SLC6A6 and SLC6A13 Contributes to the 5‐Aminolevulinic Acid (ALA)‐Induced Accumulation of Protoporphyrin IX and Photodamage,through Uptake of ALA by Cancerous Cells

δ‐Aminolevulinic acid (ALA)‐induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA‐induced accumulation of protoporphyrin by the treatment of colon cancer DLD‐1 and epithelial cancer HeLa cells with γ‐aminobutyric acid (GABA)‐related compounds. When the cells were treated with GABA, taurine and β‐alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA‐ and ALA methylester‐dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA‐treated cells were exposed to white light, the extent of photodamage increased in SLC6A6‐ and SLC6A13‐expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD‐1 and HeLa cells decreased the ALA‐induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA‐induced accumulation of protoporphyrin in cancerous cells.     

Improved In vitro and In vivo Cutaneous Delivery of Protoporphyrin IX from PLGA‐based Nanoparticles

We report the development of d , l lactic co‐glycolic acid) (PLGA)‐based nanoparticles (NPs) for topical delivery of protoporphyrin IX (PpIX), a photosensitizer (PS), in treatments like photodynamic therapy (PDT) of skin cancers. PpIX‐NPs were obtained in ~75.0% yield, encapsulation efficiency of 67.7%, drug content of 50.3 μg mg^?1, average diameter of 290 nm maintained up to 30 days and a zeta potential of 32.3 mV. Sustained in vitro release of PpIX through artificial membranes following Higuchi kinetics was kept up to 10 days. In vitro retentions of PpIX both in stratum corneum (SC) and epidermis + dermis ([EP + D]) were higher from NPs (23.0 and 10.0 times, respectively) compared to control solutions at all times. Quantification of PpIX by extraction, after in vivo skin application of NPs‐PpIX on hairless mice, showed higher retention of the PS both in SC and in [EP + D] (3.0 and 2.0 times, respectively) compared to control solutions. Taken together, the results indicate that NPs are suitable for PpIX encapsulation showing minimal permeation through the skin and a localized effect, characteristics of a potential and promising delivery system for PDT‐associated treatments of skin cancers, photodiagnosis and their off‐label uses.     

Ag Nanowire‐Ag Nanoparticle Hybrids for the Highly Enhanced Fluorescene Detection of Protoporphyrin IX Based on Surface Plasmon‐Enhanced Fluorescence

In this study, we developed an approach to fabricate novel 1D Ag NWs‐Ag NPs hybrid substrate for enhanced fluorescene detection of protoporphyrin IX (PpIX) based on surface plasmon‐enhanced fluorescence. The Ag NWs‐Ag NPs hybrid was synthesized by combining the hydrothermal method and self‐assembly method with the asisstance of polyvinylpyrrolidone (PVP). When the Ag NWs‐Ag NPs hybrid was deposited on the glass substrate and employed as active substrate to detect PpIX, the fluorescence intensity of PpIX was enhanced greatly due to the coupling effect of localized surface plasmon‐localized surface plasmon (LSP‐LSP) and localized surface plasmon‐surface plasmon propagation (LSP‐SPP) which induced great enhancement of the electromagnetic field. Furthermore, the enhancement effect was approximately linear when the concentration of PpIX was ranged from 1×10^?7 mol/L to 2×10^?5 mol/L, and the photobleaching phenomenon of PpIX was reduced greatly, indicating that the fabricated Ag NWs‐Ag NPs hybrid substrate had well performance for PpIX imaging. This work provides an effective approach to prepare highly sensitive and stable fluorescence enhancement substrate, and has great potential application in fluorescence imaging.     

Noninvasive Optical Imaging of UV‐Induced Squamous Cell Carcinoma in Murine Skin: Studies of Early Tumor Development and Vitamin D Enhancement of Protoporphyrin IX Production

Better noninvasive techniques are needed to monitor protoporphyrin IX (PpIX) levels before and during photodynamic therapy (PDT) of squamous cell carcinoma (SCC) of the skin. Our aim was to evaluate (1) multispectral fluorescent imaging of ultraviolet light (UV)‐induced cancer and precancer in a mouse model of SCC and (2) multispectral imaging and probe‐based fluorescence detection as a tool to study vitamin D (VD) effects on aminolevulinic acid (ALA)‐induced PpIX synthesis. Dorsal skin of hairless mice was imaged weekly during a 24‐week UV carcinogenesis protocol. Hot spots of PpIX fluorescence were detectable by multispectral imaging beginning at 14 weeks of UV exposure. Many hot spots disappeared after cessation of UV at week 20, but others persisted or became visible after week 20, and corresponded to tumors that eventually became visible by eye. In SCC‐bearing mice pretreated with topical VD before ALA application, our optical techniques confirmed that VD preconditioning induces a tumor‐selective increase in PpIX levels. Fluorescence‐based optical imaging of PpIX is a promising tool for detecting early SCC lesions of the skin. Pretreatment with VD can increase the ability to detect early tumors, providing a potential new way to improve efficacy of ALA‐PDT.     

Protoporphyrin IX‐Functionalized AgSiO2 Core–Shell Nanoparticles: Plasmonic Enhancement of Fluorescence and Singlet Oxygen Production

Metal‐enhanced processes arising from the coupling of a dye with metallic nanoparticles (NPs) have been widely reported. However, few studies have simultaneously investigated these mechanisms from the viewpoint of dye fluorescence and photoactivity. Herein, protoporphyrin IX (PpIX) is grafted onto the surface of silver core silica shell NPs in order to investigate the effect of silver (Ag) localized surface plasmon resonance (LSPR) on PpIX fluorescence and PpIX singlet oxygen (¹O₂) production. Using two Ag core sizes, we report a systematic study of these photophysical processes as a function of silica (SiO₂) spacer thickness, LSPR band position and excitation wavelength. The excitation of Ag NP LSPR, which overlaps the PpIX absorption band, leads to the concomitant enhancement of PpIX fluorescence and ¹O₂ production independently of the Ag core size, but in a more pronounced way for larger Ag cores. These enhancements result from the increase in the PpIX excitation rate through the LSPR excitation and decrease when the distance between PpIX and Ag NPs increases. A maximum fluorescence enhancement of up to 14‐fold, together with an increase in photogenerated ¹O₂ production of up to five times are obtained using 100 nm Ag cores coated with a 5 nm thick silica coating.     

Water‐Mediated Recognition of Simple Alkyl Chains by Heart‐Type Fatty‐Acid‐Binding Protein

Long‐chain fatty acids (FAs) with low water solubility require fatty‐acid‐binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. However, the precise mechanism by which these proteins recognize the various lengths of simple alkyl chains of FAs with similar high affinity remains unknown. To address this question, we employed a newly developed calorimetric method for comprehensively evaluating the affinity of FAs, sub‐Angstrom X‐ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. Our results clearly showed that the heart‐type FABP (FABP3) preferentially incorporates a U‐shaped FA of C10–C18 using a lipid‐compatible water cluster, and excludes longer FAs using a chain‐length‐limiting water cluster. These mechanisms could help us gain a general understanding of how proteins recognize diverse lipids with different chain lengths.     

Chiral Phosphoric‐Acid‐Catalyzed Transfer Hydrogenation of Ethyl Ketimine Derivatives by Using Benzothiazoline

Chiral phosphoric acid catalyzed transfer hydrogenation of ketimines derived from propiophenone derivatives and reductive amination of alkyl ethyl ketone derivatives were extensively examined in the presence of two representative hydrogen donors. The excellent enantioselective transfer hydrogenation was achieved by use of benzothiazoline as a hydrogen donor. The theoretical studies elucidated that the unsymmetrical structure of benzothiazoline plays an important role in high enantioselective hydrogenation.     

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.     

Amino‐Acid‐Based Polymerizable Surfactants for the Synthesis of Chiral Nanoparticles

Amino‐acid‐based chiral surfactants with polymerizable moieties are synthesized, and a versatile approach to prepare particles thereof with a chiral surface functionality is presented. As an example of an application, the synthesized particles are tested for their ability as nucleating agents in the enantioselective crystallization of amino acid conglomerate systems, taking rac‐asparagine as a model system. Particles resulting from chiral surfactants with different tail groups are compared and the results demonstrate that only the chiral nanoparticles made of the polymerizable surfactant are able to act efficiently as nucleation agent in enantioselective crystallization.