In Vitro Anti-Inflammatory and Antioxidant Activities of pH-Responsive Resveratrol-Urocanic Acid Nano-Assemblies.

Inflammatory environments provide vital biochemical stimuli (i.e., oxidative stress, pH, and enzymes) for triggered drug delivery in a controlled manner. Inflammation alters the local pH within the affected tissues. As a result, pH-sensitive nanomaterials can be used to effectively target drugs to the site of inflammation. Herein, we designed pH-sensitive nanoparticles in which resveratrol (an anti-inflammatory and antioxidant compound (RES)) and urocanic acid (UA) were complexed with a pH-sensitive moiety using an emulsion method. These RES-UA NPs were characterized by transmission electron microscopy, dynamic light scattering, zeta potential, and FT-IR spectroscopy. The anti-inflammatory and antioxidant activities of the RES-UA NPs were assessed in RAW 264.7 macrophages. The NPs were circular in shape and ranged in size from 106 to 180 nm. The RES-UA NPs suppressed the mRNA expression of the pro-inflammatory molecules inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in a concentration-dependent manner. Incubation of LPS-stimulated macrophages with RES-UA NPs reduced the generation of reactive oxygen species (ROS) in a concentration-dependent manner. These results suggest that pH-responsive RES-UA NPs can be used to decrease ROS generation and inflammation.

The Multiple Roles of Urocanic Acid in Health and Disease.

Trans-urocanic acid (trans-UCA) is synthesized in the skin, liver, and brain. It is a major natural moisturizing factor in skin and maintains its acid pH. In skin, it isomerizes to cis-UCA following exposure to UVR. Both isomers fulfill multiple roles in health and disease. Cis-UCA has immunomodulatory properties linked with several cutaneous diseases such as skin cancer, atopic dermatitis, and urticaria and associates with systemic diseases including multiple sclerosis. The levels of UCA in the skin, brain, urine, and feces reflect some physiological processes and may be disease biomarkers. Both isomers of UCA have therapeutic potential for a range of disorders.

Stepwise pH-responsive nanoparticles for enhanced cellular uptake and on-demand intracellular release of doxorubicin.

Physicochemical properties, including particle size, zeta potential, and drug release behavior, affect targeting efficiency, cellular uptake, and antitumor effect of nanocarriers in a formulated drug-delivery system. In this study, a novel stepwise pH-responsive nanodrug delivery system was developed to efficiently deliver and significantly promote the therapeutic effect of doxorubicin (DOX). The system comprised dimethylmaleic acid-chitosan-urocanic acid and elicited stepwise responses to extracellular and intracellular pH. The nanoparticles (NPs), which possessed negative surface charge under physiological conditions and an appropriate nanosize, exhibited advantageous stability during blood circulation and enhanced accumulation in tumor sites via enhanced permeability and retention effect. The tumor cellular uptake of DOX-loaded NPs was significantly promoted by the first-step pH response, wherein surface charge reversion of NPs from negative to positive was triggered by the slightly acidic tumor extracellular environment. After internalization into tumor cells, the second-step pH response in endo/lysosome acidic environment elicited the on-demand intracellular release of DOX from NPs, thereby increasing cytotoxicity against tumor cells. Furthermore, stepwise pH-responsive NPs showed enhanced antiproliferation effect and reduced systemic side effect in vivo. Hence, the stepwise pH-responsive NPs provide a promising strategy for efficient delivery of antitumor agents.

Suppression of experimental autoimmune encephalomyelitis by ultraviolet light is not mediated by isomerization of urocanic acid.

BACKGROUND: Ultraviolet B irradiation confers strong resistance against experimental autoimmune encephalomyelitis, a model of multiple sclerosis. This protection by ultraviolet B is independent of vitamin D production but causes isomerization of urocanic acid, a naturally occurring immunosuppressant. METHODS: To determine whether UCA isomerization from trans to cis is responsible for the protection against experimental autoimmune encephalomyelitis afforded by ultraviolet B, trans- or cis-urocanic acid was administered to animals and their disease progression was monitored. RESULTS: Disease incidence was reduced by 74% in animals exposed to ultraviolet B, and skin cis-urocanic acid levels increased greater than 30%. However, increasing skin cis-urocanic acid levels independent of ultraviolet B was unable to alter disease onset or progression. CONCLUSIONS: It is unlikely that urocanic acid isomerization is responsible for the ultraviolet B-mediated suppression of experimental autoimmune encephalomyelitis. Additional work is needed to investigate alternative mechanisms by which UVB suppresses disease.

In vitro and in vivo assessment of chitosan modified urocanic acid as gene carrier.

Chitosan nanoparticles modified with 10 and 30% urocanic acid (CUA) via carbodiimide crosslinking were examined for an efficient gene delivery carrier. The CUA gene carrier was characterized by FTIR, TEM, SEM and the in vitro transfection efficiency CUA polyplex was tested with HeLa and 3T3 cells. The loading efficiency of CUA complexes with DNA was assessed at different N/P ratio of 1, 2, 4, 6, 8, and 10. The DNA loading efficiency was found be to >85% for chitosan, CUA10 and CUA30% and the DNA protection ability of CUA10 and CUA30 nanoparticle complexes was confirmed upon incubation with NheI and HindIII. The cell toxicity and cell viability results have supported the non-toxic nature of CUA10 and CUA30 nanoparticles. In vitro transfection efficiency of CUA10 and CUA30 polyplex was tested for EGFP expression in 3T3 and HeLa cells and a relative maximum % transfection of about 10% was confirmed by CUA10 and CUA30 after 96h transfection. The feasibility and biocompatibility of CUA gene carrier in transgenic chickens was also demonstrated. The in vitro transfection and in vivo embryonic viability studies further confirmed the CUA as promising gene carrier because of the improved biocompatibility and DNA protection ability.

Urocanic acid-modified chitosan nanoparticles can confer anti-inflammatory effect by delivering CD98 siRNA to macrophages.

CD98 plays an important role in the development and progression of inflammation. Here, CD98 siRNA (siCD98) was complexed with urocanic acid-modified chitosan (UAC) to form nanoparticles (NPs), which were transfected into Raw 264.7 macrophages in an effort to convey anti-inflammatory effects. Characterization showed that the generated NPs had a desirable particle size (156.0-247.1nm), a slightly positive zeta potential (15.8-17.5mV), and no apparent cytotoxicity against Raw 264.7 macrophages and colon-26 cells compared to control NPs fabricated by Oligofectamine (OF) and siRNA. Cellular uptake experiments demonstrated that macrophages exhibited a time-dependent accumulation profile of UAC/siRNA NPs. Further in vitro gene silencing experiments revealed that UAC/siCD98 NPs with a weight ratio of 60:1 yielded the most efficient knockdowns of CD98 and the pro-inflammatory cytokine, TNF-α. Indeed, the RNAi efficiency obtained with our NPs was even higher than that of the positive control OF/siCD98 NPs. These results suggest that UAC/siCD98 NPs might be a safe, efficient and promising candidate for the treatment of inflammatory disease.

Protective Effect of Chitin Urocanate Nanofibers against Ultraviolet Radiation.

Urocanic acid is a major ultraviolet (UV)-absorbing chromophore. Chitins are highly crystalline structures that are found predominantly in crustacean shells. Alpha-chitin consists of microfibers that contain nanofibrils embedded in a protein matrix. Acid hydrolysis is a common method used to prepare chitin nanofibrils (NFs). We typically obtain NFs by hydrolyzing chitin with acetic acid. However, in the present study, we used urocanic acid to prepare urocanic acid chitin NFs (UNFs) and examined its protective effect against UVB radiation. Hos: HR-1 mice coated with UNFs were UVB irradiated (302 nm, 150 mJ/cm²), and these mice showed markedly lower UVB radiation-induced cutaneous erythema than the control. Additionally, sunburn cells were rarely detected in the epidermis of UNFs-coated mice after UVB irradiation. Although the difference was not as significant as UNFs, the number of sunburn cells in mice treated with acetic acid chitin nanofibrils (ANFs) tended to be lower than in control mice. These results demonstrate that ANFs have a protective effect against UVB and suggest that the anti-inflammatory and antioxidant effects of NFs influence the protective effect of ANFs against UVB radiation. The combination of NFs with other substances that possess UV-protective effects, such as urocanic acid, may provide an enhanced protective effect against UVB radiation.

Investigation of a Degradant in a Biologics Formulation Buffer Containing L-Histidine.

PURPOSE: An unknown UV 280 nm absorbing peak was observed by SEC for protein stability samples formulated in L-histidine during a stress stability study. Understanding the source would enhance the confidence in the SEC results. We identified the unknown peak, studied the cause, and evaluated ways to eliminate it. METHODS: The unknown peak was fractionated by preparative size exclusion chromatography separations, and subsequently analyzed by Hydrophilic Interaction Chromatography (HILIC) coupled with Time-of-Flight (TOF) high resolution mass spectrometry. The possible degradation was also studied with the presence of different excipients, including metal cations, chelating agents, and amino acids. RESULTS: The unknown peak was identified to be trans-urocanic acid, a degradant of histidine, based on evidences from HILIC retention time, UV profile, accurate mass measurement, trans-cis isomerization, and pI measurement. The degradation from histidine to urocanic acids was not affected by the presence of Fe(2+), but slightly activated by Mn(2+). The chelating agents, EDTA and DTPA, counteracted the Mn(2+) effects. This degradation was evidenced to be caused by contamination. Adding alanine or cysteine as an excipient was found to reduce this degradation by 97 and 98%, respectively. CONCLUSIONS: L-histidine formulation buffer can be contaminated to induce histidine degradation to trans-urocanic acid, which shows a large UV 280 nm absorbing peak at the total permeation volume under SEC conditions. Amino acids alanine and cysteine effectively inhibit this histidine degradation.

Enhanced antitumor efficacies of multifunctional nanocomplexes through knocking down the barriers for siRNA delivery.

Multifunctional nanocomplexes (NCs) consisting of urocanic acid-modified galactosylated trimethyl chitosan (UA-GT) conjugates as polymeric vectors, poly(allylamine hydrochloride)-citraconic anhydride (PAH-Cit) as charge-reversible crosslinkers, and vascular endothelial growth factor (VEGF) siRNA as therapeutic genes, were rationally designed to simultaneously overcome the extracellular, cellular, and intracellular barriers for siRNA delivery. The strong physical stability of UA-GT/PAH-Cit/siRNA NCs (UA-GT NCs) at pH 7.4 and 6.5 endowed protection from massive dilution, competitive ions, and ubiquitous nucleases in the blood and tumorous microenvironment. Their internalization into hepato-carcinoma cells was facilitated through the recognition of galactose receptors, followed by effective escape from endosomes/lysosomes owing to the strong buffering capacity of imidazole residues. At the meantime, the endosomal/lysosomal acidity triggered the charge reversal of PAH-Cit in UA-GT NCs, thus evoking their structural disassembly and subsequently accelerated release of siRNA in the cytosol. As a result, robust in vivo performance in terms of both gene silencing and tumor inhibition was achieved by UA-GT NCs at a low siRNA dose. Moreover, neither histological nor hematological toxicity was detected following repeated intravenous administration. Therefore, UA-GT NCs potentially served as an efficient and safe candidate in the treatment of hepatocellular carcinoma through knocking down the overall barriers for siRNA delivery.

Photochemical mechanisms of radiationless deactivation processes in urocanic acid.

Urocanic acid is a UV filter found in human skin that protects the skin from UV damage but has also been linked to the onset of skin cancer and to photoimmunosuppression. We report on ab initio investigations of two rotameric forms of each of the two tautomers of neutral (E)- and (Z)-urocanic acid. We have computed the vertical singlet excitation energies of eight isomers and have explored the singlet excited-state reaction paths of several photochemical processes for radiationless excited-state deactivation: the E/Z photoisomerization, an electron-driven proton transfer for an intramolecularly hydrogen-bonded Z isomer, as well as the hydrogen-atom detachment process and the ring-puckering process involving the NH group inherent to the imidazole moiety. We have optimized the S1/S0 conical intersections for each of these processes and located additional ππ*/nπ* conical intersections. Because of the reversed energetic order of the nπ* and ππ* states in the N3H and N1H tautomers, an energy window exists where the N3H tautomers can be excited to the nπ* state, from which only the photoisomerization process is accessible, while the N1H tautomers can be excited to the ππ* state, from which several deexcitation processes compete from the onset of the absorption. These results explain the unusual dependence of the quantum yield for E→Z photoisomerization on the excitation wavelength. The present work provides novel insight into the complex photochemistry of this biomolecule and paves the way for future computational studies of the photoinduced excited-state dynamics of urocanic acid.

cis-Urocanic acid as a potential nickel(II) binding molecule in the human skin.

cis-Urocanic acid, a derivative of histidine, is one of the essential components of human skin. We found that it can bind nickel(II) ions in a pH-dependent manner, with the dissociation constant in the low millimolar range, as revealed by potentiometry, and confirmed by isothermal titration calorimetry and UV-vis spectroscopy. The binding occurs within the physiological skin pH range. Considering the fact that cis-urocanic acid is present in the human skin in concentrations as high as millimolar, this molecule may be a physiologically important player in nickel trafficking in the human organism.

Galectin-7, induced by cis-urocanic acid and ultraviolet B irradiation, down-modulates cytokine production by T lymphocytes.

Urocanic acid (UCA) is an epidermal chromophore that undergoes trans to cis isomerization after UVB irradiation. cis-UCA is a potent inhibitor of cutaneous acquired immunity. The aim of this study was to explore the genes, which are upregulated by cis-UCA in normal human epidermal keratinocytes (NHEK) and investigated its role in vitro using human T-lymphocyte cell line, Jurkat cells. DNA microarray analysis and real-time PCR investigation revealed that cis-UCA, not trans-UCA, increased the expression of a gene encoding a ß-galactoside-binding lectin, galectin-7, LGALS7B. Immunohistochemical study demonstrated that galectin-7 was highly expressed in the epidermis in the patients with actinic keratosis. Galectin-7 administration upregulated apoptosis and inhibited the expression of interleukin-2 (IL2) and interferon-γ (IFNG) mRNA in Jurkat cells. Taken together, galectin-7 may play important roles in downregulating the functions of T lymphocytes after UVB irradiation and can be developed into novel immunosuppressive therapies for inflammatory skin diseases.

Microwave spectrum, structure, tautomeric, and conformational composition of 4-vinylimidazole.

The microwave spectra of the two conformers each, of the 1H and 3H tautomers of 4-vinylimidazole, have been measured in the 48-72 GHz spectral region. The 4-vinylimidazole was generated in situ by the facile decarboxylation of urocanic acid at its vaporization temperature of 220 °C. The recognition of this reaction casts doubt on the reliability of a previous published spectroscopic study apparently mistakenly thought to be of uncontaminated vaporized urocanic acid, a natural product of great interest in skin cancer etiology. Quantum chemical theoretical predictions of the structures of each of ten possible conformers∕tautomers of urocanic acid and four of 4-vinylimidazole were performed at the ab initio MP2∕cc-pVTZ level, with vibrational predictions at the B3LYP∕cc-pVTZ and M062X∕cc-pVTZ levels. The predicted values of rotational constants for all the urocanic acid species were found to be quite inconsistent with those of the four observed spectra. For the 4-vinylimidazole isomers, the calculated relative energies suggested that all four species would have substantial equilibrium mole fractions at 220 °C. The isomers were identified by matching the observed and calculated rotational constants. The resulting assignment was found to be consistent with the predicted and observed (14)N nuclear quadrupole hyperfine multiplet patterns for a suitable rotational transition, and with the observed versus empirically calculated inertial defects. With one exception, the predicted structures were found to be planar. Resembling the case of 1-vinylimidazole, where one conformer is nonplanar, one isomer of 4-vinylimidazole was found to be quasiplanar. This seems to belong to a class of spontaneous symmetry-breaking observed in the molecular structure of some otherwise planar vinyl aromatic compounds.

Molecular targets for the protodynamic action of cis-urocanic acid in human bladder carcinoma cells.

BACKGROUND: cis-urocanic acid (cis-UCA) is an endogenous amino acid metabolite capable of transporting protons from the mildly acidic extracellular medium into the cell cytosol. The resulting intracellular acidification suppresses many cellular activities. The current study was aimed at characterizing the molecular mechanisms underlying cis-UCA-mediated cytotoxicity in cultured cancer cells. METHODS: 5367 bladder carcinoma cells were left untreated or treated with cis-UCA. Cell death was assessed by measuring caspase-3 activity, mitochondrial membrane polarization, formation and release of cytoplasmic histone-associated DNA fragments, and cellular permeabilization. Cell viability and metabolic activity were monitored by colorimetric assays. Nuclear labelling was used to quantify the effects of cis-UCA on cell cycle. The activity of the ERK and JNK signalling pathways was studied by immunoblotting with specific antibodies. Phosphatase activity in cis-UCA-treated cells was determined by assay kits measuring absorbance resulting from the dephosphorylation of an artificial substrate. All statistical analyses were performed using the two-way Student's t-test (p < 0.05). RESULTS: Here we report that treatment of the 5637 human bladder carcinoma cells with 2% cis-UCA induces both apoptotic and necrotic cell death. In addition, metabolic activity of the 5637 cells is rapidly impaired, and the cells arrest in cell cycle in response to cis-UCA. Importantly, we show that cis-UCA promotes the ERK and JNK signalling pathways by efficiently inhibiting the activity of serine/threonine and tyrosine phosphatases. CONCLUSIONS: Our studies elucidate how cis-UCA modulates several cellular processes, thereby inhibiting the proliferation and survival of bladder carcinoma cells. These anti-cancer effects make cis-UCA a potential candidate for the treatment of non-muscle invasive bladder carcinoma.

Urocanic acid improves transfection efficiency of polyphosphazene with primary amino groups for gene delivery.

The biodegradable cationic poly(2-(2-aminoethoxy)ethoxy)phosphazene (PAEP) bearing primary amino groups and a new PAEP derivative, urocanic acid (UA) modified PAEP (UA-PAEP), were synthesized and investigated for gene delivery. The results indicated that PAEP was able to condense DNA into complex nanoparticles with the size around 120 nm at the polymer/DNA ratio (N/P) of 35, at which PAEP/DNA complex nanoparticles (PACNs) showed efficient transfection activity in complete medium. After conjugating with UA at the substitution degree of 7% (UA-PAEP7), UA-PAEP7/DNA complex nanoparticles (UP7CNs) exhibited higher transfection efficiency than PACNs and UA-PAEP25/DNA complex nanoparticles (UP25CNs) and much lower cytotoxicity compared with PEI/DNA complex nanoparticles (PEICNs). The transfection experiment using a proton pump inhibitor suggested that the gene expression of PACNs and UP-PAEP/DNA complex nanoparticles (UPCNs) was dependent on the endosomal acidification process. The acetate solution (20 mM, pH5.7) improved the transfection activity of UP7CNs in HeLa and COS 7 cell lines, which was almost comparable to PEICNs at the N/P ratio of 35. Therefore, the results suggested that UP7CNs could be a promising carrier for gene delivery.

Cis-urocanic acid suppresses UV-B-induced interleukin-6 and -8 secretion and cytotoxicity in human corneal and conjunctival epithelial cells in vitro.

PURPOSE: Urocanic acid (UCA) is a major ultraviolet (UV)-absorbing endogenous chromophore in the epidermis and is also an efficacious immunosuppressant. The anti-inflammatory and cytoprotective effects of cis-UCA were studied in ocular surface cell cultures exposed to UV-B irradiation. METHODS: Human corneal epithelial cells (HCE-2) and human conjunctival epithelial cells (HCECs) were incubated with 10, 100, 1,000, and 5,000 microg/ml cis-UCA with and without a single UV-B irradiation dose. The concentrations of IL-1beta, IL-6, IL-8, and TNF-alpha in the culture medium and caspase-3 activity in the cell extract sampled were measured by enzyme-linked immunosorbent assay (ELISA). Cell viability was measured by the colorimetric MTT (3-(4,5-dimethyldiazol- 2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS: UV-B irradiation multiplied interleukin IL-6 and IL-8 secretion levels in HCE-2 cells and HCECs as analyzed with ELISA. Cell viability as measured by the MTT assay declined by 30%-50% in HCE-2 cells and by 20%-40% in HCECs after UV-B irradiation. Moreover, UV-B increased caspase-3 activity in both cell types as analyzed with ELISA. Treatment with 100 microg/ml cis-UCA completely suppressed IL-6 and IL-8 secretion, decreased caspase-3 activity, and improved cell viability against UV-B irradiation. No significant effects on IL-6 or IL-8 secretion, caspase-3 activity, or viability of the non-irradiated cells were observed with 100 microg/ml cis-UCA in both cell types. The 5,000 microg/ml concentration was toxic. CONCLUSIONS: These findings indicate that cis-UCA may represent a promising anti-inflammatory and cytoprotective treatment option to suppress UV-B-induced inflammation and cellular damage in human corneal and conjunctival epithelial cells.

cis-Urocanic acid stimulates primary human keratinocytes independently of serotonin or platelet-activating factor receptors.

Urocanic acid (UCA) is a major epidermal chromophore that undergoes trans to cis isomerization after ultraviolet radiation (UVR). cis-UCA suppresses cell-mediated immunity. Recent studies suggest that cis-UCA binds to serotonin (5-hydroxytryptamine) 2A (5-HT(2A)) receptor and that antagonists of 5-HT(2A) and the platelet-activating factor (PAF) receptor can block cis-UCA-induced immune suppression in mice. Here, we examined the involvement of 5-HT(2A) and PAF receptors in the ability of cis-UCA to stimulate immunomodulatory mediator production in primary human keratinocytes. Using real-time reverse transcription-PCR (RT-PCR), PAF but not 5-HT(2A) receptor mRNA was constitutively expressed in primary human keratinocytes. Treatment with cis-UCA increased prostaglandin E(2) (PGE(2)), tumor necrosis factor-alpha (TNF-alpha), and IL-6 secretion, whereas 5-HT only stimulated IL-6 production. Pretreatment with a 5-HT receptor antagonist partially inhibited IL-6 increase by 5-HT, but did not inhibit mediator production by cis-UCA. Similarly, a PAF receptor antagonist did not inhibit cis-UCA-induced increase in PGE(2). Intracellular calcium mobilization studies using a human epithelial cell line stably transfected with PAF receptor also showed little evidence that cis-UCA stimulated PAF receptor and it did not bind to this receptor. Thus, cis-UCA stimulates mediator production by a pathway that is independent of these receptors in human keratinocytes, and these cells may not be the major target for cis-UCA-induced immune suppression.

Recent advances in urocanic acid photochemistry, photobiology and photoimmunology.

Urocanic acid (UCA), produced in the upper layers of mammalian skin, is a major absorber of ultraviolet radiation (UVR). Originally thought to be a 'natural sunscreen', studies conducted a quarter of a century ago proposed that UCA may be a chromophore for the immunosuppression that follows exposure to UVR. With its intriguing photochemistry, its role in immunosuppression and skin cancer development, and skin barrier function, UCA continues to be the subject of intense research effort. This review summarises the photochemical, photobiological and photoimmunological findings regarding UCA, published since 1998.

Screening of urocanic acid isomers in human basal and squamous cell carcinoma tumors compared with tumor periphery and healthy skin.

Trans-urocanic acid is a major chromophore for ultraviolet (UV) radiation in human epidermis. The UV induces photoisomerization of trans-urocanic acid (tUCA) form to cis-urocanic acid (cUCA) and has been reported as an important mediator in the immunosuppression induced by UV. This immunomodulation has been recognized as an important factor related to skin cancer development. This is the first time that UCA isomers have been measured in epidermis of skin biopsies from patients with squamous cell carcinoma (SCC) and with basal cell carcinoma (BCC) and compared with the tumor periphery and biopsies of healthy photoexposed and non-photoexposed skin as controls. The UCA isomers were separated and quantified by high performance liquid chromatography. Analysis of UCA in healthy skin showed significant increase in total UCA content in non-photoexposed body sites compared with highly exposed skins. In contrast, the percentage of cUCA was higher in photoexposed body sites. Maximal levels of cUCA were found in cheek, forehead and forearm and lower levels in abdomen and thigh. No differences were found in total UCA concentration between the tumor samples and healthy photoexposed skin. However, differences were found in relation between isomers. Higher levels of cUCA were detected in SCC biopsies (44% of total UCA) compared with samples of BCC and that of healthy photoexposed skin (30%). These results suggest that the UV radiation exposure, a main factor in development of SCC can be mediated, apart from direct effect to cells (DNA damage), by immunosuppression pathways mediated by high production of cUCA.