Photosensitizer Riboflavin Research Articles

A Novel Fiber-Optic Confocal Laser Caliper Approach for Non-Contact Optical Characterization of Silk Fibroin Thin Films Created by Riboflavin Photo-Crosslinking.

Silk fibroin with its attractive combination of advanced properties is promising for regenerative treatments of corneal disorders. Novel photonics approach is used to characterize the thickness and refractive index of silk fibroin thin films photo-crosslinked with a natural photosensitizer Riboflavin.

Riboflavin-induced metal-free ATRP of (meth)acrylates

Metal-free ATRP approach was improved by eliminating undesirable organic photocatalyst from the reaction medium by an application of a fully-biocompatible riboflavin (vitamin B2)-based molecule playing two crucial roles in photoinduced polymerization – as an ATRP initiator and photoinitiator simultaneously. The brominated ribitol tail constituted an ATRP initiator, while the unmodified isoalloxazine ring was excited to a highly reactive triplet excited state under blue light irradiation, and thus induced the photopolymerization of (meth)acrylates from the ribitol tail. The double initiation functionality of riboflavin provides brush-like polymers with side chains composed of poly(ethylene glycol) methyl ether methacrylate (OEGMA500) with narrow molecular weight distribution (Mw/Mn = 1.36) and preserved chain-end functionality proved by successfully conducted chain extension experiment with the final product characterized by low dispersity (Mw/Mn = 1.35). The riboflavin-induced polymerization of OEGMA500 was temporally controlled by switching on–off blue light irradiation without the loss of chain-end functionality after restarting the synthesis, resulting in well-defined polymer materials (Mw/Mn = 1.37). The results indicate precisely established reversible redox cycle of the riboflavin photosensitizer during polymerization, providing equilibrium between the active and dormant species, and thus controlled polymer products. The double-functional riboflavin molecule makes metal-free ATRP approach an efficient and green chemistry solution for the preparation of functional polymer materials. The prepared riboflavin-based brush-like polymers were visualized by atomic force microscopy (AFM).

A microvalve cell printing technique using riboflavin photosensitizer for selective cell patterning onto a retinal chip

Delivery of cells onto subretinal chips for stimulation possess a significant technical challenge. Often, such capabilities are confined by sophisticated laboratory-based commercial instrumentation, which are inherently expensive and equipped with a limited capacity. As a result, exploring new delivery techniques requires an ad hoc approach to suit the requirements of the target deposition method, material(s) and cells under investigation. For the first time, a customised 3D printer, equipped with a microvalve, developed at a low cost, and capable of high resolution placement on a retinal chip is proposed. This allowed the investigation of a pneumatically-driven and microvalve printing technique to deliver cells on a subretinal chip, thus suiting the required resolution and localization of cells. The bioink is coupled with a light-induced collagen photo-crosslinking mechanism. The collagen, mixed with the riboflavin, can be crosslinked after exposure to blue light. The cells printed on the chip remained in the collagen patterns and demonstrated good viability and growth. This technique provided a useful means of delivering cells where high precision, throughput and accuracy is required, such as onto sub-retinal chips.

Riboflavin-photosensitized thiamine oxidation in aqueous solutions on exposure to ultraviolet and visible light

It was shown the irradiation of aqueous solutions of thiamine and riboflavin with ultraviolet UVA (315–400 nm) and visible light (λ > 400 nm) leads to oxidation of thiamine to thiochrome and oxodihydrothiochrome. In absence of riboflavin in aerobic conditions thiamine oxidation to thiochrome was mainly due to reaction with singlet oxygen. Under anaerobic conditions, thiamine in the presence of riboflavin was photostable and UV and visible light irradiation of their aqueous solutions did not result in thiamine oxidation. Under aerobic conditions, thiochrome was oxidized to form oxodihydrothiohrome in reactions with singlet oxygen and riboflavin in triplet states. Under anaerobic conditions, oxidation of thiochrome to oxodihydrothiochrome occurred due to reaction with riboflavin in triplet state. Possible mechanisms of the damage of cellular eye structures due to inactivation of thiamine-dependent enzymes on exposure to intensive UVA and visible light in the presence of endogenous photosensitizer riboflavin are discussed.

Noninvasive detection of nanoscale structural changes in cornea associated with cross-linking treatment.

Corneal cross-linking (CXL) using ultraviolet-A (UVA) irradiation with a riboflavin photosensitizer has grown from an interesting concept to a practical clinical treatment for corneal ectatic diseases globally, such as keratoconus. To characterize the corneal structural changes, existing methods such as X-ray microscopy, transmission electron microscopy, histology and optical coherence tomography (OCT) have been used. However, these methods have various drawbacks such as invasive detection, the impossibility for in vivo measurement, or limited resolution and sensitivity to structural alterations. Here, we report the application of oversampling nanosensitive OCT for probing the corneal structural alterations. The results indicate that the spatial period increases slightly after 30 minutes riboflavin instillation but decreases significantly after 30 minutes UVA irradiation following the Dresden protocol. The proposed noninvasive method can be implemented using existing OCT systems, without any additional components, for detecting nanoscale changes with the potential to assist diagnostic assessment during CXL treatment, and possibly to be a real-time monitoring tool in clinics.

Application of optical coherence tomography and optical path length method for monitoring corneal thickness and refractive index change during corneal cross-linking.

Corneal cross-linking (CXL) using UVA irradiation with a riboflavin photosensitizer has emerged as a new treatment paradigm for corneal ectatic disorders. The thickness threshold for protection of intraocular structures has often been challenged with ongoing developments, and corneal thinning becomes an important safety concern, especially for patients with thin corneas. In this study with an ex vivo bovine eye model, we monitored corneal thinning and corneal refractive index changes using optical coherence tomography (OCT) integrated with an adaptation of the optical path length method. CXL experiments were performed based on the standard protocol that includes removal of the corneal epithelium to facilitate diffusion of riboflavin into the stroma. The corneal stromal thickness and group refractive index were measured by a 1310nm Fourier-domain OCT imaging system at three critical points of the procedure: immediately after epithelial removal, after 30min riboflavin instillation, and after 30min UVA irradiation with continuing instillation. We found that the refractive index of the bovine cornea changed significantly from epithelial removal to riboflavin instillation and UVA irradiation, increasing from 1.377±0.005 (mean±standard deviation) after de-epithelization to 1.387±0.003 after 30min instillation and 1.388±0.008 after subsequent irradiation. The corneas also underwent a considerable decrease (10%-20%) in stromal thickness with thinning of 95±29 μm (mean±standard deviation) after riboflavin instillation and a further decrease (∼5%) with thinning of 42±19 μm after UVA irradiation. Our study highlights the importance of corneal thickness monitoring during CXL, especially after riboflavin instillation when the decrease is the largest, to avoid delivering endothelial cytotoxic doses. An increase in refractive index heightens the concern for corneal thinning and the need for careful monitoring as a safety precaution.

An experimental study on riboflavin photosensitization treatment for inactivation of circulating HCT116 tumor cells

Surgical resection is one of the most common radical treatments for cancers. However, tumors may be compressed or the local intravascular pressure may be increased during surgical manipulation, causing the shedding and entry of tumor cells into the blood circulation and hence distant recurrence and metastasis of tumors. We have preliminarily established a method of riboflavin photosensitization treatment (RPT) for inactivation of circulating tumor cells. This technology promises to solve the problems of shedding and entry of solid tumor cells into blood circulation before surgical manipulation, and almost unavoidable hematogenous dissemination of tumor cells during surgical resection. In the present study, apoptosis detection and tumorigenicity experiment in immunodeficient mice were conducted to evaluate the effect of RPT for inactivation of circulating tumor cells respectively. Next, functional evaluation was carried out for the immune cells through detecting apoptosis rate and cytokine secretion of lymphocyte. Finally, thromboelastography (TEG) and free hemoglobin were detected to assess peripheral blood coagulation and red blood cell damage. The results showed that RPT (50 μmol/L riboflavin, 10.8 J/cm2 UV) could effectively make tumor cell lose the ability of proliferation in the peripheral blood. In the meantime, the damage caused to peripheral blood coagulation, immune cell function and red blood cells was generally acceptable. The results of the study showed that RPT had huge potential in addressing the problems of shedding and entry of solid tumor cells into blood circulation before surgical manipulation, and almost unavoidable hematogenous dissemination of tumor cells during surgical resection. This therapy is expected to be an auxiliary and supportive method to reduce the risk of hematogenous metastasis and recurrence of cancers, and to increase the surgical success rate of malignant solid tumors.

Oxidative stability of oil-in-water emulsions with α-tocopherol, charged emulsifier, and different oxidative stress.

α-Tocopherol is known to show different activity depending on the concentration and food matrix. Effects of α-tocopherol at the concentrations of 0, 0.1, 0.5, and 1.0mM were determined in oil-in-water (O/W) emulsions containing anionic, neutral, and cationic emulsifiers under different types of oxidative stress including riboflavin photosensitization, photooxidation, and autoxidation. Headspace oxygen depletion, lipid hydroperoxides, and conjugated dienes were analyzed to determine the oxidative stability of O/W emulsions. α-Tocopherol served as an antioxidant in O/W emulsion with a cationic emulsifier irrespective of oxidative stress. α-Tocopherol acted as an antioxidant in O/W emulsion with a neutral emulsifier at riboflavin photosensitization while a prooxidant at photooxidation. However, in samples with an anionic emulsifier, α-tocopherol activity differed from the concentration and types of oxidative stress. Therefore, cationic transition metals or reactive oxygen species generated from RF photosensitization could play key roles of α-tocopherol in O/W emulsion.

Recent Innovations in Collagen Corneal Cross-linking; a Mini Review.

Background:The introduction of corneal cross-linking (CXL) with ultraviolet-A (UVA) and Riboflavin photosensitizer (Vit B2) from Seiler et al., revolutionized the treatment of Keratoconus and other corneal ectatic diseases.Today, the commonly known epithelium off Dresden protocol is in clinical use for the last 15 years with great success and regarded by many as the golden standard.Methods:With several studies demonstrating its simplicity, efficacy and safety this revolutionary method, paved the way for new therapies and strategies in the treatment of corneal ectatic diseases and changed our understanding in corneal biomechanics. Recent scientific and technological advances enabled the creation of various modifications of the initial CXL protocol and the formation of new ones.Conclusion:This work highlights the recent advances of CXL, such as the role of oxygen, higher fluence and shorter irradiation times as well as the various clinical applications and updates of this method.

Oxidative stability of extracts from red ginseng and puffed red ginseng in bulk oil or oil-in-water emulsion matrix

BackgroundExplosive puffing can induce changes in the chemical, nutritional, and sensory quality of red ginseng. The antioxidant properties of ethanolic extracts of red ginseng and puffed red ginseng were determined in bulk oil and oil-in-water (O/W) emulsions. MethodsBulk oils were heated at 60°C and 100°C and O/W emulsions were treated under riboflavin photosensitization. In vitro antioxidant assays, including 2,2-diphenyl-1-picrylhudrazyl, 2,2′-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid, ferric reducing antioxidant power, total phenolic content, and total flavonoid content, were also performed. ResultsThe total ginsenoside contents of ethanolic extract from red ginseng and puffed red ginseng were 42.33 mg/g and 49.22 mg/g, respectively. All results from above in vitro antioxidant assays revealed that extracts of puffed red ginseng had significantly higher antioxidant capacities than those of red ginseng (p<0.05). Generally, extracts of puffed red and red ginseng had high antioxidant properties in riboflavin photosensitized O/W emulsions. However, in bulk oil systems, extracts of puffed red and red ginseng inhibited or accelerated rates of lipid oxidation, depending on treatment temperature and the type of assay used. ConclusionAlthough ethanolic extracts of puffed red ginseng showed stronger antioxidant capacities than those of red ginseng when in vitro assays were used, more pro-oxidant properties were observed in bulk oils and O/W emulsions.

Oxidative Stability in Oil-in-Water Emulsions with Quercetin or Rutin Under Iron Catalysis or Riboflavin Photosensitization.

The effects of quercetin and rutin on the oxidative stability of oil-in-water (O/W) emulsions were tested under riboflavin (RF) photosensitization in the presence or absence of FeCl2 . The degree of oxidation in O/W emulsions was determined by headspace oxygen content, conjugated dienes, and lipid hydroperoxides. Quercetin chelated more metal than did rutin in iron catalyzed O/W emulsions. Generally, 0.1 mM quercetin and rutin was oxidative while 0.5 and 1.0 mM quercetin and rutin was antioxidative in O/W emulsions under RF photosensitization. Depending on the analysis method, the antioxidants had different strengths. The antioxidative or oxidative properties of quercetin and rutin vary in O/W emulsions and depend the quercetin and rutin concentrations and oxidative forces like transition metals, RF photosensitization, or a combination thereof.

Photocrosslinked tyramine-substituted hyaluronate hydrogels with tunable mechanical properties improve immediate tissue-hydrogel interfacial strength in articular cartilage

Articular cartilage lacks the ability to self-repair and a permanent solution for cartilage repair remains elusive. Hydrogel implantation is a promising technique for cartilage repair; however for the technique to be successful hydrogels must interface with the surrounding tissue. The objective of this study was to investigate the tunability of mechanical properties in a hydrogel system using a phenol-substituted polymer, tyramine-substituted hyaluronate (TA-HA), and to determine if the hydrogels could form an interface with cartilage. We hypothesized that tyramine moieties on hyaluronate could crosslink to aromatic amino acids in the cartilage extracellular matrix. Ultraviolet (UV) light and a riboflavin photosensitizer were used to create a hydrogel by tyramine self-crosslinking. The gel mechanical properties were tuned by varying riboflavin concentration, TA-HA concentration, and UV exposure time. Hydrogels formed with a minimum of 2.5 min of UV exposure. The compressive modulus varied from 5 to 16 kPa. Fluorescence spectroscopy analysis found differences in dityramine content. Cyanine-3 labelled tyramide reactivity at the surface of cartilage was dependent on the presence of riboflavin and UV exposure time. Hydrogels fabricated within articular cartilage defects had increasing peak interfacial shear stress at the cartilage-hydrogel interface with increasing UV exposure time, reaching a maximum shear stress 3.5× greater than a press-fit control. Our results found that phenol-substituted polymer/riboflavin systems can be used to fabricate hydrogels with tunable mechanical properties and can interface with the surface tissue, such as articular cartilage.

Caffeine metabolites not caffeine protect against riboflavin photosensitized oxidative damage related to skin and eye health

Caffeine metabolites were found to bind riboflavin with dissociation constant in the millimolar region by an exothermic process with positive entropy of reaction, which was found by 1H NMR and fluorescence spectroscopy to occur predominantly by hydrogen bonding with water being released from riboflavin solvation shell upon caffeine metabolite binding to riboflavin. The caffeine metabolites 1-methyl uric acid and 1,7-dimethyl uric acid were shown by transient absorption laser flash photolysis to be efficient as quenchers of triplet riboflavin with second-order rate constant of 1.4 108Lmol−1s−1 and 1.0 108Lmol−1s−1, respectively, in aqueous solution of pH6.4 at 25°C and more efficient than the other caffeine metabolite 1,7-dimethyl xanthine with second-order rate constant of 4.2 107Lmol−1s−1. Caffeine was in contrast found to be non-reactive towards triplet riboflavin. Caffeine metabolites rather than caffeine seem accordingly important for the observed protective effect against cutaneous melanoma identified for drinkers of regular but not of decaffeinated coffee. The caffeine metabolites, but not caffeine, were by time resolved single photon counting found to quench singlet excited riboflavin through exothermic formation of ground-state precursor complexes indicating importance of hydrogen bounding through keto-enol tautomer's for protection of oxidizable substrates and sensitive structures against riboflavin photosensitization.

Effects of emulsifier charges on the oxidative stability in oil-in-water emulsions under riboflavin photosensitization.

The oxidative stability in oil-in-water (O/W) emulsions containing different emulsifier charges was tested under riboflavin photosensitization by analysis of headspace oxygen content and lipid hydroperoxides. Sodium dodecyl sulfate (SDS), Tween 20, and cetyltrimethylammonium bromide (CTAB) were selected as anionic, neutral, and cationic emulsifiers, respectively. The O/W emulsions containing CTAB had lower oxidative stability than those with SDS and Tween 20. The addition of ethylenediaminetetraacetic acid, a well-known metal chelator, increased the oxidative stability in O/W emulsions, irrespective of emulsifier charges. Oxidative stability in Tween 20-stabilized emulsions decreased in FeCl3 and FeCl2 concentration-dependent manner. However, oxidative stability in samples containing CTAB increased up to 0.5mM of FeCl3 and FeCl2 and then decreased, which implies that CTAB act differently during lipid oxidation compared to SDS and Tween 20.

Riboflavin Phototransformation on the Changes of Antioxidant Capacities in Phenolic Compounds.

Eight phenolic compounds including: p-coumaric acid, vanillic acid, caffeic acid, chlorogenic acid, trolox, quercetin, curcumin, and resveratrol were treated with riboflavin (RF) photosensitization and in vitro antioxidant capacities of the mixtures were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2' azino bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. Mixtures containing p-coumaric acid and vanillic acid under RF photosensitization showed increases in ferric ion reducing ability and radical scavenging activity of DPPH, whereas mixtures of other compounds had decreases in both radical scavenging ability and ferric reducing antioxidant power. Hydroxycoumaric acid and conjugated hydroxycoumaric and coumaric acids were tentatively identified from RF photosensitized p-coumaric acid, whereas dimmers of vanillic acid were tentatively identified from RF photosensitized vanillic acid. RF photosensitization may be a useful method to enhance antioxidant properties like ferric ion reducing abilities of some selected phenolic compounds.

Evaluation of Antioxidant or Prooxidant Properties of Selected Amino Acids Using In Vitro Assays and in Oil-in-Water Emulsions Under Riboflavin Sensitization.

The antioxidant properties of selected amino acids were tested using in vitro assays and oil-in-water (O/W) emulsions under riboflavin (RF) photosensitization. Headspace oxygen content, lipid hydroperoxides, and conjugated dienes were determined for the degree of oxidation. Riboflavin photosensitization was adapted as the oxidation driving force. In vitro assays showed that cysteine had the highest antioxidant properties followed by tryptophan and tyrosine. However, in O/W emulsions under RF photosensitization, tyrosine inhibited lipid oxidation whereas tryptophan acted as a prooxidant. Tryptophan accelerated the rates of oxidation in O/W emulsion without RF. The antioxidant properties of amino acids differed depending on the antioxidant determination methods, oxidation driving forces, and food matrices.

Corneal Cross-Linking: An Example of Photoinduced Polymerization as a Treatment Modality in Keratoconus.

The cornea is one of the principal refractive elements in the human eye and plays a crucial role in the process of vision. Keratoconus is the most common corneal dystrophy, found mostly among young adults. It is characterized by a reduced number of collagen cross-links in the corneal stroma, resulting in reduced biomechanical stability and an abnormal shape of the cornea. These changes lead to progressive myopia, corneal thinning, central scarring and irregular astigmatism, causing severely impaired vision. Hard contact lenses, photorefractive keratectomy or intracorneal rings are the most common treatment options for refractive error caused by keratoconus. However, these techniques do not treat the underlying cause of the corneal ectasia and therefore are not able to stop the progression of the disease. Riboflavin photoinduced polymerization of corneal collagen, also known as corneal cross-linking (CXL), has been introduced as the first therapy which, by stabilizing the structure of the cornea, prevents the progression of keratoconus. It stiffens the cornea using the photo-sensitizer riboflavin in combination with ultraviolet irradiation. This is a current review of the CXL procedure as a therapy for keratoconus, which relies on photoinduced polymerization of human tissue. We have focused on its biomechanical and physiological influences on the human cornea and have reviewed the previous and current biochemical theories behind cross-linking reactions in the cornea.

Antioxidant Properties of Aqueous Extract of Roasted Hulled Barley in Bulk Oil or Oil‐in‐Water Emulsion Matrix

Antioxidant properties of the aqueous extracts of hulled barley (Hordeum vulgare L.) that had been roasted at 210 °C for 20 min were determined in bulk oil and oil-in-water (O/W) emulsions. Bulk oils were heated at 60, 100, and 180 °C, and O/W emulsions were oxidized under riboflavin photosensitization. The content of phenolic compounds was analyzed by high-performance liquid chromatography, and in vitro antioxidant assays were also conducted. The major phenolics contained in the aqueous extract of roasted hulled barley (AERB) were p-coumaric, ferulic, protocatechuic, chlorogenic, 4-hydroxybenzoic, and vanillic acids. Depending on the concentration and oxidation temperature, AERB had antioxidant or prooxidant properties in bulk oil. At 60 °C, AERB at a concentration of 0.5% acted as a prooxidant, whereas at 1.0% it acted as an antioxidant. At 100 °C, AERB acted as an antioxidant irrespective of concentration. In 180 °C conditions, 0.5% AERB acted as a prooxidant, whereas other concentrations of AERB acted as antioxidants. In the case of riboflavin photosensitized O/W emulsions, AERB showed antioxidant properties irrespective of concentration. Antioxidant abilities of AERB are affected by the food matrix, including bulk oil and O/W emulsions, and concentrations of AERB, even though diverse phenolic compounds may display high antioxidant properties in in vitro assays. Roasted barley has been widely used as a tea ingredient in East Asian countries such as Korea, China, and Japan. The highly antioxidative properties of the aqueous extracts of roasted barley have been confirmed in bulk oil and O/W emulsions as well as in vitro assays because of the presence of phenolic compounds. The results of this study can contribute to the development of antioxidant-rich beverages using roasted barley by aiding in the selection of proper food matrix-containing extracts of high phenolic compounds, as well as by expanding consumers’ choices for healthy beverages.

Corneal collagen cross-linking (CXL) in thin corneas.

Corneal collagen cross-linking (CXL) is a therapeutic procedure aiming at increasing the corneal stiffness in the keratoconus eyes by induction of cross-links within the extracellular matrix. It is achieved by ultraviolet-A (370 nm) irradiation of the cornea after saturation with the photosensitizer riboflavin. In the conventional CXL protocol, a minimum de-epithelialized corneal thickness of 400 μm is recommended to avoid potential irradiation damage to the corneal endothelium. In advanced keratoconus, however, stromal thickness is often lower than 400 μm, which limits the application of CXL in that category. Efforts have been undertaken to modify the conventional CXL procedure to be applicable in thin corneas. The current review discusses different techniques employed to achieve this end and their results. The overall safety and efficacy of the modified CXL protocols are good, as most of them managed to halt the progression of keratectasia without postoperative complications. However, the evidence of safety and efficacy in the use of modified CXL protocols is still limited to few studies with few patients involved. Controlled studies with long-term follow-up are required to confirm the safety and efficacy of the modified protocols.

Changes in isoflavone profiles and antioxidant activities in isoflavone extracts from soybeans and soyfoods under riboflavin photosensitization

Isoflavone extracts from soybeans and soyfoods were treated with riboflavin photosensitization and changes in antioxidant activities of isoflavone extracts under riboflavin sensitization were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. Extracts rich in malonyl derivatives, β-glucosides, and aglycones were prepared from raw, ovendried, and almond-incubated soybeans, and the fermented soyfoods cheongukjang and natto. As the period of visible light irradiation increased to 120 min, the total isoflavone content determined using HPLC decreased in all samples. Aglycones in almond-incubated extracts decreased by 77.5% after 120min, and β-glucosides in oven-dried extracts decreased by 52.0%. The radical scavenging activity based on DPPH and ABTS assays significantly (p 0.05) increased after riboflavin sensitization.