Riboflavin: The Health Benefits of a Forgotten Natural Vitamin

Riboflavin (RF) is a water-soluble member of the B-vitamin family. Sufficient dietary and supplemental RF intake appears to have a protective effect on various medical conditions such as sepsis, ischemia etc., while it also contributes to the reduction in the risk of some forms of cancer in humans. These biological effects of RF have been widely studied for their anti-oxidant, anti-aging, anti-inflammatory, anti-nociceptive and anti-cancer properties. Moreover, the combination of RF and other compounds or drugs can have a wide variety of effects and protective properties, and diminish the toxic effect of drugs in several treatments. Research has been done in order to review the latest findings about the link between RF and different clinical aberrations. Since further studies have been published in this field, it is appropriate to consider a re-evaluation of the importance of RF in terms of its beneficial properties

Detection of CD33 expression on monocyte surface is influenced by phagocytosis and temperature

CD33 is a myeloid-associated marker and belongs to the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family. Such types of receptors are highly expressed in acute myeloid leukemia, which could be used in its treatment. CD33 shows high variability in its expression levels with still unknown reasons. Here, we investigated the CD33 expression of monocytes in human blood samples processed at different temperatures and in dependence on their phagocytic activity against opsonized Escherichia coli. The samples were stained by fluorescently labelled anti-human CD14 to specify the monocyte population, anti-human CD33 antibodies to evaluate CD33 expression and analyzed by flow cytometry and confocal laser scanning microscopy. In blood samples kept at 37°C or first pre-chilled at 0°C with subsequent warming up to 37°C, the percentage of CD33-positive monocytes as well as their relative fluorescence intensity was up-regulated compared to samples kept constantly at 0°C. After exposure to E. coli the CD33 relative fluorescence intensity of the monocytes activated at 37°C was 3 to 4 times higher than that of those cells kept inactive at 0°C. Microscopic analysis showed internalisation of CD33 due to its enhanced expression on the surface followed by engulfment of E. coli

Doxorubicin-Loaded Human Serum Albumin Submicron Particles: Preparation, Characterization and In Vitro Cellular Uptake

Doxorubicin (DOX) is an effective anthracycline antibiotic drug which is commonly used in a broad range cancer therapy. However, due to dose depending side effects and toxicity to non-cancerous tissues, its clinical applications are restricted. To overcome these limitations, human serum albumin (HSA) has been investigated as a biocompatible drug delivery vehicle. In this study, human serum albumin submicron particles (HSA-MPs) were fabricated by using the Co-precipitation-Crosslinking-Dissolution technique (CCD technique) and DOX was loaded into the protein particles by absorption. DOX-HSA-MPs showed uniform peanut-like shape, submicron size and negative zeta-potential (-13 mV). The DOX entrapment efficiency was 25% of the initial amount. The in vitro release in phosphate buffered saline pH 7.4 was less than 1% within 5 h. In contrast, up to 40% of the entrapped DOX was released in presence of a protein digesting enzyme mixture (Pronase®) within the same time. In addition, in vitro cytotoxicity and cellular uptake of DOX-HSA-MPs were evaluated using the lung carcinoma cell line A549. The results demonstrated that DOX-HSA-MPs reduced the cell metabolic activities after 72 h. Interestingly, DOX-HSA-MPs were taken up by A549 cells up to 98% and localized in the cell lysosomal compartment. This study suggests that DOX-HSA-MPs which was fabricated by CCD technique is seen as a promising biopolymer particle as well as a viable alternative for drug delivery application to use for cancer therapy

Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry

Methemoglobin (MetHb) is a hemoglobin (Hb) derivative with the heme iron in ferric state (Fe3+), unable to deliver oxygen. Quantification of methemoglobin is a very important diagnostic parameter in hypoxia. Recently, novel hemoglobin microparticles (Hb-MP) with a narrow size distribution around 700 nm, consisting of cross-linked Hb were proposed as artificial oxygen carriers. The cross-linking of Hb by glutaraldehyde (GA) generates a certain amount of MetHb. Due to the strong light scattering, quantitative determination of MetHb in Hb-MP suspensions by common spectrophotometry is not possible. Here, we demonstrate that 1H2O NMR relaxometry is a perfect tool for direct measurement of total Hb and MetHb concentrations in Hb-MP samples. The longitudinal relaxation rate 1/T1 shows a linear increase with increasing MetHb concentration, whereas the transverse relaxation rate 1/T2 linearly increases with the total Hb concentration. In both linear regressions the determination coefficient (R2) is higher than 0.99. The method does not require time-consuming pretreatment or digestion of the particles and is not impaired by light scattering. Therefore, it can be established as the method of choice for the quality control of Hb-MP and similar hemoglobin-based oxygen carriers in the future

Biokompatibilität von Biopolymerpartikeln

Recently, we employed the Co-precipitation Crosslinking Dissolution technique (CCD-technique) to fabricate biopolymer particles and to load them with riboflavin (RF). Moreover, we modified the CCD-technique by combining co-precipitation and cross-linking into one preparation step using a cross-linker, periodate-oxidized dextran (Odex). HSA and bovine haemoglobin (Hb) were used to demonstrate this technique. The haemocompatibilty of these submicron particles is important for their application as intravenously administered drug carriers. In vitro haemocompatibility assays including haemolysis test, platelet activation test, and phagocytosis test in human blood were studied. Our results showed that, both RF-HSA-MPs and Odex-HbMPs exhibited the percentage of hemolysis in the low range of 1% - 7%. Microparticle-platelet interaction did not activate the platelets and did not augment the platelet response to antagonists. The PMN phagocytes exposed to microparticles showed no phagocytic activity. These results suggested that our RF-HSA- MPs by CCD-technique and Odex-HbMPs by One-Pot formulation revealed a good hemocompatibility. Furthermore, the RF-HSA-MPs by CCD-technique were characterized showing a narrow size distribution in the range of 0.9 to 1 µm, uniform peanut-like morphology, and a zeta-potential of −15 mV. In vitro release studies represented biphasic release profiles of RF in a phosphate buffered saline (PBS) pH 7.4 and a cell culture medium (RPMI) 1640 medium over a prolonged period (study 1). Moreover, to assess phagocytic activity, cell surface marker, CD33 is used to identify monocytes. Here, we also investigated the CD33 expression of monocytes in human blood samples processed at different temperatures and in dependence on their phagocytic activity in the presence of opsonized Escherichia coli. The samples were then stained by fluorescently labelled anti-human CD14 and anti-human CD33 antibodies to specify the monocyte population and to evaluate CD33 expression, respectively and evaluated by flow cytometry and confocal laser scanning microscopy. We found that the percentage of CD33-positive monocytes as well as their relative fluorescence intensity was up-regulated when the blood samples were kept at 37 °C or first pre-chilled at 0 °C with subsequent warming up to 37 °C, in comparison to samples kept constantly at 0 °C was significantly lower. After exposure to E. coli the CD33 relative fluorescence intensity of the monocytes activated at 37°C was 3 to 4 times higher than that of those cells kept inactive at 0 °C. Furthermore, microscopic analysis showed internalization of CD33 due to its expression on the surface and the engulfment of E. coli (study 2).Für die Herstellung von Biopolymerpartikeln und deren Beladung mit Riboflavin (RF) wurde die Co-precipitations-Crosslinking-Dissolution-Technik (CCD-Technik) verwendet. Diese Technik wurde zu einem Einschrittverfahren modifiziert, indem als Crosslinker Periodat oxidiertes Dextran (Odex) verwendet wurde. Submikropartikel bestehend aus Humanserumalbumin (HSA-MP) und Rinderhämoglobin (HbMP) wurden hergestellt, um die Methode zu demonstrieren. Die Hämokompatibilität dieser Partikel ist für deren intravenöse Anwendung als Drug Carrier erforderlich. Die In vitro Hämokompatibilität der Partikel wurde mittels Hämolysetest, Plättchenaktivitätstests sowie des Phagozytosetest in Humanblut untersucht. Unsere Ergebnisse zeigen, dass sowohl RF-HSA-MPs als auch Odex-HbMPs eine Hämolyserate von 1 -7% aufweisen. Die Plättchen wurden durch die Mikropartikel weder aktiviert noch wurde ihre Antwort auf Agonisten beeinflusst. PMN Phagozyten zeigten bei Inkubation mit den Mikropartikeln keine Phagozytose. Diese Ergebnisse lassen den Schluss zu, dass die mittels CCD-Technik sowie der Einschrittformulierung hergestellten RF-HSA-MPs bzw. die Odex-HbMPs hämokompatibel sind. Die RF-HSA-MPs wurden zusätzlich hinsichtlich ihrer Größe und Form sowie ihres Zetapotentials untersucht. Sie weisen eine erdnussartige Form, eine enge Größenverteilung im Bereich von 0,9 und 1,0 μm sowie ein Zetapotential von -15 mV auf. Die Freisetzung von RF zeigt ein biphasisches Profil sowohl in Phosphatpuffer (pH 7,4) als auch in Zellkulturmedium RPMI 1640 über einen Zeitraum von mehr als 3 Tage (Studie 1). Um die Phagozytoseaktivität zu charakterisieren wurde der Oberflächenmarker CD33 für Monozyten verwendet. Dazu wurde die CD33 Expression auf humanen Monozyten in Blutproben, die bei verschiedenen Temperaturen prozessiert wurden sowie in Abhängigkeit von der Phagozytose der Monozyten in Gegenwart opsonierter Escherichia coli untersucht. Die CD33 Expression der Monoyten wurde nach deren Markierung mit CD14 und CD33 Antikörpern mittels Durchflusszytometer und Konfokalem Laserscanningmikroskop untersucht. Der prozentuale Anteil von CD33-positiven Monozyten sowie ihrer relative Fluoreszenzintensität waren hochreguliert, wenn die Blutproben bei 37°C gehalten oder erst auf 0°C abgekühlt und danach wieder auf 37°C erwärmt wurden im Vergleich zu den Proben, die konstant bei 0°C gehalten wurden. Nach Inkubation mit E. coli war die CD33 relative Fluoreszenzintensität der bei 37°C aktivierten Monozyten 3 bis 4 mal höher als bei den Zellen, die bei 0°C gehalten wurden. Die mikroskopischen Untersuchungen zeigten die Internalizierung von CD33 infolge ihrer Expression auf der Monozytenoberfläche und dem Einschluss der E. coli (Studie 2)

Evaluation of Anti-Hyperglycemia and Complications of Red and Black Thai Jasmine Rice Cultivars in Streptozotocin-Induced Diabetic Rats

The phytochemical constituents of red (RR) and black (BR) rice extracts were determined using high-pressure liquid chromatography (HPLC). Phytochemical screening revealed the presence of catechin, rutin, isoquercetin, cyanidin 3-glucoside, cyanidin 3-O-rutinoside, peonidin and quercetin. The anti-diabetic activities of RR and BR extracts on diabetic complications were examined in a streptozotocin-induced diabetic rat model. Rats (n = 80) were divided into 10 groups (n = 8 rats per group). Healthy and diabetic RR or BR-treated groups received 10, 50, or 200 mg of RR or BR per kg of body weight daily for 45 days. The results demonstrated significantly improved glucose control in rats administered RR or BR, while triglyceride and cholesterol levels were reduced in the diabetic groups. Moreover, RR or BR treatment led to decreased levels of malondialdehyde, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine. Further, glutathione concentration was significantly increased in both serum and liver tissue from RR- and BR-treated diabetic rats

Doxorubicin–Loaded Human Serum Albumin Submicron Particles: Preparation, Characterization and In Vitro Cellular Uptake

Doxorubicin (DOX) is an effective anthracycline antibiotic drug which is commonly used in a broad range cancer therapy. However, due to dose depending side effects and toxicity to non-cancerous tissues, its clinical applications are restricted. To overcome these limitations, human serum albumin (HSA) has been investigated as a biocompatible drug delivery vehicle. In this study, human serum albumin submicron particles (HSA-MPs) were fabricated by using the Co-precipitation–Crosslinking–Dissolution technique (CCD technique) and DOX was loaded into the protein particles by absorption. DOX-HSA-MPs showed uniform peanut-like shape, submicron size and negative zeta-potential (−13 mV). The DOX entrapment efficiency was 25% of the initial amount. The in vitro release in phosphate buffered saline pH 7.4 was less than 1% within 5 h. In contrast, up to 40% of the entrapped DOX was released in presence of a protein digesting enzyme mixture (Pronase®) within the same time. In addition, in vitro cytotoxicity and cellular uptake of DOX-HSA-MPs were evaluated using the lung carcinoma cell line A549. The results demonstrated that DOX-HSA-MPs reduced the cell metabolic activities after 72 h. Interestingly, DOX-HSA-MPs were taken up by A549 cells up to 98% and localized in the cell lysosomal compartment. This study suggests that DOX-HSA-MPs which was fabricated by CCD technique is seen as a promising biopolymer particle as well as a viable alternative for drug delivery application to use for cancer therapy

Fabrication and Characterization of Human Serum Albumin Particles Loaded with Non-Sericin Extract Obtained from Silk Cocoon as a Carrier System for Hydrophobic Substances

Non-sericin (NS) extract was produced from the ethanolic extract of Bombyx mori silk cocoons. This extract is composed of both carotenoids and flavonoids. Many of these compounds are composed of substances of poor aqueous solubility. Thus, this study focused on the development of a carrier system created from biocompatible and biodegradable materials to improve the biological activity of NS extracts. Accordingly, NS was incorporated into human serum albumin template particles with MnCO3 (NS-HSA MPs) by loading NS into the preformed HAS-MnCO3 microparticles using the coprecipitation crosslinking dissolution technique (CCD-technique). After crosslinking and template dissolution steps, the NS loaded HSA particles are negatively charged, have a size ranging from 0.8 to 0.9 mu m, and are peanut shaped. The degree of encapsulation efficiency ranged from 7% to 57% depending on the initial NS concentration and the steps of adsorption. In addition, NS-HSA MPs were taken up by human lung adenocarcinoma (A549 cell) for 24 h. The promotion of cellular uptake was evaluated by flow cytometry and the results produced 99% fluorescent stained cells. Moreover, the results from CLSM and 3D fluorescence imaging confirmed particle localization in the cells. Interestingly, NS-HSA MPs could not induce inflammation through nitric oxide production from macrophage RAW264.7 cells. This is the first study involving the loading of non-sericin extracts into HSA MPs by CCD technique to enhance the bioavailability and biological effects of NS. Therefore, HSA MPs could be utilized as a carrier system for hydrophobic substances targeting cells with albumin receptors

Fabrication and Characterization of Human Serum Albumin Particles Loaded with Non-Sericin Extract Obtained from Silk Cocoon as a Carrier System for Hydrophobic Substances

Non-sericin (NS) extract was produced from the ethanolic extract of Bombyx mori silk cocoons. This extract is composed of both carotenoids and flavonoids. Many of these compounds are composed of substances of poor aqueous solubility. Thus, this study focused on the development of a carrier system created from biocompatible and biodegradable materials to improve the biological activity of NS extracts. Accordingly, NS was incorporated into human serum albumin template particles with MnCO3 (NS-HSA MPs) by loading NS into the preformed HAS-MnCO3 microparticles using the coprecipitation crosslinking dissolution technique (CCD-technique). After crosslinking and template dissolution steps, the NS loaded HSA particles are negatively charged, have a size ranging from 0.8 to 0.9 µm, and are peanut shaped. The degree of encapsulation efficiency ranged from 7% to 57% depending on the initial NS concentration and the steps of adsorption. In addition, NS-HSA MPs were taken up by human lung adenocarcinoma (A549 cell) for 24 h. The promotion of cellular uptake was evaluated by flow cytometry and the results produced 99% fluorescent stained cells. Moreover, the results from CLSM and 3D fluorescence imaging confirmed particle localization in the cells. Interestingly, NS-HSA MPs could not induce inflammation through nitric oxide production from macrophage RAW264.7 cells. This is the first study involving the loading of non-sericin extracts into HSA MPs by CCD technique to enhance the bioavailability and biological effects of NS. Therefore, HSA MPs could be utilized as a carrier system for hydrophobic substances targeting cells with albumin receptors