Thermal-Sensitive Liposomes
Glenn J. Jaffe, Paul Ashton, P. Andrew Pearson in Intraocular Drug Delivery, 2006
Liposomes containing fluorescent dye (6-carboxyfluorescein, CF) were prepared. The temperature profile was studied by measuring the concentration of the free (unencapsulated) CF as the liposomes were incubated at various temperatures for 10 minutes in Ringer's lactate solution plus 1% human serum. Due to self-quenching at high concentrations, CF encapsulated in the liposomes does not contribute to the fluorescence of the sample. This permitted the assessment of the free CF concentration with a fluorophotometer without having to separate the supernatant from the liposomes. Complete release was defined as the fluorescence intensity after the dissolution of the liposomes with a detergent (TritonX 100). The free dose fraction was found to be 2% at room temperature, 5% at body temperature (37°C), and 83% at 41°C. This indicates that a sharp transition can be achieved in vitro at the intended temperature, yielding a 17-fold increase of free dye after release from the liposomes.
The Microenvironment in Multicellular Spheroids
Rolf Bjerkvig in Spheroid Culture in Cancer Research, 2017
The HT-29 cells were too small for microelectrode measurements. Instead, these cells were loaded with the pH-sensitive dye 4′,5′-dimethyl-5-and-6-carboxyfluorescein (Me3 CF) (Figure 6). By this method the U-118MG and HT-29 cells were compared regarding their capacity to regulate the intracellular pH by the amiloride-sensitive Na+/H+ exchanger. The pH-induced absorbance changes of the intracellular dye were measured photometrically according to the method of Chaillet and Boron,51 and the results are shown in Table 1. The U-118MG cells had an intracellular pH of about 7.18, which decreased after 1 h of amiloride treatment to 6.84. In the controls the HT-29 cells already had the low value of 6.94, which only decreased slightly (not statistically significant) during amiloride treatment. Thus, the U-118MG cells had a Na+/H+ exchanger active at physiological intracellular pH values, while this was not the case for HT-29 cells. It is known that the Na+/H+ exchanger of HT-29 cells becomes activated only at pH values below 6.52 The activity of the Na+/H+ exchanger seemed to be related to growth control, since U-118MG multicellular spheroids could be growth inhibited effectively by low doses of amiloride (0.2 mM), as shown in Figure 7, whereas HT-29 spheroids with the quiescent exchanger were not affected. The results are discussed in more detail by Szolgay-Daniel et al.53
Interaction of The ANTI-CANDIDA Amphotericin B (And Other Polyene Antibiotics) With Lipids
Rajendra Prasad, Mahmoud A. Ghannoum in Lipids of Pathogenic Fungi, 2017
Another elegant method exploits use of the fluorescence changes in the pH-sensitive fluorescent probe pyranine, encapsulated in unilamellar vesicles, to determine the permeability to saline ions.3,5,6 If, for instance, the salt is composed of K+ and an impermeant anion, an electroneutral exchange with H+ through the membrane is necessary to maintain the K+ flux. When the H+ current is facilitated by a protonophore, the drug-induced K+ flux becomes the rate-limiting step. The exchange is triggered either by a transmembrane pH gradient3 or a salt gradient.6 Another, more popular, spectrofluorimetric method involves measurement of an increase in fluorescence due to the drug-induced efflux of self-quenched encapsulated carboxyfluorescein or calcein.7 Such dyes release freely through membranes in their anionic forms and it is the efflux of their alkaline counter-ion which controls their release.8
Oregonin from Alnus incana bark affects DNA methyltransferases expression and mitochondrial DNA copies in mouse embryonic fibroblasts
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2018
Jelena Krasilnikova, Liga Lauberte, Elena Stoyanova, Desislava Abadjieva, Mihail Chervenkov, Mattia Mori, Elisa De Paolis, Vanya Mladenova, Galina Telysheva, Bruno Botta, Elena Kistanova
The purity and concentration of extracted DNA was measured using Nano Drop 1000 (Thermo Fisher Scientific, Waltham, MA USA). A ratiometric assay30 of the levels of a single mitochondrial gene cytochrome B (Cytb) against a single copy nuclear gene (β-actin) was used to estimate the average copy value of mtDNA/per cell. This technique allows the calculation of average mtDNA copy number/cell without the necessity to estimate cell number. Primers and probes were obtained from Sigma-Aldrich, Saint Louis, MA, USA on the base of sequences mouse genes used by Aiken et al.31 (Table 2). Probes were labelled fluorescently with carboxyfluorescein (FAM) (5′ end) and carboxytetramethylrhodamine (TAMRA) (3′ end). TaqMan Master Mix (Applied Biosystems, Foster City, CA, USA) was used for the reaction. The PCR conditions were following: initial steps at 50 °C for 2 min and 95 °C for 15 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 65 °C. Standard curves were created separately for every sample from a dilution series to ensure the efficiency of reaction. The cycle threshold (CT) value for β-actin was subtracted from that for Cytb to give the value ΔCt. Average mtDNA copy number30 per nuclear genome (two actin gene copies) is calculated as 2 × 2−(ΔCt).
Enhanced blood–brain barrier penetration and glioma therapy mediated by T7 peptide-modified low-density lipoprotein particles
Published in Drug Delivery, 2018
Meng Liang, Chunhong Gao, Yuli Wang, Wei Gong, Shiyao Fu, Lin Cui, Zhenhan Zhou, Xiaoyang Chu, Yue Zhang, Qianqian Liu, Xiong Zhao, Baoquan Zhao, Meiyan Yang, Zhiping Li, Chunrong Yang, Xiangyang Xie, Yang Yang, Chunsheng Gao
The carboxyl group in fluorescence probe (5-(6)-carboxyfluorescein diacetate, CFDA) was reacted with the primary amine group in the T7 of NHS-PEG3500-T7 and formed the target product NHS-PEG3500-T7(CFDA). Briefly, 5 mg of CFDA was dissolved in 1 mL of dimethyl sulfoxide, followed by adding 2.4 mg of N,N′-dicyclohexyl carbodiimide and 1.3 mg of NHS. This system was stirred for 24 h at room temperature, and then the insoluble substances were removed by centrifugation (4000 r·min−1, 15 min) from it. After that, 2 mg of NHS-PEG3500-T7 and 1 μL of triethylamine were added to the supernatant at room temperature, and reacted for 24 h avoiding light. Then, the above system was dialyzed (MWCO 3.5 kDa, Thermo Scientific, Waltham, MA) in distilled water without light for 24 h to remove the unreacted products. The dialyzed products (NHS-PEG3500-T7(CFDA)) were freeze-dried and obtained puffed solid products, which were finally characterized by MALDI-TOF MS.
Carbonic anhydrase 12 mutation modulates membrane stability and volume regulation of aquaporin 5
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Soyoung Hwang, Jung Yun Kang, Min Jae Kim, Dong Min Shin, Jeong Hee Hong
β-Actin antibody (A3854), 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS; 462268), carbachol (1092009), acetazolamide (A6011), and amiloride hydrochloride hydrate (Amiloride; A7410) were purchased from Sigma (Saint-Louis, MO). AQP5 (ab92320), Na+-K+-Cl− co-transporter1 (NKCC1, ab59791), SLC26A6 (ab123001), and AE2 (ab42687) antibodies were purchased from Abcam (Cambridge, MA). CA12 antibody was purchased from ProteinTech Inc. (15180–1-AP; Chicago, IL). 2',7'-Bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-AM was purchased from TEFlabs (0061; Austin, TX). TNF-α (210-TA-005) was purchased from R&D Systems (Minneapolis, MN). Calcein-AM was purchased from Molecular Probes (C1430; Eugene, OR). Pluronic acid (F-127, 20% in dimethyl sulphoxide, P-3000MP), pHrodo Green AM (P35373), and ZO-1 antibody (940–2200) were purchased from Invitrogen (Carlsbad, CA). All other chemicals were purchased from Sigma. GFP-tagged human AQP5 was a kind gift from Kyung Pyo Park (Seoul National University, South Korea), and DNA plasmids for pcDNA3.0-human NKCC1, mKate-human SLC26A6, HA-rabbit AE2, mKate-human CA12 wild type, and mKate-CA12 E143K mutants were generously provided by Dr. Shmuel Muallem (National Institutes of Health, Bethesda)8.
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