Uptake Of Fluorescein Research Articles

Uptake of Fluorescein via a pH-Dependent Monocarboxylate Transporter by Human Kidney 2 (HK-2) Cells.

Herein, we investigated whether a fluorescent probe for an organic anion transporter (OAT), fluorescein (FLS), could be accumulated by human kidney 2 (HK-2) cells derived from human kidney proximal tubular epithelia. HK-2 cells took up FLS in a pH-dependent and concentration-dependent manner. FLS accumulation by HK-2 cells was inhibited by monocarboxylic acids, ibuprofen, rosuvastatin, and indoleacetic acid but not by typical substrates for OATs. A typical protonophore, carbonyl cyanide p-trichloromethoxyphenylhydrazone completely abolished FLS accumulation by HK-2 cells. The FLS efflux process from the preloaded HK-2 cells exhibited substantial trans-stimulation by the excess amount of extracellular FLS transport inhibitable monocarboxylate compounds such as 2,4-dichloro phenoxyacetic acid, fluvastatin, ibuprofen, indoleacetic acid, salicylic acid and rosuvastatin, indicating that the FLS transporter can recognize and accumulate them into the cells in a pH-dependent manner. The involvement of the FLS transporter in the reabsorption of monocarboxylic compounds was indicated by demonstrating that the pH-dependent FLS uptake is inhibited by various monocarboxylates in rabbit renal brush border membrane vesicles. pH-dependent FLS uptake was trans-stimulated by the inhibitable monocarboxylates. Collectively, the present data indicate that the pH-dependent transporters expressed in HK-2 cells are involved in the reabsorption of monocarboxylates from the urinary fluid into the tubular epithelia.

Uptake of Fluorescein upon Osmotic Cell Swelling is Dependent on the Volume- Regulated Anion Channel VRAC/LRRC8

Background/Aims: The permeability of the plasma membrane for some substances increases during osmotic swelling of the cell. Transport systems for cationic dyes were identified previously, but the hypotonic uptake of anionic substances remained elusive. We aimed to test for the hypotonic uptake of the anionic dye fluorescein and to identify the transport pathway. Methods: We quantified the uptake of fluorescein in hypoosmotic medium by fluorescence microscopy in several cell lines. To investigate the uptake mechanism, we used pharmacological inhibitors for the volume-regulated anion channel VRAC and a genome-edited cell line lacking the essential LRRC8A VRAC subunit. Results: Fluorescein was specifically taken up under hypotonic conditions in HeLa, MDCK, and 3T3 cells. Both pharmacological inhibition and genetic disruption of VRAC strongly diminished hypotonicity-driven dye uptake. Conclusion: Cellular uptake of fluorescein is mediated by the volume-regulated anion channel VRAC. The measurement of fluorescein accumulation can be used as a convenient method to detect and study VRAC activation.

Development of Fluorescently Labeled Self-Emulsifying Drug Delivery Systems (SEDDS) for Prolonged Stability, In Vitro Sustained Release, and Cellular Uptake.

In this study, four fluorescein hydrophobic ionic complexes were formed with the cationic polymers Eudragit RS, Eudragit RL, Eudragit E, and polyethyleneimine (PEI) to provide fluorescein sustained release, sustained cellular uptake, and stability. Complexes were loaded in a self-emulsifying drug delivery system (SEDDS) composed of 40% Tween 80, 20% Kolliphor EL, 15% 2-n-Octyl-1-dodecanol, and 25% dipropylene glycol. SEDDS were investigated regarding their size, polydispersity index (PDI), zeta potential, and cytotoxicity. Fluorescein release from SEDDS was performed in phosphate buffer (pH 6.8 and pH 8), and the released fluorescein was evaluated for cellular uptake. Moreover, fluorescein from all of the SEDDS pre-concentrates was released at different time points to check its long-term stability over six months. The average fluorescein load in SEDDS was 0.045%. SEDDS showed an average droplet size of 24.9 ± 1.6 nm with PDI ≤ 0.3. SEDDS complexes diluted 1:100 increased the zeta potential from -7.3 mV to +3.7 mV and provided > 85% cell viability. A 92.27 ± 3.18% fluorescein exhibited a few seconds of immediate release when used as control or PEI complex in SEDDS. On the contrary, Eudragit-fluorescein complexes in SEDDS showed sustained release of 87.01 ± 5.22% fluorescein in ≤ 70 min with 22.19 ± 14.56% and 59.27 ± 16.57% released at 10 min in pH 6.8 and pH 8 release media, respectively. Comparatively, the medium at pH 6.8 maintained a significantly improved sustained fluorescein release (p ≤ 0.001). Furthermore, Eudragit RS/RL compared to Eudragit E, significantly exhibited a slower fluorescein release rate from SEDDS (p ≤ 0.01). The cellular uptake of the released fluorescein was 72.4 ± 8.2% for all SEDDS complexes after 3 h. Eudragit complexes compared to PEI complex in SEDDS significantly showed m ore sustained fluorescein cellular uptake at 1 h and 2 h (p ≤ 0.001). However, SEDDS complexes showed the longest fluorescein stability with PEI after six months, whereas fluorescein stability for SEDDS containing fluorescein as Eudragit complex and control showed 39.1% and 82.5% fluorescence decrease, respectively, after three months. In the developed SEDDS, the presence of hydrophobic ionic complexes can significantly promote longer stability and sustained cellular uptake of fluorescein while releasing in a sustained manner.

EXTH-36. ELECTROCONVULSIVE SEIZURE-INDUCED CHANGES IN THE TUMOR MICROENVIRONMENT PROMOTE SURVIVAL IN GLIOMA-BEARING MICE

Abstract PURPOSE Growing evidence indicates that the neurotransmitters dysregulated in psychiatric disorders are similarly dysregulated in glioblastoma (GBM) biology. GBM cells are dependent on bountiful neuronal glutamate, utilize elevated dopamine receptor expression to augment progression, and catabolize serotonin to drive proliferation and inhibit anti-tumor immunity. The clinical induction of seizure, known as electroconvulsive therapy (ECT), has been used by psychiatrists since the 1930s to correct these dysregulations and can additionally improve medication blood-brain barrier (BBB) penetrance. We hypothesized that seizure-induced changes in the glioma microenvironment occur with ECT, slowing tumor progression, increasing BBB permeability, and prolonging overall survival in glioma-bearing mice. METHODS C57BL6 mice were orthotopically injected with CT-2A-Luc mouse glioma cells. Mice were randomized to receive ECT via ear-clip electrodes or sham treatment daily up to five times per week. Intracranial progression was monitored via bioluminescent signal from CT-2A-Luc xenografts. BBB permeability was assessed by subjecting mice to ECT or sham treatment immediately following intravenous injection of sodium fluorescein. RESULTS Intracranial progression was maximally reduced in ECT-treated mice relative to sham-treated mice after 17 ECT treatments (ECT radiance 2.6 x 109 photons/second versus sham 4.7 x 109 photons/second, p=0.013), which was further confirmed by both decreased tumor weight and tumor size on histologic evaluation. This translated into an improvement in overall survival from median 29 days in sham-treated mice to 38 days in ECT-treated mice (p=0.0018). Mean seizure duration was 41.8 seconds and positively correlated with overall survival (Pearson coefficient r=0.63, p=0.028). Brain parenchymal uptake of sodium fluorescein was significantly higher in ECT-treated mice (mean relative increase in ECS to sham radiance of 1.47, p< 0.05). CONCLUSION Repeated ECT slows tumor progression and prolongs overall survival in C57BL6 mice bearing CT-2A-Luc xenografts. The BBB is compromised immediately following ECT. ECT merits further oncologic investigation as a potential therapeutic in GBM.

Characterizing Seizure-Induced Changes in the Glioma Microenvironment

Growing evidence indicates that the neurotransmitters dysregulated in psychiatric disorders are similarly dysregulated in glioblastoma (GBM) biology. Patients with psychiatric disorders are classically thought of having excessive glutamate (major depression, bipolar depression), excessive dopamine (mania, psychosis), and/or insufficient serotonin (major depression, anxiety). Analogously, GBM cells are dependent on bountiful neuronal glutamate, utilize elevated dopamine receptor expression to activate the hypoxic response and augment progression, and catabolize serotonin to drive proliferation. The clinical induction of seizure, known as electroconvulsive therapy (ECT), has been used by psychiatrists since the 1930s to treat severe cases of depression, mania, psychosis, and catatonia. ECT has been shown to decrease extracellular glutamate and glutamate receptor expression in vivo and increase blood-brain barrier (BBB) permeability. ECT, then, may have previously unexplored oncologic value. We hypothesized that seizure-induced changes in the glioma microenvironment occur with ECT, increasing permeability of the BBB, slowing tumor progression, and prolonging overall survival in glioma-bearing mice.C57BL6 mice were orthotopically injected with CT-2A-Luc mouse glioma cells. Mice were randomized to receive ECT via ear-clip electrodes or sham treatment daily up to five times per week until survival endpoints were reached. Seizure duration and rates of tonic hindlimb extension were recorded. Intracranial progression was monitored via bioluminescent signal from CT-2A-Luc xenografts. BBB permeability was assessed by subjecting mice to ECT or sham treatment immediately following tail vein injection of sodium fluorescein. Mice were saline perfused and brains were harvested for fluorescent spectroscopy.Intracranial progression was maximally reduced in ECT-treated mice relative to sham-treated mice after 17 treatments (mean sham radiance 4.7 × 109 photons/s versus mean ECT radiance 2.6 × 109 photon/s, P = 0.013). This translated into an improvement in overall survival from median 29 days in sham-treated mice to 34.5 days in ECT-treated mice (P = 0.0018). Mean seizure duration was 41.8 seconds and positively correlated with overall survival (Pearson coefficient r = 0.63, P = 0.028). Tonic hindlimb extension occurred in 68% of seizures and positively correlated with overall survival (Pearson coefficient r = 0.72, P = 0.0078). Brain parenchymal uptake of sodium fluorescein was significantly higher in ECT-treated mice, with a mean relative increase in ECS to sham radiance of 1.47 (P < 0.05).Repeated ECT slows tumor progression and prolongs overall survival in C57BL6 mice bearing CT-2A-Luc xenografts. The BBB is compromised immediately following ECT. ECT merits further oncologic investigation as a potential therapeutic in GBM.T. Sita: None. L. Hurley: None. C. Dussold: None. S. Sachdev: None. R. Stupp: Research Grant; CarThera, Paris. Consultant/ad hoc advisor for research protocol development; GT Medical Technologies, Insightec; Northwest Biotherapeutics. Scientific Advisory Board; CranioVation, TriAct, Hemispherian. IDMC of an ongoing clinical trial; Celularity. A. Stegh: None.

M01 as a novel drug enhancer for specifically targeting the blood-brain barrier.

Drug delivery to the brain is limited for most pharmaceuticals by the blood-brain barrier (BBB) where claudin-5 dominates the paraendothelial tightening. For circumventing the BBB, we identified the compound M01 as a claudin-5 interaction inhibitor. M01 causes transient permeabilisation of the BBB depending on the concentration of small molecules in different cell culture models within 3 to 48 h. In mice, brain uptake of fluorescein peaked within the first 3 h after M01 injection and normalised within 48 h. Compared to the cytostatic paclitaxel alone, M01 improved delivery of paclitaxel to mouse brain and reduced orthotopic glioblastoma growth. Results on interactions of M01 with claudin-5 were incorporated into a binding model which suggests association of its aromatic parts with highly conserved residues of the extracellular domain of claudin-5 and adjacent transmembrane segments. Our results indicate the following mode of action: M01 preferentially binds to the extracellular claudin-5 domain, which weakens trans-interactions between adhering cells. Further decrease in membranous claudin-5 levels due to internalization and transcriptional downregulation enables the paracellular passage of small molecules. In summary, the first small molecule is introduced here as a drug enhancer, which specifically permeabilises the BBB for a sufficient interval for allowing neuropharmaceuticals to enter the brain.

Screening of Polymer-Based Drug Delivery Vehicles Targeting Folate Receptors in Triple-Negative Breast Cancer

PurposeTo compare cellular uptake and cytotoxicity of fluorescein (FL)-labeled polyethylene glycols (PEGs) carrying 2 folate groups (targeted delivery vehicles [TDVs]) to non-PEGylated molecules with 1 or 2 folate groups. Materials and MethodsThree PEGylated TDVs and 2 non-PEGylated folic acid (FA)–fluorescein (FL) conjugates (FA-FL and FA-FL-FA) were synthesized. Two triple-negative breast cancer cell lines (MDA-MB-231and MDA-MB-468) were cultured to 70% confluency and incubated for 2 h in a folate-depleted medium. Folate receptor (FR) expression was confirmed by immunocytochemistry. Cellular uptake and cytotoxicity of compounds were measured by flow cytometry. Intracellular localization was confirmed using confocal microscopy. ResultsMDA-MB-231 demonstrated 40% more FR staining than MD-MB-468. Intracellular localization of the 2 non-PEGylated molecules (FA-FL and FA-FL-FA) and the 3 PEGylated TDVs was confirmed with confocal microscopy. Cellular uptake was independent of concentration for FA-FL, but there was 26.8% more cytotoxicity at 30 μg/mL compared with no treatment (P ≤ .05). Uptake was > 90% for FA-FL-FA at 10 μg/mL and 30 μg/mL without significant cytotoxicity (P ≤ .005). Cellular uptake was > 80% for all TDVs. The molecule containing monodispersed PEG with Mn = 1,000 g/mol had the highest uptake in both cell lines without cytotoxicity. Maximum toxicity was demonstrated by the molecule containing PEG2,000 only at the highest dose of 30 μg/mL (8.66% ± 3.94% cytotoxicity; cut-off was 20%). ConclusionsThe molecule containing monodispersed PEG with Mn = 1,000 g/mol and 2 FA targeting groups demonstrated better targetability and cellular uptake as a TDV.

Design and Characterization of a Cancer-Targeted Drug Co-Delivery System Composed of Liposomes and Selenium Nanoparticles.

A drug co-delivery system composed of selenium nanoparticles (SeNPs) has attracted increasing interest due to its ability to increase the anticancer efficacy against multidrug-resistant cancer cells. In this study, a cancer-targeted drug co-delivery system combining fluorescein-loaded liposomes and SeNPs was designed and evaluated. The system was developed by coating SeNPs and fluorescein-loaded liposomes with folic acid-chitosan conjugates (FA-CS-SeNPs-Lips). Folic acid-chitosan conjugates (FA-CS) were synthesized by coupling folic acid (FA) with chitosan (CS), and the structure was confirmed by performing Fourier transform spectroscopy (FT-IR) and nuclear magnetic resonance (1H-NMR) spectroscopy. Dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM) were used to evaluate the particle size, Zeta potential, and morphology. The cytotoxicity of SeNPs coated with FA-CS conjugates (FA-CS-SeNPs) toward A549 cells and HeLa cells was examined using the MTT assay. The cancer-targeting ability and drug release behaviors were evaluated in vitro by measuring the cellular uptake of fluorescein and dialysis, respectively. The FA-CS-SeNPs were uniform, spherical particles with a ~50 nm diameter and high positive Zeta potential (+57.7 mV). Based on the results of the MTT assay, FA-CS-SeNPs displayed a more significant increase in the anticancer efficacy in HeLa cells than CS-SeNPs. FA-CS-SeNPs-Lips not only slowly released fluorescein but also specifically targeted HeLa cells through selective binding between folate and folate receptors to increase the cellular uptake of fluorescein.

Surface-Modified Chitosan: An Adsorption Study of a “Tweezer-Like” Biopolymer with Fluorescein

Tweezer-like adsorbents with enhanced surface area were synthesized by grafting aniline onto the amine sites of a chitosan biopolymer scaffold. The chemical structure and textural properties of the adsorbents were characterized by thermogravimetric analysis (TGA) and spectral methods, including Fourier transform infrared (FT-IR), nuclear magnetic resonance (1H- and, 13C-NMR) and scanning electron microscopy (SEM). Equilibrium solvent swelling results for the adsorbent materials provided evidence of a more apolar biopolymer surface upon grafting. Equilibrium uptake studies with fluorescein at ambient pH in aqueous media reveal a high monolayer adsorption capacity (Qm) of 61.8 mg·g−1, according to the Langmuir isotherm model. The kinetic adsorption profiles are described by the pseudo-first order kinetic model. 1D NMR and 2D-NOESY NMR spectra were used to confirm the role of π-π interactions between the adsorbent and adsorbate. Surface modification of the adsorbent using monomeric and dimeric cationic surfactants with long hydrocarbon chains altered the hydrophile-lipophile balance (HLB) of the adsorbent surface, which resulted in attenuated uptake of fluorescein by the chitosan molecular tweezers. This research contributes to a first example of the uptake properties for a tweezer-like chitosan adsorbent and the key role of weak cooperative interactions in controlled adsorption of a model anionic dye.

64Cu and fluorescein labeled anti-miRNA peptide nucleic acids for the detection of miRNA expression in living cells

MiRNAs are single stranded RNAs of 18–22 nucleotides. They are promising diagnostic and prognostic markers for several pathologies including tumors, neurodegenerative, cardiovascular and autoimmune diseases. In the present work the development and characterization of anti-miRNA radiolabeled probes based on peptide nucleic acids (PNAs) for potential non-invasive molecular imaging in vivo of giant cell arteritis are described. MiR-146a and miR-146b-5p were selected as targets because they have been found up-regulated in this disease. Anti-miR and scramble PNAs were synthesized and linked to carboxyfluorescein or DOTA. DOTA-anti-miR PNAs were then labelled with copper-64 (64Cu) to function as non-invasive molecular imaging tools. The affinity of the probes for the targets was assessed in vitro by circular dichroism and melting temperature. Differential uptake of fluorescein and 64Cu labeled anti-miRNA probes was tested on BCPAP and A549 cell lines, expressing different levels of miR-146a and -146b-5p. The experiments showed that the anti-miR-146a PNAs were more effective than the anti-miR-146b-5p PNAs. Anti-miR-146a PNAs could bind both miR-146a and miR-146b-5p. The uptake of fluorescein and 64Cu labeled anti-miR-146a PNAs was higher than that of the negative control scramble PNAs in miRNA expressing cells in vitro. 64Cu-anti-miR-146a PNAs might be further investigated for non-invasive PET imaging of miR-146 overexpressing diseases.

Cellular fluorescein hyperfluorescence is dynamin-dependent and increased by Tetronic 1107 treatment.

Sodium fluorescein ('fluorescein') staining of the ocular surface is frequently an indicator of compromised ocular health, and increases in the presence of certain contact lens multi-purpose solutions (MPS), a phenomenon known as solution induced corneal staining (SICS). The mechanism(s) underpinning fluorescein hyperfluorescence are uncertain, though may reflect increased cellular uptake of fluorescein by corneal epithelial cells. We have developed an in vitro model to study fluorescein uptake in both 'generic' mammalian cells (murine fibroblasts) and human corneal cells. Fluorescein hyperfluorescence increased after treatment with two MPS associated with clinical corneal fluorescein staining, yet there was no cellular hyperfluorescence for two MPS that do not cause this staining. Increased fluorescein uptake did not correlate with presence of a necrotic or an apoptotic marker (propidium iodide and caspase-3 respectively). Incubation of MPS-treated cells with dynasore (an inhibitor of dynamin, implicated in endocytic pathways) reduced fluorescein uptake irrespective of MPS treatment. The non-ionic surfactant Tetronic 1107 (present in both MPS associated with corneal fluorescein staining) increased uptake of fluorescein for both cell types, whereas an unrelated surfactant (Triton X-100) did not. We conclude that the clinical hyperfluorescence profile observed after exposure to four MPS can be reproduced using a simple model of cellular fluorescein uptake, suggesting this is the biological basis for SICS. Fluorescein entry does not correlate with necrosis or apoptosis, but instead involves a dynamin-dependent active process. Moreover the surfactant Tetronic 1107 appears to be a key MPS constituent triggering increased fluorescein entry, and may be the major factor responsible for SICS.

Insulin- and quercetin-loaded liquid crystalline nanoparticles: implications on oral bioavailability, antidiabetic and antioxidant efficacy.

The present study reports insulin (INS)- and quercetin (QT)-lyotropic liquid crystalline nanoparticles (LCNPs) with improved bioavailability, antidiabetic and antioxidant efficacy following oral administration. The developed INS-QT-LCNPs were evaluated for simulated gastric fluid stability. In vitro Caco-2 uptake studies were also performed. Furthermore, in vivo pharmacokinetics and pharmacodynamics of INS-QT-LCNPs were evaluated. INS entrapped within LCNPs demonstrated excellent stability in simulated gastric fluid. Higher uptake of fluorescein isothiocyanate-INS-LCNPs were observed in Caco-2 cells. INS-LCNPs demonstrated approximately 20% relative bioavailability compared with subcutaneously administered INS. Significant decrease in oxidative stress was confirmed by reduction in malondialdehyde level. Overall, combination strategy not only overcomes poor oral bioavailability of INS and QT, but also prevents the generation of reactive oxygen species, responsible for diabetes-mediated complications.

Photoperiodic Regulation of Growth-Dormancy Cycling through Induction of Multiple Bud-Shoot Barriers Preventing Water Transport into the Winter Buds of Norway Spruce.

Whereas long days (LDs) sustain shoot elongation, short days (SDs) induce growth cessation and formation of dormant buds in young individuals of a wide range of temperate and boreal tree species. In specific conifers, including Norway spruce, photoperiodic control of bud development is associated with the formation of a plate of thick-walled cells, denoted as the crown, at the base of the bud. Information about cellular characteristics of this crown region is limited. We aimed to test whether the crown region is an important SD-induced barrier ensuring dehydration of the developing winter bud by preventing water influx. Using microscopy and synchrotron techniques, we show here that under LD, cell walls in growing shoot tips had highly methyl-esterified homogalacturonan pectin. During SD-induced bud development, the homogalacturonan in the crown region was de-methyl-esterified, enabling Ca2+ binding and crosslinking, a process known to decrease cell wall water permeability by reducing pectin pore size. In addition, there was abundant callose deposition at plasmodesmata in the crown region, and xylem connections between the bud and the subtending shoot were blocked. Consistent with reduced water transport across the crown region into the bud, uptake of fluorescein in shoot tips was blocked at the base of the bud under SD. Upon transfer from SD to bud-break-inducing LD, these processes were reversed, and aquaporin transcript levels significantly increased in young stem tissue after 4 weeks under LD. These findings indicate that terminal bud development is associated with reduced water transport through decreased cell wall permeability and blocking of plasmodesmata and xylem connections in the crown structure. This provides further understanding of the regulatory mechanism for growth-dormancy cycling in coniferous tree species such as Norway spruce.

Substrate-dependent effects of molecular-targeted anticancer agents on activity of organic anion transporting polypeptide 1B1

1. Organic anion-transporting polypeptide 1B1 (OATP1B1) plays an important role in the hepatic uptake of a broad range of substrate drugs. In vitro experiments show that molecular-targeted agents do not always have similar effects on OATP1B1 activity.2. The purpose of this study was to clarify whether the effects of molecular-targeted agents on OATP1B1 are substrate-dependent. We used OATP1B1-transfected cells to compare the effects of molecular-targeted agents on OATP1B1-mediated uptake of fluorescein (FL), 2′,7′-dichlorofluorescein (DCF), atorvastatin, SN-38 and valsartan.3. Cabozantinib, cediranib, neratinib, pazopanib, regorafenib, sorafenib and tivantinib did not affect or only slightly affected OATP1B1-mediated substrate uptake. Nilotinib and lenvatinib moderately and strongly inhibited OATP1B1-mediated substrate uptake, respectively. In contrast, afatinib stimulated OATP1B1-mediated uptake of FL and SN-38, ceritinib stimulated that of valsartan, and nintedanib stimulated that of FL and valsartan. In addition, the effects of afatinib, ceritinib and nintedanib on OATP1B1 activity differed markedly depending on the type of substrate. Afatinib, ceritinib and nintedanib had a substrate-dependent effect on OATP1B1 activity.4. We conclude that the evaluation of OATP1B1 activity using only a single probe substrate for some molecular-targeted agents may lead to a faulty understanding of their mechanisms of drug interactions.

Effect of molecular weight on sonoporation-mediated uptake in human cardiac cells

Sonoporation of cells induced by ultrasound-driven microbubble cavitation has been utilized for intracellular delivery of molecular therapeutics. The molecular weight of therapeutic agents can vary significantly, with DNA plasmids often larger than 5 MDa, siRNAs and miRNAs ~10 kDa, and other drugs often less than 1 kDa. Some studies have suggested that sonoporation-mediated uptake may decrease at higher molecular weights due to slower diffusion rates, but experiments with equal molar concentrations have not been reported. Therefore, the objective of this study was to explore the effect of molecular weight on sonoporation-mediated uptake of fluorescein (0.33 kDa) or fluorescent dextrans (10 kDa, 2 MDa) using equal molar concentrations (100 nM). Sonoporation was induced in cultured human cardiac mesenchymal cells using lipid microbubbles and 2.5 MHz B-mode ultrasound (P4-1 transducer, Verasonics Vantage ultrasound system) and uptake by viable cells was measured with flow cytometry. No significant differences in sonoporation-mediated uptake were measured between molecules of different sizes at equal molar concentrations (ANOVA p = 0.92). However, significant differences in uptake were observed at different molar concentrations and acoustic pressures (ANOVA p = 0.02). In summary, these results suggest that the effect of molecular weight on sonoporation-mediated uptake is minimal compared to drug concentration or acoustic pressure.

Kinetic Analysis of the Uptake and Release of Fluorescein by Metal-Organic Framework Nanoparticles.

Metal-organic framework nanoparticles (MOF NPs) are promising guest-host materials with applications in separation, storage, catalysis, and drug delivery. However, on- and off-loading of guest molecules by porous MOF nanostructures are still poorly understood. Here we study uptake and release of fluorescein by two representative MOF NPs, MIL-100(Fe) and MIL-101(Cr). Suspensions of these MOF NPs exhibit well-defined size distributions and crystallinity, as verified by electron microscopy, dynamic light scattering, and X-ray diffraction. Using absorbance spectroscopy the equilibrium dissociation constants and maximum numbers of adsorbed fluorescein molecules per NP were determined. Time-resolved fluorescence studies reveal that rates of release and loading are pH dependent. The kinetics observed are compared to theoretical estimates that account for bulk diffusion into NPs, and retarded internal diffusion and adsorption rates. Our study shows that, rather than being simple volumetric carriers, MOF-NPs are dominated by internal surface properties. The findings will help to optimize payload levels and develop release strategies that exploit varying pH for drug delivery.

Designing Silica‐Based Hybrid Polymers and Their Application in the Loading and Release of Fluorescein as a Model Drug and Diagnostic Agent

ABSTRACTPolymeric hybrid materials are interesting functional materials with many diverse properties. Two new hybrid nanomaterials, poly(methacrylic acid‐co‐(2‐dimethylamino)ethyl methacrylate)/silica (P(MAA‐DMAEM)/SiO2) and poly(methacrylic acid)/silica (P(MAA)/SiO2) were prepared via the emulsifier‐free emulsion polymerization in which 3‐aminopropyltriethoxysilane was used as a silane‐coupling agent, tetraethoxysilane and polyvinyl alcohol were used as a silica component precursor and polymeric colloid stabilizer, respectively. The synthesized nanomaterials were characterized by various techniques and were used as drug delivery systems. Fluorescein, mainly used in angiography, was chosen as a candidate drug/diagnostic agent to investigate its loading and release behavior from the hybrid nanomaterials. The uptake studies were carried out at room temperature, whereas the release studies were carried out at 37ºC under phosphate buffer solution at pH 7.4. The release kinetics was studied by subjecting the obtained data to different kinetic models. The nanohybrid materials exhibited a structure–property relationship in the uptake and release of fluorescein as higher adsorption capacity was observed with a P(MAA)/SiO2 hybrid nanomaterial, whereas more drug release was observed from a P(MAA‐DMAEM)/SiO2 nanomaterial than the former.

Effects of Antiviral Drugs on Organic Anion Transport in Human Placental BeWo Cells.

Placental drug transfer is important for achieving better pharmacotherapy in pregnant women and in fetuses. In the present study, we examined the effects of anti-hepatitis C virus (HCV) and anti-HIV drugs on organic anion transport in human placental BeWo cells. The cellular uptake of two fluorescence organic anions, 8-(2-[fluoresceinyl]aminoethylthio)adenosine-3',5'-cyclic monophosphate (8-FcAMP) and fluorescein, was temperature and concentration dependent. The Michaelis constant (Km) and the maximum uptake rate (Vmax) for 8-FcAMP transport in BeWo cells were estimated to be 6.45 ± 0.75 μM and 25.55 ± 5.93 pmol/mg protein/10 min, respectively. The Km and Vmax values for fluorescein uptake were estimated to be 31.2 ± 11.8 μM and 510.9 ± 90.6 pmol/mg protein/10 min, respectively. Several known substrates of organic anion transporters in human placenta, including atorvastatin, glibenclamide, estrone-3-sulfate, and rifampin, inhibited cellular uptake of 8-FcAMP and fluorescein in BeWo cells. Transport of 8-FcAMP and fluorescein was inhibited by the antiviral drugs boceprevir, telaprevir, elvitegravir, and maraviroc. These findings suggest that some antiviral drugs are sufficiently potent to influence placental drug transfer and cause drug-drug interactions.

Study of the biodistribution of fluorescein in glioma-infiltrated mouse brain and histopathological correlation of intraoperative findings in high-grade gliomas resected under fluorescein fluorescence guidance.

Intravenous fluorescein sodium has been used during resection of high-grade gliomas to help the surgeon visualize tumor margins. Several studies have reported improved rates of gross-total resection (GTR) using high doses of fluorescein sodium under white light. The recent introduction of a fluorescein-specific camera that allows for high-quality intraoperative imaging and use of very low dose fluorescein has drawn new attention to this fluorophore. However, the ability of fluorescein to specifically stain glioma cells is not yet well understood. The authors designed an in vitro model to assess fluorescein uptake in normal human astrocytes and U251 malignant glioma cells. An in vivo experiment was also subsequently designed to study fluorescein uptake by intracranial U87 malignant glioma xenografts in male nonobese diabetic/severe combined immunodeficient mice. A genetically induced mouse glioma model was used to adjust for the possible confounding effect of an inflammatory response in the xenograft model. To assess the intraoperative application of this technology, the authors prospectively enrolled 12 patients who underwent fluorescein-guided resection of their high-grade gliomas using low-dose intravenous fluorescein and a microscope-integrated fluorescence module. Intraoperative fluorescent and nonfluorescent specimens at the tumor margins were randomly analyzed for histopathological correlation. The in vitro and in vivo models suggest that fluorescein demarcation of glioma-invaded brain is the result of distribution of fluorescein into the extracellular space, most likely as a result of an abnormal blood-brain barrier. Glioblastoma tumor cell-specific uptake of fluorescein was not observed, and tumor cells appeared to mostly exclude fluorescein. For the 12 patients who underwent resection of their high-grade gliomas, the histopathological analysis of the resected specimens at the tumor margin confirmed the intraoperative fluorescent findings. Fluorescein fluorescence was highly specific (up to 90.9%) while its sensitivity was 82.2%. False negatives occurred due to lack of fluorescence in areas of diffuse, low-density cellular infiltration. Margins of contrast enhancement based on intraoperative MRI-guided StealthStation neuronavigation correlated well with fluorescent tumor margins. GTR of the contrast-enhancing area as guided by the fluorescent signal was achieved in 100% of cases based on postoperative MRI. Fluorescein sodium does not appear to selectively accumulate in astrocytoma cells but in extracellular tumor cell-rich locations, suggesting that fluorescein is a marker for areas of compromised blood-brain barrier within high-grade astrocytoma. Fluorescein fluorescence appears to correlate intraoperatively with the areas of MR enhancement, thus representing a practical tool to help the surgeon achieve GTR of the enhancing tumor regions.

Inducible nitric oxide synthase in the placenta of pregnant women with preeclampsia

Preeclampsia (PEC) is the cause of fetal and maternal death. Inducible nitric oxide synthase (iNOS) found in macrophages, smooth muscle cells and endothelial cells. The main objective was to detect the fluorescence iNOS in placenta. Cotyledons cuts were made, they were placed in 10% formalin. And on the slides washed with buffer solution, oxygenated water with methanol, using 10% pig serum, anti iNOS Rabbit pAb solution, streptavidin-fluorescein. Microscope Carl Zeiss and Image-Pro 7.0 software was used. Placentas of pregnant women with PEC without pathology were analyzed. Increased uptake of fluorescein in erythrocytes of women without PEC was found, patients who developed PEC was remarkable decreased uptake of fluorescein. Decreasing iNOS activity is evident in preeclampsia.