Addition Of Fluorescein Research Articles

Implementation and validation of an in-house combined fluorescein/media-fill test to qualify radiopharmacy operators

BackgroundThe purpose of this work was to design, validate and implement a media-fill test combined with fluorescein (MFT-F) for the specific qualification and training of radiopharmacy operators, in accordance with United States Pharmacopeia General Chapter 797 and European Good Manufacturing Practices. MFT-F was embedded in the quality management system of our radiopharmacy unit. Its validation involved fluorescein concentration choice, media growth promotion test and evaluation protocol controls (with or without intentional aseptic mistakes). Each operator was evaluated following a three-part evaluation form. Evaluation criteria related to garbing and hygiene, fluorescent contamination and bacteriological contamination (pre- and post-evaluation environment controls and MFT-F samples). Combined MFT-F allowed the assessment of aseptic compounding skills and non-contamination of the working area through a single evaluation. It was also designed to fit the constraints of radiopharmacy common practice related to radiation protection equipment and to the small volumes handled.ResultsA 0.01% fluorescein concentration was chosen to prepare MFT-F. Addition of fluorescein in the culture medium did not jeopardize its growth properties according to growth promotion test. Eleven operators were evaluated and carried out 3 MFT-F over 3 successive days. Pre- and post-evaluation bacteriological controls of every session showed no CFU of microbiological contaminant above 5. All operators validated the garbing and hygiene evaluation, with an average score of 92.7%. All operators validated the fluorescent contamination evaluation, with an average score of 29.4 out of 30. None of the MFT-F samples showed any visible bacterial growth after incubation.ConclusionsCombined MFT-F, as a part of a comprehensive sterile compounding training program, appeared as a convenient and promising tool to increase both the sterile compounding safety and awareness of radioactive contamination in radiopharmacy.

Processing and characterization of MAGIC-f gel with ultraviolet/visible light spectrophotometry

The use of dosimetric gel in radiotherapy for quality control and verification of the dose delivered to the patient has gained prominence, since it is still the only three-dimensional dosimetry method. Based on this concept, the present work had the objective of processing the MAGIC-f polymeric gel with addition of fluorescein and characterizing it with UV/Vis (ultraviolet/visible light) spectrophotometry. The characterization of the samples was done 2, 7 and 34 days after irradiation with 6 MV photons and wavelengths between 300 and 600 nm. The results showed that the higher the dose, the lower the absorbance intensity measured by the equipment due to the increased polymerization degree, a satisfactory and expected result. It could also be concluded that the higher the irradiation dose of the MAGIC-f gel, the lower the fluorescein absorbance intensity – even a few days after irradiation– due to the continuity of polymerization. The MAGIC-f dosimeter with fluorescein has shown to be very promising, but further tests will be necessary to evaluate other dosimetric properties, such as energetic and dose rate dependence.

Real-time molecular optical micro-imaging of EGFR mutations using a fluorescent erlotinib based tracer

BackgroundEGFR mutations are routinely explored in lung adenocarcinoma by sequencing tumoral DNA. The aim of this study was to evaluate a fluorescent-labelled erlotinib based theranostic agent for the molecular imaging of mutated EGFR tumours in vitro and ex vivo using a mice xenograft model and fibred confocal fluorescence microscopy (FCFM).MethodsThe fluorescent tracer was synthesized in our laboratory by addition of fluorescein to an erlotinib molecule. Three human adenocarcinoma cell lines with mutated EGFR (HCC827, H1975 and H1650) and one with wild-type EGFR (A549) were xenografted on 35 Nude mice. MTT viability assay was performed after exposure to our tracer. In vitro imaging was performed at 1 μM tracer solution, and ex vivo imaging was performed on fresh tumours excised from mice and exposed to a 1 μM tracer solution in PBS for 1 h. Real-time molecular imaging was performed using FCFM and median fluorescence intensity (MFI) was recorded for each experiment.ResultsMTT viability assay confirmed that addition of fluorescein to erlotinib did not suppress the cytotoxic of erlotinib on tumoral cells. In vitro FCFM imaging showed that our tracer was able to distinguish cell lines with mutated EGFR from those lines with wild-type EGFR (p < 0.001). Ex vivo FCFM imaging of xenografts with mutated EGFR had a significantly higher MFI than wild-type (p < 0.001). At a cut-off value of 354 Arbitrary Units, MFI of our tracer had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours.ConclusionReal time molecular imaging using fluorescent erlotinib is able to identify ex vivo tumours with EGFR mutations.

A nanodiamond-fluorescein conjugate for cell studies

The use of nanodiamonds in studies with living systems generally involves the modification of their surfaces with functional groups. Fluorescent molecules can be attached to these groups, so that one can know the exact position of the particles in each moment of the interaction with the cells. Here we modify the surface of detonation nanodiamonds and nitrogen-vacancy center nanodiamonds using carboxylation and hydroxylation procedures. Subsequent reactions with silicates and cysteine, before addition of fluorescein allow to obtain fluorescent nano-conjugates. We used confocal microscopy to observe the position of nanodiamonds interacting with HeLa cells. At 3 h post-incubation the green fluorescence is localized in extracellular rounded like-vesicles assemblies while at 24 h the conjugates can be observed inside the cells. The measurement of the fluorescence emitted by both conjugates allowed to find an enhanced emission of fluorescein isothiocyanate (FITC) when the nitrogen-vacancy center is present. We propose the existence of a fluorescence enhancement by electron transference process. The procedure described in this work allows the functionalization of nanodiamonds with FITC and other molecules using functional surface groups and small size mediators. Also, as was proved in our work, the nanodiamond-fluorescein conjugates can be used to track nanoparticles position within the cell. Localization studies are particularly important for drug delivery applications of nanodiamonds.

Targeted cutaneous delivery of ciclosporin A using micellar nanocarriers and the possible role of inter-cluster regions as molecular transport pathways

Oral administration of ciclosporin A (CsA) is indicated in the treatment of severe recalcitrant plaque psoriasis. However, CsA is both nephro- and hepatotoxic and its systemic administration also exposes the patient to other severe side effects. Although topical delivery of CsA, targeted directly to psoriatic skin, would offer significant advantages, there are no topical formulations approved for dermatological use. The aim of this work was to formulate CsA loaded polymeric micelles using the biodegradable and biocompatible MPEG-dihexPLA diblock copolymer and to evaluate their potential for delivering the drug selectively into the skin without concomitant transdermal permeation. Micelle formulations were characterised with respect to drug content, size and morphology. Micelle and drug penetration pathways were subsequently visualised with confocal laser scanning microscopy (CLSM) using fluorescein labelled CsA (Fluo-CsA) and Nile-Red (NR) labelled copolymer. Visualisation studies typically use fluorescent dyes as “model drugs”; however, these may have different physicochemical properties to the drug molecule under investigation. Therefore, in this study it was decided to chemically modify CsA and to use this structurally similar fluorescent analogue to visualise molecular distribution and transport pathways. Molecular modelling techniques and experimental determination of log D served as molecular scale and macroscopic methods to compare the lipophilicity of CsA and Fluo-CsA. The spherical, homogeneous and nanometre-scale micelles (with Zav from 25 to 52nm) increased the aqueous solubility of CsA by 518-fold. Supra-therapeutic amounts of CsA were delivered to human skin (1.4±0.6μg/cm2, cf. a statistically equivalent 1.1±0.5μg/cm2 for porcine skin) after application of the formulation with the lowest CsA and copolymer content (1.67±0.03mg/ml of CsA and 5mg/ml of copolymer) for only 1h without concomitant transdermal permeation. Fluo-CsA was successfully synthesised, characterised and incorporated into fluorescent NR-MPEG-dihexPLA micelles; its conformation was not modified by the addition of fluorescein and its log D, measured from pH4 to 8, was equivalent to that of CsA. Fluo-CsA and NR-MPEG-dihexPLA copolymer were subsequently visualised in skin by CLSM. The images indicated that micelles were preferentially deposited between corneocytes and in the inter-cluster regions (i.e. between the clusters of corneocytes). Fluo-CsA skin penetration was deeper in these structures, suggesting that inter-cluster penetration is probably the preferred transport pathway responsible for the increased cutaneous delivery of CsA.

Crystallization of dicalcium phosphate dihydrate with presence of glutamic acid and arginine at 37 °C.

The formations of non-metabolic stones, bones and teeth were seriously related to the morphology, size and surface reactivity of dicalcium phosphate dihydrate (DCPD). Herein, a facile biomimetic mineralization method with presence of glutamic acid and arginine was employed to fabricate DCPD with well-defined morphology and adjustable crystallite size. In reaction solution containing more arginine, crystallization of DCPD occurred with faster rate of nucleation and higher density of stacked layers due to the generation of more OH(-) ions after hydrolysis of arginine at 37 °C. With addition of fluorescein or acetone, the consumption of OH(-) ions or desolvation reaction of Ca(2+) ions was modulated, which resulted in the fabrication of DCPD with adjustable crystallite sizes and densities of stacked layers. In comparison with fluorescein-loading DCPD, dicalcium phosphate anhydrate was prepared with enhanced photoluminescence properties due to the reduction of self-quenching effect and regular arrangement of encapsulated fluorescein molecules. With addition of more acetone, DCPD was prepared with smaller crystallite size via antisolvent crystallization. The simulated process with addition of amino acids under 37 °C would shed light on the dynamic process of biomineralization for calcium phosphate compounds.

Inter‐ and intra‐individual variability of non‐invasive tear break‐up time in Hong Kong Chinese

Non-invasive tear break-up time (NITBUT) has been proposed as a measure of tear film integrity which is superior to the more commonly used tear break-up time (TBUT), since it does not alter the volume or the physicochemical properties of the tear layer by the addition of fluorescein. We measured NITBUT by measuring the time taken for distortions or discontinuities to appear in the reflected image of a grid pattern which covered about 80 per cent of the corneal surface. NITBUT measures were made 100 times on seven Hong Kong Chinese subjects with up to 20 consecutive measures being made on a single day. We also measured NITBUT on one occasion on an unselected population of 52 Hong Kong Chinese subjects. NITBUT shows a skewed distribution in all subjects, with many shorter values and some extremely long values. There are statistically significant variations in NITBUT from day to day, and from subject to subject. The group of 52 subjects also had a skewed NITBUT distribution with many short values and some very long values. The arithmetic mean does not adequately represent NITBUT data, either for individual subjects or for this group of subjects. As many as five to eight measures may be necessary to gain a stable estimate of the NITBUT and stability of the measure is improved if extreme values are omitted. We recommend the use of nonparametric statistics to compare NITBUT values from day to day in or between subjects.

Estimation of irrigant absorption during transurethral resection of the prostate

Absorption of irrigating solution may complicate transurethral resection of the prostate (TURP), and a system which warns of fluid overload reliably would be of benefit in the prevention of these complications. Fluorescein can easily be detected at very low concentrations in blood and can be added to the irrigating solution in amounts invisible to the naked eye, providing a possible means of easily monitoring the absorption of irrigant solution during TURP. To test this hypothesis, the plasma concentration of fluorescein was determined at intervals after intraperitoneal injection in rats. Although the published data on fluorescein suggest that it meets the criteria for a suitable marker substance to be introduced into the irrigant solution, the results show that plasma fluorescein is constant and not dose related. The addition of fluorescein to the irrigant solution would not provide a quantitative means of determining the volume of irrigant absorbed. The use of other substances may provide the answer to this major clinical problem. We have defined a set of criteria which such a substance should fulfil.