Laser flow cytometry as a tool for the advancement of clinical medicine.

Flow cytometry is a classic laser technology. With the discovery of the cytometer, flow cytometry has become a primary tool in biodiagnostic research. This review focuses on current applications of flow cytometry to the diagnosis of disease and treatment monitoring at the single-cell level. A description of the principles of flow cytometry and a brief overview of the major applications are presented. Our criteria for selecting research papers for this review are those that show advances in biomedicine and pharmacotherapy achieved by using non-invasive flow cytometry. New concepts for diagnosis and classification based on quantitative measurements of cellular parameters and the expression of specific differentiation antigens on the surface of cells will be discussed herein.

(19)F applications in drug development and imaging - a review.

To control drugs in vivo, new approaches are needed. Considerable progress has been made towards the applications of fluorine ((19)F) in pharmacotherapy in this regard. To date, many authors have showed that by using (19)F labelled drugs and non-invasive magnetic resonance imaging (MRI) techniques together, drug biodistribution can be tracked. This review presents methods for (19)F incorporation into pharmaceuticals by forming C-F bonds and drug fluorine oil-water emulsions. Inadequate drug delivery is a major cause of drug resistance, which can be improved using approaches discussed herein aided by (19)F MRI.

Singlet oxygen generation on porous superhydrophobic surfaces: effect of gas flow and sensitizer wetting on trapping efficiency.

We describe physical-organic studies of singlet oxygen generation and transport into an aqueous solution supported on superhydrophobic surfaces on which silicon-phthalocyanine (Pc) particles are immobilized. Singlet oxygen ((1)O2) was trapped by a water-soluble anthracene compound and monitored in situ using a UV-vis spectrometer. When oxygen flows through the porous superhydrophobic surface, singlet oxygen generated in the plastron (i.e., the gas layer beneath the liquid) is transported into the solution within gas bubbles, thereby increasing the liquid-gas surface area over which singlet oxygen can be trapped. Higher photooxidation rates were achieved in flowing oxygen, as compared to when the gas in the plastron was static. Superhydrophobic surfaces were also synthesized so that the Pc particles were located in contact with, or isolated from, the aqueous solution to evaluate the relative effectiveness of singlet oxygen generated in solution and the gas phase, respectively; singlet oxygen generated on particles wetted by the solution was trapped more efficiently than singlet oxygen generated in the plastron, even in the presence of flowing oxygen gas. A mechanism is proposed that explains how Pc particle wetting, plastron gas composition and flow rate as well as gas saturation of the aqueous solution affect singlet oxygen trapping efficiency. These stable superhydrophobic surfaces, which can physically isolate the photosensitizer particles from the solution may be of practical importance for delivering singlet oxygen for water purification and medical devices.

Superhydrophobic photosensitizers. Mechanistic studies of (1)O2 generation in the plastron and solid/liquid droplet interface.

We describe here a physical-organic study of the first triphasic superhydrophobic sensitizer for photooxidations in water droplets. Control of synthetic parameters enables the mechanistic study of "borderline" two- and three-phase superhydrophobic sensitizer surfaces where (1)O2 is generated in compartments that are wetted, partially wetted, or remain dry in the plastron (i.e., air layer beneath the droplet). The superhydrophobic surface is synthesized by partially embedding silicon phthalocyanine (Pc) sensitizing particles to specific locations on polydimethylsiloxane (PDMS) posts printed in a square array (1 mm tall posts on 0.5 mm pitch). In the presence of red light and oxygen, singlet oxygen is formed on the superhydrophobic surface and reacts with 9,10-anthracene dipropionate dianion (1) within a freestanding water droplet to produce an endoperoxide in 54-72% yields. Control of the (1)O2 chemistry was achieved by the synthesis of superhydrophobic surfaces enriched with Pc particles either at the PDMS end-tips or at PDMS post bases. Much of the (1)O2 that reacts with anthracene 1 in the droplets was generated by the sensitizer "wetted" at the Pc particle/water droplet interface and gave the highest endoperoxide yields. About 20% of the (1)O2 can be introduced into the droplet from the plastron. The results indicate that the superhydrophobic sensitizer surface offers a unique system to study (1)O2 transfer routes where a balance of gas and liquid contributions of (1)O2 is tunable within the same superhydrophobic surface.

Autocatalytic-assisted photorelease of a sensitizer drug bound to a silica support.

The photorelease of a sensitizer from a fluorinated silica surface occurs by a reaction of singlet oxygen with the vinyl ether bond linker with scission of a dioxetane intermediate. Irradiation of the released sensitizer generates singlet oxygen, which accelerates the release of more sensitizer via an autocatalytic reaction. Sigmoidal behavior of sensitizer release in n-butanol and n-octanol occurs at an optimal temperature of 20 °C. The photorelease efficiency was reduced at low temperatures, where the sensitizer was retained on the surface due to a long-lived dioxetane with inefficient scission, and also reduced at high temperatures, due to a slower reaction of (1)O2 with the vinyl ether bond. Immediate acceleration is a result of released sensitizer being used as a dopant to eliminate the induction step, further implicating an autocatalytic mechanism. However, the sigmoidal sensitizer release was not correlated to solvent viscosity, heat, or light from the dioxetane decomposition or to minor O2 solubility enhancements caused by the fluorinated silica. The mechanistic information collected here can be used to help control the pace of drug release; however, it remains to be seen whether an autocatalytic-based drug delivery system has an advantage to those with non-sigmoidal kinetics.

Detection of (19)F-labeled biopharmaceuticals in cell cultures with magnetic resonance.

Magnetic resonance (MR) studies of the therapeutic efficacy of fluorinated drugs have recently become possible due to improvements in detection including the application of very strong magnetic fields up to 9.4Tesla (T). These advances allow tracking, identification, and quantification of (19)F-labeled biopharmaceuticals using (19)F MR imaging ((19)F MRI) and spectroscopy ((19)F MRS). Both techniques are noninvasive, are nondestructive, and enable serial measurements. They also allow for controlled and systematic studies of cellular metabolism in cancerous tissue in vivo (small animals and humans) and in vitro (body fluids, cells culture, tissue extracts and isolated tissues). Here we provide an overview of the (19)F MRI and (19)F MRS techniques used for tracking (19)F labeled anticancer chemotherapeutics and antibodies which allow quantification of drug uptake in cancer cells in vitro.

A fiberoptic (photodynamic therapy type) device with a photosensitizer and singlet oxygen delivery probe tip for ovarian cancer cell killing.

A portable "fiber optic-based sensitizer delivery" (FOSD) device has been developed and studied. Before there might be success in photodynamic therapy (PDT) and antibacterial ambitions, an understanding of basic factors on device performance was needed. Thus, the device was examined for the localized delivery of sensitizer molecules in ovarian cancer cells and production of high concentrations of singlet oxygen for their eradication in vitro. The device tip releases stored pheophorbide by attack of singlet oxygen from sensitized oxygen gas delivered through the hollow fiber using 669 nm laser light. The performance of the device was enhanced when configured with a fluorosilane tip by virtue of its Teflon-like property compared with a conventional glass tip (greater sensitizer quantities were photoreleased and laterally diffused, and greater amounts of ovarian OVCAR-5 cancer cells were killed). No cell damage was observed at 2.2 N of force applied by the probe tip itself, an amount used for many of the experiments described here.

Synthesis and characterization of mono-, di-, and tri-poly(ethylene glycol) chlorin e6 conjugates for the photokilling of human ovarian cancer cells.

PEGylated chlorin e(6) photosensitizers were synthesized with tri(ethylene glycol) attached at the ester bond(s) for a 1:1 conjugate at the 17(3)-position, a 2:1 conjugate at the 15(2)- and 17(3)-positions, and a 3:1 conjugate at the 13(1)-, 15(2)-, and 17(3)-positions. These chlorin sensitizers were studied for hydrolytic stability and solubility, as well as ovarian OVCAR-5 cancer cell uptake, localization, and phototoxicity. Increasing numbers of the PEG groups in the mono-, di-, and tri-PEG chlorin conjugates increased the water solubility and sensitivity to hydrolysis and uptake into the ovarian cancer cells. The PEG chlorin conjugates accumulated in the cytoplasm and mitrochondria, but not in lysosomes. Higher phototoxicity was roughly correlated with higher numbers of PEG groups, with the tri-PEG chlorin conjugate showing the best overall ovarian cancer cell photokilling of the series. Singlet oxygen lifetimes, solvent deuteration, and the effects of additives azide ion and d-mannitol were examined to help clarify the photokilling mechanisms. A Type-II (singlet oxygen) photosensitized mechanism is suggested for the di- and tri-PEG chlorin conjugates; however, a more complicated process based in part on a Type-I (radicals or radical ions) mechanism is suggested for the parent chlorin e(6) and the mono-PEG chlorin conjugate.

Bacterial inactivation by a singlet oxygen bubbler: identifying factors controlling the toxicity of (1)O2 bubbles.

A microphotoreactor device was developed to generate bubbles (1.4 mm diameter, 90 µL) containing singlet oxygen at levels toxic to bacteria and fungus. As singlet oxygen decays rapidly to triplet oxygen, the bubbles leave behind no waste or byproducts other than O(2). From a comparative study in deaerated, air saturated, and oxygenated solutions, it was reasoned that the singlet oxygen bubbles inactivate Escherichia coli and Aspergillus fumigatus, mainly by an oxygen gradient inside and outside of the bubble such that singlet oxygen is solvated and diffuses through the aqueous solution until it reacts with the target organism. Thus, singlet oxygen bubble toxicity was inversely proportional to the amount of dissolved oxygen in solution. In a second mechanism, singlet oxygen interacts directly with E. coli that accumulate at the gas-liquid interface although this mechanism operates at a rate approximately 10 times slower. Due to encapsulation in the gaseous core of the bubble and a 0.98 ms lifetime, the bubbles can traverse relatively long 0.39 mm distances carrying (1)O(2) far into the solution; by comparison the diffusion distance of (1)O(2) fully solvated in H(2)O is much shorter (~150 nm). Bubbles that reached the outer air-water interface contained no (1)O(2). The mechanism by which (1)O(2) deactivated organisms was explored through the addition of detergent molecules and Ca(2+) ions. Results indicate that the preferential accumulation of E. coli at the air-water interface of the bubble leads to enhanced toxicity of bubbles containing (1)O(2). The singlet oxygen device offers intriguing possibilities for creating new types of disinfection strategies based on photodynamic ((1)O(2)) bubble carriers.

Fluorine end-capped optical fibers for photosensitizer release and singlet oxygen production.

The usefulness of a fiber optic technique for generating singlet oxygen and releasing the pheophorbide photosensitizer has been increased by the fluorination of the porous Vycor glass tip. Singlet oxygen emerges through the fiber tip with 669-nm light and oxygen, releasing the sensitizer molecules upon a [2 + 2] addition of singlet oxygen with the ethene spacer and scission of a dioxetane intermediate. Switching from a nonfluorinated to a fluorinated glass tip led to a clear reduction of the adsorbtive affinity of the departing sensitizer with improved release into homogeneous toluene solution and bovine tissue, but no difference was found in water since the sensitizer was insoluble. High surface coverage of the nonafluorohexylsilane enhanced the cleavage efficiency by 15% at the ethene site. The fluorosilane groups also caused crowding and seemed to reduce access of (1)O(2) to the ethene site, which attenuated the total quenching rate constant k(T), although there was less wasted (1)O(2) (from surface physical quenching) at the fluorosilane-coated than the native SiOH silica. The observations support a quenching mechanism that the replacement of the SiOH groups for the fluorosilane C-H and C-F groups enhanced the (1)O(2) lifetime at the fiber tip interface due to less efficient electronic-to-vibronic energy transfer.

Generating singlet oxygen bubbles: a new mechanism for gas-liquid oxidations in water.

Laser-coupled microphotoreactors were developed to bubble singlet oxygen [(1)O(2) ((1)Δ(g))] into an aqueous solution containing an oxidizable compound. The reactors consisted of custom-modified SMA fiberoptic receptacles loaded with 150 µm silicon phthalocyanine glass sensitizer particles, where the particles were isolated from direct contact with water by a membrane adhesively bonded to the bottom of each device. A tube fed O(2) gas to the reactor chambers. In the presence of O(2), singlet oxygen was generated by illuminating the sensitizer particles with 669 nm light from an optical fiber coupled to the top of the reactor. The generated (1)O(2) was transported through the membrane by the O(2) stream and formed bubbles in solution. In solution, singlet oxygen reacted with probe compounds (9,10-anthracene dipropionate dianion, trans-2-methyl-2-pentanoate anion, N-benzoyl-D,L-methionine, or N-acetyl-D,L-methionine) to give oxidized products in two stages. The early stage was rapid and showed that (1)O(2) transfer occurred via bubbles mainly in the bulk water solution. The later stage was slow; it arose only from (1)O(2)-probe molecule contact at the gas/liquid interface. A mechanism is proposed that involves (1)O(2) mass transfer and solvation, where smaller bubbles provide better penetration of (1)O(2) into the flowing stream due to higher surface-to-volume contact between the probe molecules and (1)O(2).

Alterations in glycopeptides associated with herceptin treatment of human breast carcinoma mcf-7 and T-lymphoblastoid cells.

The therapeutic humanized monoclonal antibody IgG1 known as Herceptin® has shown remarkable antitumor effects. Although this type of therapy has increased the cancer-free survival of patients, not all tumors respond to this treatment and cancers often develop resistance to the antibody. Despite the fact that Herceptin function has been extensively studied, the precise mechanism underlying its antitumor activity still remains incompletely defined. We previously demonstrated on human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells that monoclonal antibody in combination with Lipoplex consisting of Lipofectamine mixed with plasmid DNA showed a more profound effect on cancer cell viability than antibody alone. The analyses of N-glycans isolated from cancer cells showed dramatic differences in profiles when cells were exposed to Herceptin. Moreover, the investigation of glycosylated peptides from the same cancer cell models after treatment revealed further alterations in the post-translational modifications. Tandem mass spectra obtained from the samples treated confirmed the presence of a series of glycopeptides bearing characteristic oligosaccharides as described in IgG1. However some of them differed by mass differences that corresponded to peptide backbones not described previously and more of them were detected from Herceptin treated samples than from cells transfected with Heceptin/Lipoplex. The results indicate that the presence of Lipoplex prevents antibody transformation and elongates its proper function. The better understanding of the multipart changes described in the glycoconjugates could provide new insights into the mechanism by which antibody induces regression in cancers.

19F MRI of 3D CEM cells to study the effects of tocopherols and tocotrienols.

Oximetry of the human T-Lymphoblastoid (CEM) cells was measured using (19)F magnetic resonance imaging ((19)F MRI). The cells were treated with the analogues of vitamin E, alpha-, gamma-, delta-tocopherols and corresponding tocotrienols, ex vivo in three-dimensional (3D) cell culture. The study showed that (19)F MRI allows to measure the effect of the analogues due to changes of oxygenation, which were detected using MRI. Hexafluorobenzene was used as a (19)F MRI probe sensitive to oxygen concentrations. After 72h of treatment in HFBR with alpha-, gamma-, delta-tocopherols the oxygen concentration was 19.9+/-0.8%, 19.3+/-1.4%, 16+/-3.5%, respectively. The oxygen concentration in cells treated with alpha-, gamma-, delta-tocotrienols was found to be 14+/-1.5%, 10+/-1.2% and 8.8+/-1.1%, respectively whereas for the control cells it was 22.1+/-1%. The results show that delta-tocopherol and delta-tocotrienol are the most effective treatments in CEM cells among all the tested analogues.

Ex vivo assays of CEM cells cultured and treated in the three dimensional cultures.

This study was aimed at the applications of an ex vivo assays to characterization of CEM (Human T-Lymphoblastoid) cells. CEM cells were cultured in three dimensional (3-D) geometry in the Hollow Fibre Bioreactor (HFB) device. The cells were treated with Herceptin, anti-HER-2 (clone CB-11) and lipoplex containing lipofectamine (LipA) and plasmid DNA. To identify the response to treatment, the viability was established using Trypan blue assays. Magnetic resonance imaging (MRI) at 9.4Tesla (T) was applied for localization of the cells in the HFB device. The structural changes in the cells associated with treatment were examined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The tryptic peptides and glycopeptides detected in treated cells provided evidence of the efficacy of antibody binding to the receptor. The results of the study confirmed that cells growth significantly decreased after treatment with antibodies and transfection with lipoplex.

Characterization of ex vivo-generated bovine and human cartilage by immunohistochemical, biochemical, and magnetic resonance imaging analyses.

Osteoarthritis (OA) is a prevalent age-associated disease involving altered chondrocyte homeostasis and cartilage degeneration. The avascular nature of cartilage and the altered chondrocyte phenotype characteristic of OA severely limit the capacity for in vivo tissue regeneration. Cell- and tissue-based repair has the potential to revolutionize treatment of OA, but those approaches have exhibited limited clinical success to date. In this study, we test the hypothesis that bovine and human chondrocytes in a collagen type I scaffold will form hyaline cartilage ex vivo with immunohistochemical, biochemical, and magnetic resonance (MR) endpoints similar to the original native cartilage. Chondrocytes were isolated from 1- to 3-week-old calf knee cartilage or from cartilage obtained from human total knee arthroplasties, suspended in 2.7 mg/mL collagen I, and plated as 300 microL spot cultures with 5 x 10(6) each. Medium formulations were varied, including the amount of serum, the presence or absence of ascorbate, and treatments with cytokines. Bovine chondrocytes generated metachromatic territorial and interstitial matrix and accumulated type II collagen over time. Type VI collagen was confined primarily to the pericellular region. The ex vivo-formed bovine cartilage contained more chondroitin sulfate per dry weight than native cartilage. Human chondrocytes remained viable and generated metachromatic territorial matrix, but were unable to support interstitial matrix accumulation. MR analysis of ex vivo-formed bovine cartilage revealed evidence of progressively maturing matrix, but MR-derived indices of tissue quality did not reach those of native cartilage. We conclude that the collagen-spot culture model supports formation and maturation of three-dimensional hyaline cartilage from active bovine chondrocytes. Future studies will focus on determining the capacity of human chondrocytes to show comparable tissue formation.

N-glycomic changes in human breast carcinoma MCF-7 and T-lymphoblastoid cells after treatment with herceptin and herceptin/Lipoplex.

The humanized monoclonal antibody IgG1 in combination with chemotherapy has been demonstrated to enhance survival benefit in cancer treatment. Despite positive outcomes, some cancer cells develop multidrug resistance. Numerous mechanisms in cancers can be involved in the process of treatment therapy and most of them are not still well understood. To address how the carbohydrate moieties of cells are affected during treatment, the glycan profiles from the two most common cancer cell lines - human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells - were studied here and compared with profiles after treatment with Herceptin alone or in combination with Lipofectamine mixed with plasmid DNA to form Lipoplex. N-Glycans were released from total cells by digestion with PNGaseF and analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In summary, both original cell lines showed a dominant occurrence of high-mannose glycans. After treatment, these structures were suppressed and biantennary core-fucosylated glycans originating from IgG1 were the major carbohydrate products identified in cells. The high incidence of additional fucosylated or nonfucosylated galactosylated oligosaccharides, which were not detected in original cells or Herceptin, varied with conditions and time of exposure of cells to the antibody. The results presented in this study provide strong evidence for a role of glycosylation during antibody treatment.

Combined treatment of human MCF-7 breast carcinoma with antibody, cationic lipid and hyaluronic acid using ex vivo assays.

The effective targeting of malignant cell surface antigens is essential in cancer therapy. Resistance to treatment and rapid invasion of cancer cells are the main causes of cancer mortality. Despite intense research efforts, treatments often have demonstrated insufficient outcomes in clinical applications. The aim of the present study was to determine whether combined administration of monoclonal antibody (Herceptin, trastuzumab) and anti-HER-2 (clone CB11) with hyaluronic acid (HA) and lipoplex (containing lipofectamine (LipA) and plasmid DNA) can produce a synergistic reaction to increase the therapeutic effect of monoclonal antibodies. To assess the treatment response, we cultured a 3-D MCF-7 cell line overexpressing HER-2 and CD44 receptors. The high density 3-D cell aggregation in the hollow fiber bioreactor (HFB) used for the cell culture was monitored with the use of proton magnetic resonance imaging ((1)H MRI). In addition, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used in combination with HPLC (high performance liquid chromatography) to evaluate structural changes in the proteins contained in treated cells. The study showed that incorporation of antibodies into targeted lipoplex results in more efficient delivery of the complex to tumor cells. The viability of cells decreased mostly due to cellular uptake of lipoplex and binding of the antibodies to the cellular surface receptor. The data also demonstrate that HA could be used to enhance treatment efficacy of trastuzumab and anti-HER-2 (clone CB11) in breast cancer cell cultures.

Detection of fluorine labeled herceptin using cellular (19)F MRI ex vivo.

The aim of this study was to assess the herceptin efficacy in ex vivo cultures of MCF-7 breast carcinoma cells. Herceptin was used with perfluorooctyl bromide (PFOB) and conjugated with lipoplex, containing plasmid DNA and lipofectamine (LipA), to allow fluorine-19 magnetic resonance imaging ((19)F MRI) study. Treatments such as herceptin, herceptin/PFOB and herceptin/PFOB/lipoplex were used for ex vivo targeting of MCF-7 cells cultured in three-dimensional (3D) geometry using hollow fiber bioreactor (HFB) device. The viability of MCF-7 cells after 72h treatments decreased to 54+/-2%, 50+/-3% and 45+/-1% for herceptin, herceptin/PFOB and herceptin/PFOB/Lipoplex, respectively. The EC(50) values were 1000microg/ml, 930microg/ml and 730microg/ml, respectively. The significant correlation between the treatment concentration and efficacy was observed in MCF-7 cell cultures.

Application of 19F magnetic resonance to study the efficacy of fluorine labeled drugs in the three-dimensional cultured breast cancer cells.

The cellular monitoring of tumor response to treatments is important for drug discovery and drug development in cancer therapy. We studied efficacy of Herceptin, a common breast cancer drug conjugated with a fluorine organic compound, perfluoro-15-crown-5-ether (PFCE) which easily forms biocompatible emulsions. Three new pharmaceutical forms of Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink were synthesized for the ex vivo study of their efficacy in breast cancer treatment. The emulsions were administered to 10(9)cells mL(-1) of HER-2 positive human adenocarcinoma (MCF-7) cells and the same amount of human mammary epithelial cells (HMEC) cultured in three-dimensional (3D) geometry using hollow fiber bioreactor (HFB) device. Following drugs administration ex vivo, fluorine-19 magnetic resonance imaging ((19)F MRI) was applied for cells imaging to measure their viability and to study drug efficacy over 72h. To ensure optimum drug tracking, HydraLink was used to provide stable binding affinity of emulsified Herceptin to receptor while cationic lipid (Lipofectamine) was used to enhance lipophilicity of the emulsions. After 72h of treatment with Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink the viability of cells was 54+/-2%, 49+/-3%, 43+/-5% and 42+/-1%, respectively, as compared with control 93+/-2%. The efficacy (EC(50)) of Herceptin conjugated with emulsions was found to be 970+/-13 microg mL(-1) for Herceptin/PFCE, 645+/-11 microg mL(-1) for Herceptin/PFCE/Lipoplex, 678+/-7 microg mL(-1) for Herceptin/PFCE/HydraLink and 1000+/-3 microg mL(-1) for Herceptin. The results show that fluorine emulsions improved the efficacy of Herceptin and (19)F signal intensity changes validated drug efficiency. The significant correlations between duration of treatments and MCF-7 cells viability were observed. While we studied breast cancer cells, the fluorine emulsions could be applied for treatment of other cancer cells overexpressing HER-2.

Derivatives of thiocolchicine and its applications to CEM cells treatment using 19F magnetic resonance ex vivo.

It was shown, that cultured ex vivo human T-Lymphoblastoid (CEM) cells respond to synthesized thiocolchicine and fluorine thiocolchicine derivatives. The preparation of derivatives with substitution at C-3 and C-7 is described. All compounds were used at concentration from 1 nM to 1000 nM. Inhibitory effects of these compounds were examined in the three-dimensional (3-D) culture and cells morphology during treatment was monitored using 9.4 T MRI system. We performed studies of these compounds in CEM cells ex vivo using 1H and 19F Magnetic Resonance Imaging (MRI), 19F Magnetic Resonance Spectroscopy (MRS), High Performance Liquid Chromatography coupled with Ultra Violet (HPLC-UV) and Electron Impact Mass Spectrometry (EIMS). The results of the multi-technique approach are consistent with the fact that the new derivatives are more efficient than colchicine and thiocolchicine ex vivo.