Can image analysis provide evidence that lysosomal sequestration mediates daunorubicin resistance?

Altered intracellular distribution of weak base anticancer drugs owing to lysosomal sequestration is one purported mechanism contributing to chemotherapy resistance. This has often been demonstrated with the example of daunorubicin (DNR), chemotherapy with its characteristic red fluorescence used to trace it in cellular compartments. Here we addressed the question whether image analysis of DNR fluorescence can reflect its real intracellular distribution. We observed that the relationship between the intensity of the DNR fluorescence and its concentration in water solutions with or without proteins is far from linear. In contrast, nucleic acids, RNA and DNA in particular, dramatically diminish the DNR fluorescence, however, the intensity was proportional to the amount. Therefore, image analysis reflects the composition of different cell compartments (i.e., the presence of proteins and nucleic acids) rather than the actual concentration of DNR in these compartments. In line with these results, we observed highly fluorescent lysosomes and low fluorescent nucleus in sensitive cancer cells treated with low DNR concentrations, a fluorescence pattern thought to be found only in resistant cancer cells. Importantly, LC/MS/MS analysis of extracts from sensitive cells treated with DNR or DNR in combination with an inhibitor of vacuolar ATPase, concanamycin A, indicated that lysosomal accumulation of DNR increased with increasing extracellular concentration. However, even the highest lysosomal accumulation of DNR failed to reduce its extralysosomal concentration and thus change the cell sensitivity to the drug. In conclusion, our results strongly suggest that DNR fluorescence within cells does not indicate the real drug distribution. Further they suggested that lysosomal sequestration of DNR can hardly contribute to its resistance in cancer cells in vitro.

Binding affinity data of DNA aptamers for therapeutic anthracyclines from microscale thermophoresis and surface plasmon resonance spectroscopy.

Anthracyclines like daunorubicin (DRN) and doxorubicin (DOX) play an undisputed key role in cancer treatment, but their chronic administration can cause severe side effects. For precise anthracycline analytical systems, aptamers are preferable recognition elements. Here, we describe the detailed characterisation of a single-stranded DNA aptamer DRN-10 and its truncated versions for DOX and DRN detection. Binding affinities were determined from surface plasmon resonance (SPR) and microscale thermophoresis (MST) and combined with conformational data from circular dichroism (CD). Both aptamers displayed similar nanomolar binding affinities to DRN and DOX, even though their rate constants differed as shown by SPR recordings. SPR kinetic data unravelled a two-state reaction model including a 1 : 1 binding and a subsequent conformational change of the binding complex. This model was supported by CD spectra. In addition, the dissociation constants determined with MST were always lower than that from SPR, and especially for the truncated aptamer they differed by two orders of magnitude. This most probably reflects the methodological difference, namely labelling for MST vs. immobilisation for SPR. From CD recordings, we suggested a specific G-quadruplex as structural basis for anthracycline binding. We concluded that the aptamer DRN-10 is a promising recognition element for anthracycline detection systems and further selected aptamers can be also characterised with the combined methodological approach presented here.

Overcharging Effect in Electrospray Ionization Mass Spectra of Daunomycin-Tuftsin Bioconjugates.

Peptide-based small molecule drug conjugates for targeted tumor therapy are currently in the focus of intensive research. Anthracyclines, like daunomycin, are commonly used anticancer drug molecules and are also often applied in peptide-drug conjugates. However, lability of the O-glycosidic bond during electrospray ionization mass spectrometric analysis hinders the analytical characterization of the constructs. "Overprotonation" can occur if daunomycin is linked to positively charged peptide carriers, like tuftsin derivatives. In these molecules, the high number of positive charges enhances the in-source fragmentation significantly, leading to complex mass spectra composed of mainly fragment ions. Therefore, we investigated different novel tuftsin-daunomycin conjugates to find an appropriate condition for mass spectrometric detection. Our results showed that shifting the charge states to lower charges helped to keep ions intact. In this way, a clear spectrum could be obtained containing intact protonated molecules only. Shifting of the protonation states to lower charges could be achieved with the use of appropriate neutral volatile buffers and with tuning the ion source parameters.

An electrochemical daunorubicin sensor based on the use of platinum nanoparticles loaded onto a nanocomposite prepared from nitrogen decorated reduced graphene oxide and single-walled carbon nanotubes.

A glassy carbon electrode (GCE) was modified with a nanocomposite prepared from nitrogen-doped reduced graphene oxide (N-rGO) and single walled carbon nanotubes (SWCNTs), and then loaded with platinum nanoparticles (Pt NPs) to obtain a voltammetric sensor for daunorubicin (DNR). Reductive doping of GO and the crystallization of the Pt NPs were carried out in a one-step hydrothermal process. The modified electrode was characterized by cyclic voltammetry and differential pulse voltammetry. It exhibited high sensitivity compared with unmodified electrode. Some experimental parameters which affected sensor response were optimized. Under optimum conditions and at a working voltage of typically -0.56 V (vs. Ag/AgCl), the sensor has a low detection limit (3 ng mL-1), a wide linear range (0.01-6 µg mL-1) and good long-term stability. The method was successfully applied to the sensitive and rapid determination of DNR in spiked human serum samples. Graphical abstract Platinum nanoparticles were loaded onto a nanocomposite prepared from nitrogen decorated reduced graphene oxide and single-walled carbon nanotubes (N-rGO-SWCNTs-Pt) and then used for electrochemical determination of daunorubicin (DNR).

Qualitative and quantitative analysis of therapeutic solutions using Raman and infrared spectroscopy.

Anticancer drugs are prescribed and administrated to an increasing number of patients on a daily basis. As a consequence, a number of concerns have been raised about the patient health and safety in the case that the drugs administered are not at the required concentration or even worse not the correct ones. Quality control of therapeutic solutions has therefore been extensively implemented in hospital environments, in order to avoid any failure in the intense workflow faced by administering pharmacists. In the present study, infrared (IR) and Raman spectroscopy have been employed for the analysis of 3 commercially available therapeutic solutions TEVA®, MYLAN®, CERUBIDINE®, respectively containing doxorubicin, epirubicin and daunorubicin. They perfectly illustrate the analytical difficulties encountered, as these 3 chemotherapeutic drugs are isomers, hardly distinguishable with conventional approaches such as UV/VIS spectrometry. Any analytical failure to identify these molecules can lead to delays in patient treatment. While Partial Least Squares Regression analysis demonstrates that both Raman and IR can deliver satisfactory quantitative analysis in the clinical range, with respective Root Mean Square Error of Cross Validation (RMSECV) between 0.0127 - 0.0220 g·L-1 and 0.0573 - 0.0759 g·L-1, the identification rate between the 2 techniques differs substantially. Indeed, Principal Component Analysis - Factorial Discriminant Analysis (PCA-FDA) highlights that, depending on the data preprocessing applied to Raman spectra, the discrimination between the 3 drugs is decreased, with in some cases specificity and sensitivity below 50%. However, IR analysis displays encouraging results with an overall specificity and sensitivity between 99 and 100%, suggesting that reliable validation of the therapeutic solution for administration to patients can be achieved. IR and Raman spectroscopy could assist and support quality control of chemotherapeutic solutions prepared in personalised concentrations for each patient. The effective and reliable characterisation of therapeutic solutions could have a lot to offer to improve current practices in a near future.

Optimization of LC method for the quantification of doxorubicin in plasma and urine samples in view of pharmacokinetic, biomedical and drug monitoring therapy studies.

A simple, rapid, reliable and sensitive method based on liquid chromatography with fluorescence detection (LC-FL) for the quantification of doxorubicin (DOX) in human plasma and urine samples was developed. The assay was carried out after the solid-phase extraction procedure (SPE) with hydrophilic-lipophilic balance (HLB) cartridges, and with daunorubicin hydrochloride (DAU) used as the internal standard. Chromatographic separation was performed on a Discovery HS C18 column in isocratic elution mode, and the detection of the analytes set at excitation and emission wavelengths of 487 and 555 nm, respectively. The developed LC-FL method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection and quantification for DOX were 0.5 and 1 ng/mL in both biological fluids, respectively. Linearity was confirmed in the range of 1-1000 ng/mL and 0.001-25 µg/mL in plasma and urine samples, respectively, with a correlation coefficient greater than 0.9994. The proposed LC-FL method is selective, precise and accurate, and has been successfully applied for drug monitoring in pediatric cancer patients treated with DOX as a component of OEPA (Oncovin (Vincristine)-Etoposide-Prednisone-Adriamycin) and IOA (Ifosfamide-Oncovin-Adriamycin) chemotherapeutic schemes. Moreover, real exposure of hospital personnel to the anthracycline drugs in plasma and urine was evaluated in clinical practice.

Ag-4-ATP-MWCNT electrode modified with dsDNA as label-free electrochemical sensor for the detection of daunorubicin anticancer drug.

This paper describes the synthesis of Ag-4-ATP-MWCNT nanocomposite and its use as a modifier of working electrode. The surface of the electrochemical Ag-4-ATP-MWCNT electrode was modified with a double-stranded DNA (dsDNA) to detect daunorubicin-DNA interactions. Differential pulse voltammetry was applied to develop an electroanalytical procedure for the determination of daunorubicin and evaluate its interaction with dsDNA immobilized on the electrode surface. After the optimization of operational parameters, the linear dependence of the peak current on the daunorubicin concentration was observed in the range of 0.10×10-8 to 1.00×10-5molL-1, with the detection and quantification limits of 3.00×10-10 and 1.00×10-9molL-1, respectively. The proposed biosensor was successfully applied to validate its capability for the determination of daunorubicin in human serum and urine samples.

A novel aptamer functionalized CuInS2 quantum dots probe for daunorubicin sensing and near infrared imaging of prostate cancer cells.

In this paper, a novel daunorubicin (DNR)-loaded MUC1 aptamer-near infrared (NIR) CuInS2 quantum dot (DNR-MUC1-QDs) conjugates were developed, which can be used as a targeted cancer imaging and sensing system. After the NIR CuInS2 QDs conjugated with the MUC1 aptamer-(CGA)7, DNR can intercalate into the double-stranded CG sequence of the MUC1-QDs. The incorporation of multiple CG sequences within the stem of the aptamers may further increase the loading efficiency of DNR on these conjugates. DNR-MUC1-QDs can be used to target prostate cancer cells. We evaluated the capacity of MUC1-CuInS2 QDs for delivering DNR to cancer cells in vitro, and its binding affinity to MUC1-positive and MUC1-negative cells. This novel aptamer functionalized QDs bio-nano-system can not only deliver DNR to the targeted prostate cancer cells, but also can sense DNR by the change of photoluminescence intensity of CuInS2 QDs, which concurrently images the cancer cells. The quenched fluorescence intensity of MUC1-QDs was proportional to the concentration of DNR in the concentration ranges of 33-88 nmol L(-1). The detection limit (LOD) for DNR was 19 nmol L(-1). We demonstrate the specificity and sensitivity of this DNR-MUC1-QDs probe as a cancer cell imaging, therapy and sensing system in vitro.

Fluorescence quenching and spectrophotometric methods for the determination of daunorubicin with meso-tera (4-sulphophenyl) porphyrin as probe.

In this work, a synthetic meso-tera (4-sulfophenyl) porphyrin (TPPS4) was used as a probe to determine daunorubicin (DNR) by fluorescence quenching and spectrophotometric methods. At pH 4.6 potassium acid phthalate-NaOH buffer solution, a 1:1 complex of DNR interacted with TPPS4 formed via the electrostatic attractions and hydrophobic interactions, thus resulted in TPPS4 fluorescence quenching and absorption spectra change. The maximum excitation wavelength (λex) and the maximum emission wavelength (λem) are 435 nm and 672 nm, respectively. The fluorescence quenching values (ΔF) are the good linear relationship to the concentration of DNR in the range of 0.8-6.0 mgL(-1). The method exhibits high sensitivity with the detection limit (3σ) being 27.0 ng mL(-1). Meanwhile, a decrease of absorbance is detected at 433 nm with the appearance of a new absorption peak at 420 nm. The optimum reaction conditions, influencing factors and the effect of coexisting substances have been investigated in our experiment. The results showed that the method had a good selectivity and could be applied to determine DNR in serum and urine samples. In addition, the combine ratio between DNR and TPPS4 was measured and the charge distribution before and after reaction was calculated by quantum chemistry calculation AM1 method. The type of fluorescence quenching was discussed by the absorption spectra change, Stern-Volmer plots and fluorescence lifetime determination.

Stability of [(N-morpholine)metylene]daunorubicin hydrochloride in solid state.

The influence of temperature and relative air humidity on the stability of the novel derivative of daunorubicin: [(N-morpholine)metylene]daunorubicin hydrochloride was investigated. The process of degradation was studied by using high-performance liquid chromatography with ultraviolet (UV) detection. In dry air, the degradation of [(N-morpholine)metylene]daunorubicin hydrochloride was a first-order reaction depending on the substrate concentration, while at relative air humidity from 60.5 to 90.0% it was an autocatalytic reaction of the first order with respect to MMD concentration. The kinetic and thermodynamic parameters of degradation were calculated.

Assay for determination of daunorubicin in cancer cells with multidrug resistance phenotype.

A sensitive assay for direct determination of intracellular level of daunorubicin (DRN) in resistant leukemia cells with overexpressed P-glycoprotein has been developed. This assay is based on a rapid separation of cells from media and fast cut-off of DRN transportation by centrifugation of cells through a layer of silicone oil. Cell pellets were extracted using 1% (v/v) formic acid in 50% (v/v) ethanol in water. The cell extracts were subsequently analysed by liquid chromatography (HPLC) coupled a low-energy collision tandem mass spectrometer equipped with an electrospray ionization source (ESI-CID-MS/MS) operated in the multiple-reaction monitoring (MRM) mode. Calibration curve was linear from 0.4 to 250nM with correlation coefficient (r²) better than 0.998. The limit of quantitation (LOQ) was 0.4 nM. The assay has been successfully applied to a determination of intracellular content of daunorubicin in sensitive K562 and resistant K562/Dox and K562/HHT300 cells.

Detection of daunomycin using phosphatidylserine and aptamer co-immobilized on Au nanoparticles deposited conducting polymer.

A highly sensitive and selective sensor for daunomycin was developed using phosphatidylserine (PS) and aptamer as bioreceptors. The PS and aptamer were co-immobilized onto gold nanoparticles modified/functionalized [2,2':5',2″-terthiophene-3'-(p-benzoic acid)] (polyTTBA) conducting polymer. Direct electrochemistry of daunomycin was used to fabricate a label free sensor that monitors current at -0.61 V. The formation of each layer was confirmed with XPS, SEM, and QCM. Response of the sensor was compared with and without PS in terms of sensitivity and selectivity. Interaction between the sensor probe and daunomycin was determined with DPV. The experimental parameters affecting sensor performance were optimized in terms of concentration of immobilized aptamer, PS:aptamer ratio, temperature, pH, and reaction times. The dynamic range for daunomycin analysis ranged between 0.1 and 60.0 nM with a detection limit of 52.3 ± 2.1 pM. Sensor was also examined for interference effect of other drugs. The present sensor exhibited long term stability and successfully detected daunomycin in a real human urine spiked with daunomycin.

Dendrimer modified graphite sensors for detection of anticancer drug Daunorubicin by voltammetry and electrochemical impedance spectroscopy.

The development of amino-terminated G4 PAMAM dendrimer (PDR) modified disposable electrodes were developed as the first time in our study by using the dendrimer modified disposable graphite (PDR-PGE) and multiwalled carbon nanotube based screen-printed graphite (PDR-MWCNT-SPE) electrodes. Firstly, the microscopic characterization of bare PGEs and PDR modified PGEs was performed. These sensors were then applied for electrochemical monitoring of an anticancer drug, Daunorubicin (DNR). The enhanced oxidation signal of DNR was measured at +0.50 V by using differential pulse voltammetry (DPV) in combination with the PDR-PGEs. The detection limit, estimated from S/N = 3, corresponds accordingly to 317 nM and 128 nM for DNR respectively at the PGE and PDR-PGE. The voltammetric results were consistent with electrochemical impedance spectroscopy (EIS) that was used to characterize the successful modification of PDR onto the surface of PGE and MWCNT-SPE.

Micellar electrokinetic chromatographic analysis for in vitro accumulation of anthracyclines enhanced by inhibitors of cell membrane transporter-proteins in cancer cells.

Cell membrane transporter-proteins have been partly implicated in lowering the accumulation of drugs in cancer cells, leading to multidrug resistance (MDR). Two cancer cell lines, A549 and RDES, were continuously exposed to subclinical concentration (250 nM) of anthracyclines and micellar electrokinetic chromatography was used to investigate their in vitro accumulation after treatment with inhibitors of membrane transporter-proteins. The four anthracylines [doxorubicin (DOX), epirubicin (EPI), daunorubicin (DNR), and idarubicin (IDA)] were separated within a short analysis time of less than 15 min in borate buffer (80 mM, pH 9.22) containing sodium taurodeoxycholate (35 mM), 2-hydroxypropyl-γ-cyclodextrin (3.5% wt/v), and sodium dodecylsulfate (20 mM). Laser-induced fluorescence was used for detection of the anthracyclines. Three inhibitors, verapamil, cyclosporine A and probenecid, were examined by adding each inhibitor independently or two inhibitors simultaneously to the culture medium. It was found that independent use of each inhibitor leads to more efficient accumulation than combined use of verapamil and probenecid. In addition, the results show that effect of inhibitors on the accumulation of anthracyclines depended on type of cell: in RDES, inhibitors enhanced accumulation of all four anthracyclines, while in A549, inhibitors showed different accumulation behavior for each anthracycline. Generally higher accumulation of anthracyclines was observed in RDES cells than A549, as evidenced by dead cells (7-16%) after 24 h of continuous exposure to subclinical concentration.

Stability of epidoxorubicin in solid state.

The influence of temperature and relative air humidity on the stability of epidoxorubicin hydrochloride (EP) was investigated. The degradation of the substance studied was determined: (a) in dry air at 393K, (b) at relative air humidity ~76% at 333K, 343K, 353K, 363K and 373K, (c) in the relative air humidity range 50-90% at 363K. The degradation of EP in the atmosphere of increased relative air humidity was a first-order reaction relative to substance concentration and in dry, hot air (RH 0%; 393K) is a reversible first-order reaction relative to substance concentration. The dependences lnk=f(1/T) and lnk=f(RH%) were described by the equation: lnk=(35.1±10.9)-(16,250±3823)(1/T) and lnk=(3.79±3.34) × 10(-2) (RH%)-(12.9±2.4), respectively. The kinetic and thermodynamic parameters of EP degradation were calculated. The parameters of separation were following: LiChrospher RP-18 column, 5 µm, 250 mm × 4 mm; mobile phase: the mixture of equal volume of acetonitrile and the solution containing 2.88 gl(-1) of sodium laurisulfate and 2.25 ml l(-1) of phosphoric acid (V) 85%; flow rate: 1.0 ml min (-1); UV detection - 254 nm.

Sensitive absorption-based wave-mixing detector for anthracycline drugs separated by capillary electrophoresis.

Sensitive absorption-based detection of anthracycline antibiotics, daunorubicin and doxorubicin is demonstrated using a capillary electrophoresis system interfaced to a nonlinear wave-mixing detection system. Unlike conventional absorption methods, this nonlinear absorption method can detect very thin analytes (50 microm) efficiently. At the same peak height, the wave-mixing CE peak is narrower than a conventional CE peak, and hence, compared to other laser-based or non-laser-based CE on-column detection methods, our wave-mixing detection method offers intrinsically enhanced separation resolution even when using identical CE separation conditions. In this unusually sensitive "absorbance" detection method, two input laser beams interact to produce a thermally induced grating from which coherent laser-like wave-mixing signal beams are created. Using our sensitive "absorbance" on-column CE detector, we report a preliminary concentration detection limit of 9.9x10(-10)M using a 50-mum i.d. capillary column. The corresponding "injected" mass detection limit is 9.1x10(-18)mol using an injection volume of 9.2nL. The corresponding preliminary "detected" mass detection limit inside the 12-pL detector probe volume is 1.2x10(-20)M.

An integrative approach for the isolation, screening and analysis of antitumor agents by liquid chromatography combined with mass spectrometry.

A rapid approach has been developed for screening trace level compounds with antitumor activities based on their interactions with microtubules. This interaction can be quantified with liquid chromatography (LC) by measuring the difference of bioactive compound's concentration before and after the formation of compound-microtubule complexes in the fast dialyzers. To test the effectiveness of this approach, several antitumor drugs such as colchicine, taxol, daunorubicin, and a non-antitumor reagent ketoprofen were used. Results indicate that the antitumor constituents can be identified without any disturbance, and the inactive components can be excluded. This screening method was then successfully applied to some potential antitumor compound mixtures, and the active compounds could be separated and screened rapidly. The binding activities measured were consistent with their cytotoxicity assays. This integrative approach is rapid and convenient for screening, isolating, and analyzing potential antitumor active compounds from a mixture.

Direct determination of intracellular daunorubicin in intact confluent monolayers of AT1 prostate carcinoma cells using a multiwell-multilabel counter.

The cytostatic drug daunorubicin exerts its toxic action by intercalating into the DNA. The efficacy of daunorubicin depends on the intracellular amount in the tumor cell. Here we have evaluated the use of a multiwell-multilabel reader for the direct determination of the fluorescent cytostatic drug daunorubicin in a prostate carcinoma cell line (AT1 R-3327 Dunning prostate carcinoma cells) grown on 24-well plates. We present evidence that this simple fluorescent parameter is a good measure for the toxicologically relevant amount of the drug intercalated into the DNA and, therefore, is a good predictor for the drug's cytotoxicity. The amount of cationic cytostatics in a tumor cell is primarily a function of the efflux pump protein p-gycoprotein (pGP). Therefore, it is of great value that the assay is also suitable for the estimation of the multidrug resistance efflux pump (pGP) activity.

CE-LIF method for the separation of anthracyclines: application to protein binding analysis in plasma using ultrafiltration.

Anthracyclines are chemotherapeutic drugs that are widely used in the treatment of cancers such as lung and ovarian cancers. The simultaneous determination of the anthracyclines, daunorubicin, doxorubicin and epirubicin, was achieved using CE coupled to LIF, with an excitation and emission wavelength of 488 and 560 nm, respectively. Using a borate buffer (105 mM, pH 9.0) and 30% MeOH, a stable and reproducible separation of the three anthracyclines was obtained. The method developed was shown to be capable of monitoring the therapeutic concentrations (50-50 000 ng/mL) of anthracyclines. LODs of 10 ng/mL, calculated at an S/N = 3, were achieved. Using the CE method developed, the in vitro protein binding to plasma was measured by ultrafiltration, and from this investigation the estimated protein binding was determined to be in the range of 77-94%.

Immunocytochemical studies on the distribution pattern of daunomycin in rat gastrointestinal tract.

The cancer drug daunomycin is used in treatment of leukemia but possesses severe side effects that involve the gastrointestinal tract. We therefore used a newly developed immunocytochemical procedure to determine the distribution of DM in the gastrointestinal tracts of rats after i.v. injection. Two hours after injection, DM was diffusely distributed in nuclei and most parts of the cytoplasm of intestinal epithelial cells. The cytoplasmic immunoreactivity for DM was most pronounced in small granules of the apical cytoplasm. Sixteen hours after injection, DM immunostaining was by and large absent in the villous epithelium but persisted in the intestinal crypts. In addition, staining was also detected in endothelial cells, scattered cells of the lamina propria and in smooth muscle cells. After 5 days, only little staining for DM remained. Similar findings were made in the colon. In the gastric mucosa, DM accumulation persisted at 16 h in some glandular cells but was lost from the surface epithelium. No staining was detected in saline-injected control rats. The distribution of DM accumulation correlated partially with the distribution of apoptotic cells as detected by the TUNEL procedure. Our results pinpoint that DM may exert prolonged effects on glandular and regenerative cells of the gastrointestinal tract-an observation that may explain the gastrointestinal toxicity of the drug. It seems possible that DM accumulation in surface epithelial cells is rapidly cleared through drug transporters.