Hydrogel-Coated SERS Microneedles for Drug Monitoring in Dermal Interstitial Fluid.

In vivo drug monitoring is crucial for evaluating the effectiveness and safety of drug treatment. Blood sampling and analysis is the current gold standard but needs professional skills and cannot meet the requirements of point-of-care testing. Dermal interstitial fluid (ISF) showed great potential to replace blood for in vivo drug monitoring; however, the detection was challenging, and the drug distribution behavior in ISF was still unclear until now. In this study, we proposed surface-enhanced Raman spectroscopy (SERS) microneedles (MNs) for the painless and real-time analysis of drugs in ISF after intravenous injection. Using methylene blue (MB) and mitoxantrone (MTO) as model drugs, the innovative core-satellite structured Au@Ag SERS substrate, hydrogel coating over the MNs, rendered sensitive and quantitative drug detection in ISF of mice within 10 min. Based on this technique, the pharmacokinetics of the two drugs in ISF was investigated and compared with those in blood, where the drugs were analyzed via liquid chromatography-mass spectrometry. It was found that the MB concentration in ISF and blood was comparable, whereas the concentration of MTO in ISF was 2-3 orders of magnitude lower than in blood. This work proposed an efficient tool for ISF drug monitoring. More importantly, it experimentally proved that the penetration ratio of blood to ISF was drug-dependent, providing insightful information into the potential of ISF as a blood alternative for in vivo drug detection.

H2O2-assisted photoelectrocatalytic degradation of Mitoxantrone using CuO nanostructured films: Identification of by-products and toxicity.

CuO nanostructured thin films supported on silicon with 6.5 cm2 area (geometric area greater than the studies reported in the literature) were synthesized by a chemical bath deposition technique. The electrodes were characterized by MEV, XRD, XPS, contact angle, cyclic voltammetry and electrochemical impedance spectroscopy analyses. To evaluate the photoelectrochemical properties of the CuO films, photocurrent-voltage measurements were performed using linear voltammetry. The catalytic activities of CuO nanostructures were evaluated by monitoring photodegradation of Mitoxantrone (MTX) under UV-A light irradiation. The method of photoelectrocatalysis (PEC), applying a voltage of 1.5 V and assisted by adding H2O2, was undertaken. To the best of our knowledge, no studies on the degradation of anticancer agents using PEC process have been found in the literature. For comparison purposes, experiments were performed under the same conditions by assisted photocatalysis (PC) with H2O2 and direct photolysis. CuO deposits consist of a needle-like morphology. The presence of CuO in the tenorite phase was evidenced by XRD and the XPS spectra showed the presence of copper(II) oxide. The increase in current under illumination shows that CuO exhibits photoactivity. The PEC system showed a 75% level of MTX degradation, while the level achieved using PC was 50%. Under UV-A light alone only 3% removal was obtained after 180 min. Up to 10 by-products were identified by chromatography-mass spectrometry (LC-MS) with m/z values ranging between 521 and 285 and a plausible degradation route has been proposed. It is worth mentioning that 9 by-products identified in this work, were not found in the literature in other studies of degradation or products generated as metabolites. The toxicity tests of MTX before and after PEC treatment with Artemia Salina and Allium cepa showed a decrease in the acute toxicity of the medium as the antineoplastic was degraded.

Nonidentical intracellular drug release rates in Raman and fluorescence spectroscopic determination.

Intracellular drug release rates were measured by monitoring mitoxantrone (MTX) on gold nanoparticle (AuNP) carriers by means of real-time label-free bimodal imaging with confocal Raman and fluorescence spectroscopy. The quenching nature of the MTX-AuNPs by nanometal surface energy transfer (NSET) was analyzed using the determined Stern-Volmer constant of KSV = 2.28 × 10(9) M(-1). The amount of MTX released was estimated by both the decrease in the surface-enhanced resonance Raman scattering (SERRS) signal and the increase in the fluorescence intensity. Both SERRS and NSET provide quantitative relationships between the spectral intensities of MTX concentrations in solution. Inside live cells, the signal decay profiles of the drug release from AuNPs appeared to be faster at the beginning of the bond-breaking drug release for the SERRS (R(-12)) than the recovery time of the NSET (R(-4) or R(-6)). In the first 45 min, a rather fast decay rate k of 0.0252 min(-1) with a short half-life t1/2 of 27.5 min was observed, whereas the rate became significantly slower in a diffusion process, 0.0093 min(-1) with a longer half-life of 101.4 min, after 45 min.

On-line chemiluminescence determination of mitoxantrone by capillary electrophoresis.

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0×10(-8)-1.0×10(-6)M with a detection limit of 1.0×10(-8)M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0×10(-8), 5.0×10(-7) and 1.0×10(-6)M MTX (n=11), respectively. In laboratory-built CE-CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.

HPLC analysis of mitoxantrone in mouse plasma and tissues: application in a pharmacokinetic study.

A simple, fast and economical HPLC assay for the determination of mitoxantrone in mouse plasma and tissue homogenates is described. Protein precipitation with sequential addition of sulfosalicylic acid and acetonitrile was used for sample preparation. The resolution of mitoxantrone and the I.S. were achieved by using acetonitrile and 10mM sodium phosphate buffer with 0.1% TEA. The separation was performed on a Nucleosil C18, 250mmx4mm I.D. column with UV detection at 610nm. The inter-day and intra-day precision and accuracy of quality control (QC) samples, evaluated both in plasma and tissue homogenates, were all within 15%. The lower limit of quantification (LLOQ) was 5ng/ml in plasma, 25ng/ml in liver homogenate and 12.5ng/ml in other tissue homogenates. This assay was successfully applied in a pharmacokinetic and tissue distribution study of mitoxantrone in mice.

Isolation and identification of a new impurity in mitoxantrone hydrochloride.

An unknown impurity in mitoxantrone hydrochloride bulk drug was detected by HPLC at levels around 0.5%. This impurity was isolated from a sample of crude mitoxantrone using preparative HPLC and was characterized as 1,4-dihydroxy-5-2-2-hydroxyethyl-amino-ethyl-amino-8-2-bis 2-hydroxyethyl-amino-ethyl-amino-9,10-anthracenedione based on its spectral data (NMR, IR and MS). The origin and structural elucidation of this impurity are discussed in the paper.

Quantitative online detection of low-concentrated drugs via a SERS microfluidic system.

Herein, quantitative online monitoring of concentration fluctuations of different interesting drugs, namely, the phenothiazine promethazine as well as the anti-cancer agent mitoxantrone via surface enhanced Raman scattering assay based on a microfluidic device is demonstrated. With the applied liquid/liquid two-phase-segmented flow system we succeed in preventing the adhesion of nanoparticle aggregates to the channel walls which is necessary for a quantitative analysis. Even after repeated cycles no carry-over due to sedimentation of colloid particles is observed. To the best of our knowledge these are the first measurements applying a combination of a microfluidic device with SERS detection for quantitative online monitoring of fluctuations in drug concentrations over hours without use of aggressive chemicals for rinsing the chip surfaces prior to each measurement.

Mitoxantrone-loaded BSA nanospheres and chitosan nanospheres for local injection against breast cancer and its lymph node metastases. II: Tissue distribution and pharmacodynamics.

Bovine serum albumin (BSA) and chitosan (CS) nanospheres of mitoxantrone (MTO) were comparatively evaluated in terms of tissue distribution, acute toxicity and therapeutic efficiency against breast cancer and its lymph node metastases. After local injection in rats, MTO nanospheres showed a slower elimination rate and a much higher drug concentration in lymph nodes compared with MTO solution, and a lower drug concentration in other tissues. There was no observed acute toxicity to the main tissues of Kunming mice after local injection of MTO-BSA-NS. Mild toxicity to liver and lung was observed for MTO-CS-NS, but, for MTO solution, severe toxicity to liver and lung and much lower number of white blood cells were observed. Human MCF-7 breast cancer in nude mice and animal model of P388 lymph node metastases in Kunming mice were applied to investigate the therapeutic efficiency. The inhibition rate of the nanospheres against breast cancer was much higher than that of MTO solution, and lymph node metastases were efficiently inhibited by the nanospheres, especially MTO-BSA-NS.

Quantitative analysis of mitoxantrone by surface-enhanced resonance Raman scattering.

Mitoxantrone is an anticancer agent for which it is important to know the concentration in blood during therapy. Current methods of analysis are cumbersome, requiring a pretreatment stage. A method based on surface-enhanced resonance Raman scattering (SERRS) has been developed using a flow cell and silver colloid as the SERRS substrate. It is simple, sensitive, fast, and reliable. Both blood plasma and serum can be analyzed directly, but fresh serum is preferred here due to reduced fluorescence in the clinical samples available. Fluorescence is reduced further by the dilution of the serum in the flow cell and by quenching by the silver of surface-adsorbed material. The effectiveness of the latter process is dependent on the contact time between the serum and the silver. The linear range encompasses the range of concentrations detected previously in patient samples using HPLC methods. In a comparative study of a series of samples taken from a patient at different times, there is good agreement between the results obtained by HPLC and SERRS with no significant difference between them at the 95% limit. The limit of detection in serum using the final optimized procedure for SERRS was 4.0 x 10(-11) M (0.02 ng/mL) mitoxantrone. The ease with which the SERRS analysis can be carried out makes it the preferred choice of technique for mitoxantrone analysis.

Expression and activity of breast cancer resistance protein (BCRP) in de novo and relapsed acute myeloid leukemia.

Overexpression of the breast cancer resistance protein (BCRP) efflux pump in human cancer cell lines results in resistance to a variety of cytostatic agents. The aim of this study was to analyze BCRP protein expression and activity in acute myeloid leukemia (AML) samples and to determine whether it is up-regulated due to clonal selection at relapse/refractory disease. BCRP protein expression was measured flow cytometrically with the monoclonal antibodies BXP-34 and BXP-21 in 20 paired samples of de novo and relapsed/refractory AML. BXP-34/immunoglobulin G1 ratios were observed of 1.6 +/- 0.5 (mean +/- SD, range 0.8-2.7) and BXP-21/immunoglobulin G2a ratios of 4.9 +/- 3.0 (range 1.1-14.5) in the patient samples versus 9.8 +/- 6.8 and 6.5 +/- 2.4, respectively, in the MCF-7 cell line. BCRP activity was determined flow cytometrically by measuring mitoxantrone accumulation in absence and presence of the inhibitor fumitremorgin C. Mitoxantrone accumulation, expressed as mean fluorescence intensity (MFI), varied between 44 and 761 MFI (227 +/- 146 MFI) and correlated inversely with BCRP expression (r = -0.58, P <.001). Addition of fumitremorgin C showed a small increase in mitoxantrone accumulation (11 +/- 29 MFI, n = 40) apart from the effect of PSC833 and MK-571. No consistent up-regulation of BCRP expression or activity was observed at relapse/refractory disease; some cases showed an increase and other cases a decrease at relapse. Relatively high BCRP expression correlated with immature immunophenotype, as determined by expression of the surface marker CD34 (r = 0.54, P =.001). In conclusion, this study shows that BCRP protein is expressed at low but variable levels in AML, especially in immature CD34(+) cells. BCRP was not consistently up-regulated in relapsed/refractory AML.

Noninvasive, in-situ measurement of drug concentrations in tissue using optical spectroscopy.

A small, lightweight system capable of noninvasive measurement of drug concentrations in tissue, without the use of reagents, would be advantageous for the study of the drug pharmacokinetics in space-travel applications. We have applied elastic-scattering spectroscopy (ESS) for noninvasive, real-time in vivo measurement of the concentrations of certain drugs in tissue, utilizing a simple fiber-optic-probe spectroscopic system. The system uses a broadband light source, enabling the detection of compounds with absorption bands in most regions of the visible, and potentially in the near-infrared (NIR) to 1700 nm. Subcutaneous tumors were grown in 4 Nude mice; the mice were treated with one of two chemotherapy agents, and the ESS system was used to perform pharmacokinetic measurements on the tumors following drug administration. Time histories of the drug concentrations in the tumors agreed with the known pharmacokinetics of the two drugs, and HPLC assays following sacrifice showed good relative correlation with the ESS values. Most photodynamic therapy agents and many chemotherapy drugs, including some that are not fluorescent, are ideal candidates for the ESS system. Importantly, the method can provide absolute measurements of compound concentrations.

Voltammetric behaviour of mitoxantrone at a DNA-biosensor.

The surface of an electrochemical glassy carbon electrode was modified with a layer of double-stranded DNA (dsDNA) or with double-stranded DNA conditioned in single-stranded DNA (ssDNA) and was used to investigate mitoxantrone-DNA interactions. Differential pulse and square wave voltammetry were applied to develop an electroanalytical procedure for the determination of mitoxantrone and evaluate its interaction with dsDNA or ssDNA immobilized on the electrode surface. The results demonstrate that MTX interaction with DNA is not specific to either guanine or adenine bases. The kinetics of the mitoxantrone-DNA interaction is slow and damage to DNA was followed with time.

Determination of mitoxantrone in mouse whole blood and different tissues by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method was developed for the specific determination of mitoxantrone (MTO) in whole blood and different tissues of mice (liver, heart, spleen, kidneys). MTO was extracted into dichloromethane with ametantrone (AMT) as internal standard. The different tissues were homogenised in citrate buffer (pH 3.0) containing 20% ascorbic acid. Separation of MTO and AMT was carried out using a Nucleosil C18 column. The mobile phase consisted of acetonitrile (33%) and 0.16 M ammonium formate buffer, pH 2.7. UV detection was used at 658 nm. Baseline separation of AMT and MTO was achieved in all matrices. The calibration curves were linear in all matrices (r > 0.999) in the concentration range of 2-200 micrograms/l for whole blood and 2-700 micrograms/l for tissue homogenates, respectively. The within-day and between-day precision studies showed good reproducibility with coefficients of variation below 4.5% for whole blood and below 10% for tissue homogenates, respectively. The extraction efficiencies of MTO are 60% in whole blood and 38% in tissue homogenates. The method described is suitable for pharmacokinetic studies on the distribution of MTO in different tissues of mice.

Polarographic and absorptive stripping voltammetric determination of mitoxantrone, emodin and chrysarobin using mercury and glassy carbon electrodes.

Polarographic and voltammetric methods were used to study drugs of anthrone derivatives: mitoxandrone (antineoplastic), emodin (cathartic) and chrysarobin (antipsoriatic) on mercury and glassy carbon electrodes. The surface-active properties were exploited for developing a highly sensitive adsorptive stripping voltammetric method for determination of trace amounts of these compounds. Effective pre-concentration was obtained at glassy carbon electrode, with the surface species being measured via its reduction or oxidation, respectively. It was concluded that observed cathodic and anodic currents had the diffusive character. A simple, quick and accurate methods for the determination of all studied compounds in pure form and mitoxantrone in vials have been developed. The acetate buffer pH 4.6 containing 5 to 30% ethanol was used as a supporting electrolyte. The linear calibration range was 0.5 = 100 microg cm-3 on mercury and 25 - 250 ng cm-3 on glassy carbon electrode.

Evidence for persistence of mitoxantrone within the peritoneal cavity following intraperitoneal delivery.

In clinical trials examining the intraperitoneal (ip) administration of mitoxantrone as therapy of platinum-refractory small-volume residual ovarian cancer, the characteristic "blue color" of the agent has been demonstrated to stain the surface of the peritoneal cavity and to persist for > or = 1 month following the last course of therapy. To determine if this blue staining material contains potentially cytotoxic concentrations of mitoxantrone, we analyzed tissue obtained at exploratory laparotomy in six women who had last received the agent administered ip from 6-22 weeks prior to surgery. Concentrations of mitoxantrone ranged from < 0.1 to 13.8 micrograms/g of tissue examined. Since any mitoxantrone present on the peritoneal surface will be highly protein bound, any residual drug may not have cytotoxic potential. The dose-response curves of mitoxantrone in a human clonogenic cytotoxicity assay against the RPMI 2780/S human ovarian cell line were virtually identical when the cells were incubated in either 5 or 50% fetal bovine serum, suggesting that protein binding will not significantly impair mitoxantrone-induced tumor cell killing. We conclude that the ip administration of mitoxantrone may lead to prolonged exposure of surface tumor to the high local concentrations of the active cytotoxic agent. This effect may contribute significantly to the antineoplastic potential of ip mitoxantrone in patients with small-volume residual ovarian cancer.

A novel HPLC method for the determination of mitoxantrone in pharmaceutical formulations.

An HPLC method, using reversed phase chromatography, for the quantitative determination of mitoxantrone in pharmaceutical formulations has been developed. Rhein has been used as internal standard.

Determination of mitoxantrone by flow injection analysis using an amperometric detector.

Mitoxantrone was determined by flow injection analysis using a flow cell modified in the laboratory and fitted with carbon paste as an amperometric detector. The sample solution (100 microliters, 5 x 10(-8)-1 x 10(-5) M) was injected into the carrier stream of 0.1 M perchloric acid (pH 1.12). Mitoxantrone was determined by oxidation at the carbon paste electrode (CPE) at +0.90 V. A 60-cm delay coil (0.5 mm i.d.) was incorporated just before the detector (a canal thin layer) and a flow rate of about 4 ml min-1 was used. The system was successfully applied to the determination of mitoxantrone in a pharmaceutical preparation; the method was fast and reproducible.

The stability of carboplatin, diamorphine, 5-fluorouracil and mitozantrone infusions in an ambulatory pump under storage and prolonged 'in-use' conditions.

Drug infusions can be exposed for prolonged periods to 'in-use' conditions where the temperature of an infusion in a holster-worn infusion pump may reach 37 degrees C. In this study, the stability of three cytotoxic drug infusions (carboplatin, 5-fluorouracil and mitozantrone) and one analgesic infusion (diamorphine HCl) was determined in Parker Micropump medication reservoirs under refrigerated storage and prolonged in-use conditions. The stability of the three cytotoxic drug infusions was unaffected by 14 days storage at either 4 or 37 degrees C. The diamorphine HCl infusion was stable over 14 days storage at 4 degrees C but under in-use conditions at 37 degrees C, drug degradation became significant (greater than 5%) if storage exceeded 7 days.

Quantification of mitoxantrone in bone marrow by high-performance liquid chromatography with electrochemical detection.

An analytical method for the determination of mitoxantrone in bone marrow was developed using high-performance liquid chromatography with electrochemical detection. The extraction procedure was optimized by investigating several factors which potentially could influence the recovery of mitoxantrone from bone marrow cells. The mean recovery of mitoxantrone from rat bone marrow was found to be 81.7% with a coefficient of variation 3.8%. High-performance liquid chromatography was carried out to quantitate mitoxantrone using ametantrone as internal standard. The detection limit of our analytical method amounts to 100 pg on-column, corresponding to 1 ng/ml of cell suspension containing 2 x 10(7) cells and a day-to-day variation of maximally 8%. Storage of bone marrow samples, containing mitoxantrone, for one to fourteen days resulted in a mean recovery of 94%, as compared to freshly analysed samples. Subsequently we studied the pharmacokinetics of mitoxantrone in rat bone marrow. It appeared that after an intravenous bolus injection of mitoxantrone (2.5 mg/kg) in rats, the drug accumulated in the femoral bone marrow for about four days, and thereafter gradually declined.