Dual-mode visible light-induced aptasensing platforms for bleomycin detection based on CdS-In2S3 heterojunction.

CdS-In2S3 heterojunction with enhanced photoelectrochemical (PEC) performance was synthesized to construct dual-mode visible light-induced biosensors for highly sensitive and selective detection of bleomycin (BLM). Due to improved absorption in the visible region and suppressed recombination of electron-hole pairs in the heterojunction, CdS-In2S3 composite exhibited enhanced photocurrent response under visible light illumination. Using CdS-In2S3 as photoactive materials and BLM-binding aptamer as recognition element, a PEC aptasensor displaying a declined photocurrent response to BLM was facilely constructed, which was linear to BLM concentration in the range of 5.0-250 nM. On the other hand, the CdS-In2S3 photoanode was employed to construct a photofuel cell (PFC). In such a PFC, the oxidation of water on CdS-In2S3 photoanode under visible light illumination and the reduction of oxygen on Pt cathode led to the generation of electricity. When BLM-binding aptamer was immobilized on CdS-In2S3 photoanode, the output power of the PFC was inversely proportional to the logarithm of BLM concentration from 10 to 250 nM, offering a visible light-induced self-powered sensing platform for BLM detection. Both of the proposed sensors showed high selectivity, good reproducibility and high stability. They were successfully applied to the determination of BLM in human serum samples.

Plasma Bleomycin Concentrations during Electrochemotherapeutic Treatment of Fibropapillomas in Green Turtles Chelonia mydas.

Fibropapillomatosis of sea turtles is traditionally treated with surgical debulking techniques that are often associated with prolonged healing and tumor recurrence. Electrochemotherapy was recently described for green turtles Chelonia mydas and can be an alternative to surgery and even general anesthesia. The objectives of this study were to replicate an electrochemotherapy protocol from a previous report and add plasma bleomycin analysis to the treatment. After bleomycin injection into similarly sized tumors of two green turtles and immediate electroporation at two time points, plasma bleomycin reached detectable concentrations that were considerably lower than those found in human studies. At 3 months posttherapy, no healing complications or recurrences were encountered and only scar tissue remained. This study adds further support that electrochemotherapy with bleomycin has the potential to be used as an effective alternative treatment for this complex disease.

A G-triplex based molecular beacon for label-free fluorescence "turn-on" detection of bleomycin.

Since bleomycins (BLMs) play a prominent role in the clinical treatment of various cancers, the development of convenient and sensitive detection assays for BLM is of great significance in cancer therapy and related biological mechanism research. Here, taking advantage of the easily controllable and excitation of the G-triplex DNA structure, we reported a facile, label-free G-triplex based functional molecular beacon (G3MB) sensing system for fluorescence "turn-on" detection of BLM based on BLM-Fe(ii) mediated DNA strand scission. In the presence of BLM, the stable hairpin structure of G3MB undergoes an irreversible cleavage in the loop region that contains a 5'-GT-3' recognition site for BLM. The released G-tract DNA fragment self-assembles into a G-triplex-ThT complex showing a strong fluorescence. Owing to the effective locking of G-tracts in the stem of the G3MB and the specific DNA strand scission by BLM which is like a key for the release of G-tracts, the assay shows high sensitivity and selectivity with a detection limit of 0.2 nM. In addition, satisfactory results were obtained for the detection of BLM in human serum samples. Critically, the convenient "mix-and-detect" protocol, fast response and no need for modifying DNA offered a potential application of the proposed strategy for BLM assay in biomedical and clinical studies.

Target-induced diffusivity enhancement for rapid and highly sensitive homogeneous electrochemical detection of BLM in human serum.

A simple, rapid, and sensitive homogeneous electrochemical bleomycin (BLM) bioassay has been successfully developed through the target-induced specific/efficient cleavage reaction. The designed probe, denoted as MB-DNA, contains both methylene blue (MB) and target recognizable sequences, and presents relatively low electrochemical signal. Upon the addition of BLM, the recognition/cleavage reaction occurs and leads to the in-situ generation of MB tag (MB-DNA-1), leading to the reduced electrostatic repulsive force. As a result, an obvious enhancement in differential pulse voltammetry (DPV) current is determined, which is relied on the amount of BLM. Thus, a turn on homogeneous electrochemical method for BLM is really achieved, and exhibits high sensitivity of 33 pM, and the shortest response time of 20 min. Furthermore, this electrochemical bioassay presents excellent sensing performance in the analysis of BLM in real samples. Comparing with other sensing strategies for BLM, this proposed electrochemical platform is just consisted of one DNA probe alone, and affords a really rapid and sensitive strategy for BLM analysis.

A novel label-free fluorescent sensor for highly sensitive detection of bleomycin based on nitrogen-doped graphene quantum dots.

In this work, we presented a novel label-free biosensor for rapid detection of bleomycinsulphate (BLM). The biosensor was based on the fluorescent "turn off-on" of nitrogen-doped graphene quantum dots (N-GQDs), which was prepared in a green way from citric acid and ammonia. The richness of carboxyl groups on the N-GQDs enabled strong adsorption of ssDNA to the surface of N-GQDs through π-π stacking interactions, resulting in the effective fluorescence quenching of N-GQDs system. The ssDNA underwent an irreversible cleavage event via the oxidative effect of BLM with Fe(II) as a cofactor, thus a turn-on fluorescence signal was observed. Thereby, the concentration of BLM can be quantitatively determined in a broad range from 0.34 nmol/L to 1300 nmol/L with a detection limit of 0.34 nmol/L. The presented method was applied to the determination of BLM in human serum samples with satisfactory results.

Identification and quantification of bleomycin in serum and tumor tissue by liquid chromatography coupled to high resolution mass spectrometry.

Bleomycin is a cytotoxic antibiotic available as a compost of structurally strongly related glycopeptides, which is in vivo found chelated with several metals. Its pharmacotherapy has merely been based on experimental dose - response data, whereas its biodistribution and pharmacokinetics remain fundamentally unknown. This is reasoned by an absence of a specific and sensitive mass spectrometry-based analytical method for its determination in biological tissues. We herein reveal the results of our study on the mass spectrometric behavior of two main bleomycin fractions A2 and B2, including their metal complexes, particularly the predominant copper chelates. In the electrospray ion source bleomycin forms double charged species, where for the metal-free fraction A2 and its copper complex m/z 707.76 and m/z 707.21 are seen, respectively. Hence, the second isotopic ion of the chelate (m/z 707.71) nearly coincides with the first isotopic ion of the metal-free fraction. This phenomenon can only be followed by high-resolution mass spectrometry, and is considered the plausible reason, why the attempts to determine bleomycin with mass spectrometry have been so scarce. The presented paper further describes a sensitive and selective liquid chromatography - mass spectrometry analytical method for determination of bleomycin in serum and tumor tissues. This newly developed method was employed for bleomycin pharmacokinetic studies in serum and tumors of laboratory animals. Additionally, the method was employed for determination of bleomycin pharmacokinetic parameters in elderly patients in order to determine the effective therapeutic window of electrochemotherapy with bleomycin.

Niche nanoparticle-based FRET assay for bleomycin detection via DNA scission.

We describe a highly sensitive nanoparticle-based fluorescence resonance energy transfer (FRET) probe developed without using molecular fluorophores as donors and acceptors. The success of this work relies on the strategy that DNA scission was designed to occur to the probe when target presented, which enabled the fluorescence signal "turn-on" of graphene quantum dots (GQDs) and thus quantitative analysis. In particular, amino-modified SiO2 NPs were initially coated by GQDs to form highly emitting SiO2/GQDs, followed by conjunction with DNA functionalized gold nanoparticles (Au NPs-DNA) to form SiO2/GQDs-DNA-Au NPs composite. Owing to the FRET interactions between the GQDs and Au NPs, the fluorescence of GQDs was effectively quenched by Au NPs. When bleomycin (BLM), a model analyte, was mixed with the probe, the fluorescence signal of GQDs would be restored due to the removal of Au NPs from the SiO2/GQDs surface by DNA scission treatment with BLM in the presence of Fe (II). The current FRET probe shows a good linear relationship between the fluorescence intensity and the concentration of BLM in the range from 0.5nM to 1µM with a detection limit of 0.2nM. The probe also shows satisfactory results for the analysis of clinical serum samples. This method provides versatility to the application of GQDs in FRET biosensing and could be potentially extended to other similar systems by replacing the linker between the GQDs and Au NPs.

Bleomycin pharmacokinetics of bolus bleomycin dose in elderly cancer patients treated with electrochemotherapy.

PURPOSE: With the aim to determine effective therapeutic window of electrochemotherapy, we analyzed bleomycin pharmacokinetic parameters in elderly patients. METHODS: In prospective clinical study in the treatment of tumors with electrochemotherapy, blood samples of patients older than 65 years were collected after the bolus intravenous injection of bleomycin (15,000 IU/m(2)). In serum samples, quantitative analysis was performed with liquid chromatography coupled to high-resolution mass spectrometry. Based on the data, the pharmacokinetic parameters of bleomycin elimination were determined. RESULTS: Pharmacokinetic analysis of the data revealed a monophasic serum clearance curve, which demonstrates slow elimination of bleomycin, being less than 500 ml/min and a half-time of 30 min. CONCLUSIONS: Slow monophasic elimination of bleomycin from serum in elderly patients implies on the longer therapeutic window, from 8 to up to 40 min or even longer post-bleomycin injection for electrochemotherapy. However, prolonged therapeutic bleomycin serum concentrations may also affect the possible adverse effects, such as lung fibrosis and extensive necrosis of tumors due to the uptake of toxic bleomycin concentrations into the tumors. This may imply on lowering of bleomycin dosage, in particular in the elderly patients.

Bleomycin sulphate loaded nanostructured lipid particles augment oral bioavailability, cytotoxicity and apoptosis in cervical cancer cells.

In present investigation, bleomycin sulphate loaded nanostructured lipid particles (BLM-NLPs) were constructed to enhance the oral bioavailability by overwhelming the first pass hepatic metabolism. The particles size and nanoencapsulation efficiency of BLM-NLPs were measured to be 17.4±5.4nm and 45.3±3.4%, respectively. Our studies indicated that the drug was molecularly dispersed in the lipid nanocoacervates, with amorphous geometry, without altering the chemical structure, as ascertained by spectral studies. The nanoformulation, BLM-NLPs was analyzed for dissolution testing, cytotoxicity, apoptosis and cellular uptake in human cervical cancer cell line, HeLa cells. BLM-NLPs released the drug with first order kinetic in simulated intestinal fluid (pH∼6.8±0.1), characterized by initial burst and followed by slow release. Further, an enhanced cytotoxicity (∼5.6 fold lower IC50), improved intracellular concentration (∼4.38 fold) and greater degree of apoptosis was induced by BLM-NLPs in HeLa cells, as compared to BLM alone. Moreover, BLM-NLPs also showed dose-dependent internalization, as evinced by cellular uptake study. The in vivo study indicated a significantly (P<0.0001) smaller elimination rate constant (KE), volume of distribution (Vd) and clearance rate (CLTotal) for BLM-NLPs, as compared to BLM solution in post-oral administrations. This clearly depicts the retention and stability of tailored nanoformulation in intestinal absorption pathway. In addition, our nanoformulation, BLM-NLPs documented significantly (P<0.0001)∼3.4 fold (66.20±2.57%) higher bioavailability than BLM solution (19.56±0.79%). In conclusion, our in vitro and in vivo results warrant the safety, efficacy and potency of tailored nanoformulation in clinical settings.

Simple colorimetric sensing of trace bleomycin using unmodified gold nanoparticles.

A simple colorimetric sensing platform for trace bleomycin (BLM) was proposed with the unmodified gold nanoparticles (AuNPs) as the sensing element. BLM has multiple N-donor functionality and exhibited strong coordination effect on AuNPs, which made it possible for the occurrence of ligand exchange of BLM with the weakly surface-bound citrate ions on AuNPs. Meanwhile, the positively charged BLM molecules further neutralized the surface charge, leading to increased van der Waals attractive force among AuNPs for rapid aggregation. This was reflected by the obvious color change from wine red to blue and rapid aggregation kinetics within 7.5 min. The BLM sensing based on unmodified AuNPs can be seen with the naked eye and monitored by UV-vis extinction spectra. The linear range of the colorimetric sensor for BLM was from 2 to 150 nM. The as-established colorimetric strategy opened a new avenue for trace BLM determination.

Transplacental transfer of anthracyclines, vinblastine, and 4-hydroxy-cyclophosphamide in a baboon model.

OBJECTIVE: The paucity of data on the fetal effects of prenatal exposure to chemotherapy prompted us to study transplacental transport of chemotherapeutic agents. METHODS: Fluorouracil-epirubicin-cyclophosphamide (FEC) and doxorubicin-bleomycin-vinblastine-dacarbazine (ABVD) were administered to pregnant baboons. At predefined time points over the first 25 h after drug administration, fetal and maternal blood samples, amniotic fluid (AF), urine, fetal and maternal tissues, and cerebrospinal fluid (CSF) were collected. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) were used for bioanalysis of doxorubicin, epirubicin, vinblastine, and cyclophosphamide. RESULTS: In nine baboons, at a median gestational age of 139 days (range, 93-169), FEC 100% (n = 2), FEC 200% (n=1), ABVD 100% (n = 5), and ABVD 200% (n = 1) were administered. The obtained ratios of fetal/maternal drug concentration in the different simultaneously collected samples were used as a measure for transplacental transfer. Fetal plasma concentrations of doxorubicin and epirubicin averaged 7.5 ± 3.2% (n = 6) and 4.0 ± 1.6% (n = 8) of maternal concentrations, respectively. Fetal tissues contained 6.3 ± 7.9% and 8.7 ± 8.1% of maternal tissue concentrations for doxorubicin and epirubicin, respectively. Vinblastine concentrations in fetal plasma averaged 18.5 ± 15.5% (n=9) of maternal concentrations. Anthracyclines and vinblastine were neither detectable in maternal nor in fetal brain/CSF. 4-Hydroxy-cyclophosphamide concentrations in fetal plasma and CSF averaged 25.1 ± 6.3% (n = 3) and 63.0% (n = 1) of the maternal concentrations, respectively. CONCLUSION: This study shows limited fetal exposure after maternal administration of doxorubicin, epirubicin, vinblastine, and 4-hydroxy-cyclophosphamide.

DNA as a target for anticancer compounds screening directly by resonance light scattering technique.

Anticancer drugs which selectively interact with DNA can change DNA's conformation and inhibit the duplication or transcription of DNA. An instrument-based assay for directly screening DNA-targeted anticancer drugs using resonance light scattering (RLS) technique with a common spectrofluorometer was proposed. To monitor the proposed screening method, the interactions between three anticarcinogens (Adriamycin (ADM), Bleomycin A (BLMA), Actinomycin D (ACTD)) and DNA were studied. The sequence of binding constants for the three anticarcinogens obtained from RLS spectra is: K(RLS) (ACTD, 9.43 × 10(5) L mol(-1)) > K(RLS) (ADM, 6.67 × 10(5) L mol(-1)) > K(RLS) (BLMA, 8.88 × 10(3) L mol(-1)) and of binding numbers is: N(RLS) (ACTD, 3.36 mmol g(-1)) < N(RLS) (ADM, 3.81 mmol g(-1)) < N(RLS) (BLMA, 57.44 mmol g(-1)). From the results we got the sequence of combination intensity between these three drugs and DNA as follows: ACTD-DNA > ADM-DNA > BLMA-DNA, which was completely consistent with drug activity. The conclusion indicated that the present method was direct, rapid, reliable and was another important innovation of the application of RLS technique.

Fluorescence quenching method for the determination of bleomycins A5 and A2 with halofluorescein dyes.

In weak acidic medium, the anticancer antibiotics bleomycin A5 (BLMA5) and bleomycin A2 (BLMA2) bind with halofluorescein dyes, such as erythrosin (Ery), eosin Y (EY) and eosin B (EB), to form ion-association complexes, which causes fluorescence quenching of halofluorescein dyes. The quenching values (DeltaF) are directly in proportional to the concentrations of bleomycins over the range 0.09-2.5 microg/mL. Based on this, a fluorescence quenching method for the determination of BLMA5 and BLMA2 has been developed. The dynamic range is 0.12-2.5 microg/mL for the determination of BLMA5 and 0.09-2.0 microg/mL for BLMA2, with detection limits (3sigma) of 0.04 microg/mL for BLMA5, 0.03 microg/mL for BLMA2, respectively. It has been applied to determine the two antibiotics in human serum, urine and rabbit serum samples. The recovery is in the range 90-102%. In this work, the optimum reaction conditions and the spectral characteristics of the fluorescence are investigated. The reasons for fluorescence quenching are discussed, based on the fluorescence theory.

[Pharmacokinetics of pingyangmycin hydrochloride in rabbits determined by microdialysis coupled with RP-HPLC].

Microdialysis coupled with RP-HPLC was used to study the blood pharmacokinetics of pingyangmycin hydrochloride in rabbits. Supelco RP-amide C16 column was adopted for the analysis of pingyangmycin hydrochloride. The data was analyzed with 3P87 program. The calibration curve was linear in the concentration range from 1.04 to 66.56 microg x mL(-1) (r2 = 0.999 4). The in vivo recovery of microdialysis probe was (42.8 +/- 3.4)% (n = 4). The concentration-time curve of pingyangmycin hydrochloride was fitted to two-compartment model. T1/2 alpha and T1/2 beta were 14.9 and 60.3 min, respectively. The method is proved to be accurate, simple and suitable for the pharmacokinetics study of pingyangmycin hydrochloride in rabbits.

The determination of in vitro pingyangmycin hydrochloride plasma protein binding by microdialysis.

Microdialysis sampling was used to study the binding of pingyangmycin hydrochloride (PYM) to plasma proteins in canis familiaris blood. In vitro plasma protein binding fractions were evaluated in a series of PYM concentration. The results showed decreased protein binding with increased concentration. The data was analyzed using the Scatchard analysis and Klotz plot. The results showed that the Scatchard plot and Klotz plot were linear with good correlation coefficient, indicating a good agreement of the experimental data to the theoretical equation.

The encapsulation of bleomycin within chitosan based polymeric vesicles does not alter its biodistribution.

Polymeric vesicles have recently been developed from an amphiphilic chitosan derivative--palmitoyl glycol chitosan. Their potential as a drug delivery system was evaluated using the anti-cancer compound bleomycin as a model drug. Palmitoyl glycol chitosan (GCP41) was synthesised by conjugation of palmitoyl groups to glycol chitosan. Bleomycin-containing vesicles (669 nm diameter) were prepared from a mixture of GCP41 and cholesterol by remote loading. The vesicles were imaged by freeze-fracture electron microscopy and their in-vitro stability tested. Incubation of the larger vesicles with plasma in-vitro led to a reduction of mean size by 49%, a reaction not seen with control sorbitan monostearate niosomes (215 nm in size). They also showed a higher initial drug release (1 h), but GCP41 and sorbitan monostearate vesicles retained 62% and 63% of the encapsulated drug after 24h, respectively. The biodistribution of smaller vesicles (290 nm) prepared by extrusion through a 200-nm filter was also studied in male Balb/c mice. Encapsulation of bleomycin into polymeric vesicles did not significantly alter the pharmacokinetics of biodistribution of bleomycin in male Balb/c mice although plasma and kidney levels were slightly increased. It is concluded that the extruded GCP41 vesicles break down in plasma in-vivo and hence are unlikely to offer any therapeutic advantage over the free drug.

Kinetics of 111In-labeled bleomycin in patients with brain tumors: compartmental vs. non-compartmental models.

The kinetics of an indium-111 labeled bleomycin complex (111In-BLMC) after rapid intravenous injection in patients with brain tumors was quantified by using compartmental and non-compartmental models. The models were applied to data obtained from 10 glioma, one meningioma, and one adenocarcinoma brain metastasis patients. Blood and urine samples from all the patients and tumor samples from three patients were collected. The mean transit time of 111In-BLMC in the plasma pool was 14 +/- 7 min without and 1.8 +/- 0.6 h when accounting for recirculation, and 13 +/- 4 h in the total body pool. The mean plasma clearance of 111In-BLMC was 0.3 +/- 0.1 m/blood/min and the mean half-life in urine was 3.5 +/- 0.6 h. The mean transfer coefficients for the open three-compartmental model were: excretion from plasma = 0.02 +/- 0.01, from depot to plasma = (12 +/- 9)*10(-4), from plasma to depot = 0.01 +/- 0.01, from tumor to plasma = 0.39 +/- 0.19 and from plasma to tumor = 1.11 +/- 0.57, all in units minute-1. The mean turnover time from the tumor was 4.5 +/- 2.7 min and from the depot 20 +/- 8 h. It is concluded that both compartmental and non-compartmental models are sufficient to describe the kinetics of indium-111 labeled bleomycin complex. The non-compartmental model is more practical and to some extent more efficient in describing the in vivo behaviors of 111In-BLMC than the compartmental model. The compartmental model used provides estimates of both extraction and excretion from the plasma and tumor.