Intramolecular Enhancement of a Zirconium-Based Metal-Organic Framework for Coordination-Induced Electrochemiluminescence Bleomycin Analysis.

It is significantly vital to develop a convenient assay method in clinical treatment due to an atypically low abundance (∼5 µM) of bleomycin (BLM) used in clinics. Herein, an electrochemiluminescence (ECL) biosensor using a zirconium-based metal-organic frameworks (Zr-MOFs) as an intramolecular coordination-induced electrochemiluminescence (CIECL) emitter was proposed for sensitive detection of BLM. Zr-MOFs were synthesized using Zr(IV) as metal ions and 4,4',4″-nitrilotribenzoic acid (H3NTB) as ligands for the first time. The H3NTB ligand not only acts as coordination units bonding with Zr(IV) but functions as a coreactant to enhance ECL efficiency rooted in its tertiary nitrogen atoms. Specifically, a long guanine-rich (G-rich) single-stranded DNA (ssDNA) was released by the target-BLM-controlled DNA machine that could perform π-π stacking with another G-quadruplex, ssDNA-rhodamine B (S-RB), by shearing DNA's fixed sites 5'-GC-3' and the auxiliary role of exonuclease III (Exo III). Finally, due to the quenching effect of rhodamine B, a negative correlation trend was obtained between ECL intensity and BLM concentration in the range from 5.0 nM to 50 µM and the limit of detection was 0.50 nM. We believe that it is a promising approach to guide the preparation of CIECL-based functional materials and establishment of analytical methods.

An effective validation of analytical method for determination of a polar complexing agent: the illustrative case of cytotoxic bleomycin.

The effectiveness of highly polar agents in cancer treatment is well recognized, but their physicochemical properties make their analytical determination a demanding task. Their analysis requires peculiar sample preparation and chromatographic separation, which heavily impacts the precision of such an analytical method. As a case study, we chose a polar cytotoxic bleomycin, which is a mixture of complexing congeners with relatively high molecular mass, a fact that creates an added challenge in regard to its detection via electrospray mass spectrometry. These issues combined lead to a deprived method performance, so the aim of this study is manifold, i.e., to optimize, validate, and establish quality performance measures for determination of bleomycin in pharmaceutical and biological specimens. Quantification of bleomycin is done at diametrically different concentration levels: at the concentrations relevant for analysis of pharmaceutical dosage forms it is based on a direct reversed-phase HPLC-UV detection, involving minimum sample pretreatment. On the contrary, analysis of bleomycin in biological specimens requires phospholipid removal and protein precipitation followed by HILIC chromatography with MS/MS detection of bleomycin A2 and B2 copper complexes being the predominant species. This study further attempts to solve the traceability issue in the absence of certified reference standards, determines measurement uncertainty, investigates BLM stability and method performance characteristics, and, last but not least, provides an explanatory example of how a method quality assurance procedure should be established in case of an exceedingly complex analytical method.

Metabolomic study on bleomycin and polyhexamethylene guanidine phosphate-induced pulmonary fibrosis mice models.

INTRODUCTION: Polyhexamethylene guanidine phosphate (PHMG) has been used as a disinfectant and biocide, and was known to be harmless and non-toxic. However, in 2011, PHMG used as a humidifier disinfectant was reported to be associated with lung diseases, such as, fibrosis in the toxicant studies on pulmonary fibrosis by PHMG. However, no metabolomics study has been performed in PHMG-induced mouse models of pulmonary fibrosis. OBJECTIVES: We performed a metabolomic study to understand the biochemical events that occur in bleomycin (BLM)- and PHMG-induced mouse models of pulmonary fibrosis using gas chromatography-mass spectrometry (GC-MS), LC-tandem MS, and GC-tandem MS. RESULTS: The levels of 61 metabolites of 30 amino acids, 13 organic acids, 12 fatty acids, 5 polyamines, and oxidized glutathione were determined in the pulmonary tissues of mice with BLM- and PHMG-induced pulmonary fibrosis and in normal controls. Principal component analysis and partial least squares discriminant analysis used to compare level of these 61 metabolites in pulmonary tissues. Levels of metabolites were significantly different in the BLM and PHMG groups as compared with the control group. In particular, the BLM- and PHMG-induced pulmonary fibrosis models showed elevated collagen synthesis and oxidative stress and metabolic disturbance of TCA related organic acids including fumaric acid by NADPH oxidase. In addition, polyamine metabolism showed severe alteration in the PHMG group than that of the BLM group. CONCLUSION: This result suggests PHMG will be able to induce pulmonary fibrosis by arginine metabolism and NADPH oxidase signaling.

Molecular beacon-templated silver nanoclusters as a fluorescent probe for determination of bleomycin via DNA scission.

The authors describe a molecular beacon-based fluorescent probe for the determination of the cancer drug bleomycin (BLM). The probe was tagged with DNA-templated silver nanoclusters (DNA-AgNCs) and guanine-rich sequences (GRSs) at two terminals serving as signal reporter with a loop. In the absence of the BLM-iron(II) complex [BLM-Fe(II)], the probe has a hairpin shape and displays strong fluorescence because the AgNCs are close to the GRSs. In the presence of the BLM-Fe(II) complex, it will selectively cleave the probe at the 5'-GC-3' scission site of the loop. This displaces the AgNCs away from the GRSs and causes a decrease in fluorescence, best measured at excitation/emission wavelengths of 565/623 nm. This effect enables BLM to be detected with a detection limit as low as 33 pM, which was 1-3 orders of magnitude more sensitive than most of the previous reports. The probe was applied for the determination of BLM in spiked human serum samples, and excellent performance was achieved. In our perception, the method described here represents a promising tool for highly sensitive and specific analysis of BLM during cancer treatment. Graphical abstract Schematic of a highly sensitive fluorometric assay forbleomycin. It is based on molecular beacon-templated silver nanoclusters and DNA scission.

Target-initiated synthesis of fluorescent copper nanoparticles for the sensitive and label-free detection of bleomycin.

Fluorescent copper nanoparticles (CuNPs) have received great attention due to their distinct characteristics of facile synthesis, tunable fluorescence emission, high photostability, and biological compatibility, and they have been widely used for chemical and biological analyses. Bleomycins (BLMs) are widely used antitumor agents for the clinical treatment of various cancers. Here, we develop a sensitive and label-free fluorescence method for the quantitative detection of BLM on the basis of BLM-initiated enzymatic polymerization-mediated synthesis of fluorescent CuNPs. We design two hairpin DNAs: one (Hp1) for the recognition of BLM and the other (Hp2) for signal amplification. In the presence of BLM, it may recognize and cleave the 5'-GC-3' site of the Hp1 stem, releasing the 8-17 DNAzyme fragment. The resultant 8-17 DNAzyme fragments may bind with the loop of Hp2 to form a partial double-stranded DNA (dsDNA) duplex, initiating the cyclic cleavage of Hp2 in the presence of Zn2+-dependent DNAzymes and generating numerous new DNA fragments with the free 3'-OH terminal, which can induce the formation of a poly(thymine) (poly-T) sequence with the assistance of terminal deoxynucleotidyl transferase (TdTase). Subsequently, the ploy-T sequence may function as the template for the synthesis of CuNPs with strong fluorescence emission. This method shows good selectivity and high sensitivity with a detection limit as low as 8.1 × 10-16 M, and it exhibits good performance in serum samples. Moreover, this method has distinct advantages of simplicity and low cost, holding great potential in clinical diagnosis and biomedical research.

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.

A sensitive fluorescence turn-on assay of bleomycin and nuclease using WS2 nanosheet as an effective sensing platform.

As an important antitumor drug, bleomycin (BLM) is widely used in the treatment of a variety of cancers. In addition, nucleases play a crucial role in DNA replication, recombination and repair which are associated with cancer development. Thus, the development of BLM and nuclease detection methods is of great significance in cancer therapy and related biological mechanism research. Here, a WS2 nanosheet-based turn-on fluorescent sensing platform for simple, fast and sensitive detection of BLM and nuclease was reported. WS2 nanosheet exhibits different affinity toward ssDNA with different length and excellent fluorescence quenching ability. A fluorescein (FAM)-labeled long ssDNA could be adsorbed on the surface of WS2 nanosheet and the fluorescence was therefore quenched. In the presence of BLM·Fe(II) or S1 nuclease (a ssDNA-specific nuclease which was used as a model enzyme), an irreversible scission of long ssDNA was underwent through the BLM-induced oxidation cleavage or S1 nuclease-induced enzymatic hydrolysis. Short FAM-linked oligonucleotide fragments which could not be adsorbed on the nanosheet surface were then produced, resulting in a weak fluorescence quenching after mixing WS2 nanosheets. Thus, the fluorescence signal was restored. The proposed sensor displays a wide linear range and a high sensitivity with a detection limit of 0.3 nM for BLM and 0.01 U mL(-1) for S1 nuclease. It also exhibits a good performance in complex biological samples. This method not only provides a strategy for BLM or S1 nuclease assay but also offers a potential application in biomedical and clinical study.

Ultrasensitive fluorescence detection of bleomycin via exonuclease III-aided DNA recycling amplification.

A "signal on" approach for ultrasensitive detection of bleomycin was developed by bleomycin-Fe(II) induced hairpin DNA scission to release its loop, which was subsequently recycled with the aid of exonuclease III and a probe to amplify the detectable fluorescent signal.

Label-free electrogenerated chemiluminescence biosensing method for trace bleomycin detection based on a Ru(phen)3(2+)-hairpin DNA composite film electrode.

A novel label-free electrogenerated chemiluminescence (ECL) DNA-based biosensing method for the determination of trace bleomycin (BLM) was developed on basis of Fe(II)·BLM-mediated DNA strand scission and Ru(phen)3(2+) as an ECL probe. A thiolated ss-DNA, as substrate for BLMs, was self-assembled onto surface of a gold electrode to form a hairpin structure. Ru(phen)3(2+) was intercalated into the hairpin DNA structure. In the presence of Fe(II)·BLM, the hairpin DNA sequence undergoes the irreversible cleavage event under the oxidative effect of BLM with Fe(II) as a cofactor and the intercalated Ru(phen)3(2+) released from the gold electrode, which can be transduced into a significant decrease in ECL intensity. The ECL intensity versus the concentration of BLMs was linear in the range from 0.1 pM to 50 pM. The detection limit was 0.03 pM. This work demonstrates that using the sequence selectivity of DNA cleavage strategy for the fabrication of the label-free ECL biosensing method is a promising approach for the determination of antitumor drugs.

Ultrasensitive electrogenerated chemiluminescent DNA-based biosensing switch for the determination of bleomycin.

An ultrasensitive electrogenerated chemiluminescent (ECL) DNA-based biosensing switch for the determination of bleomycin (BLM) was developed based on Fe(II) · BLM-mediated hairpin DNA strand cleavage and a structure-switching ECL-dequenching mechanism. A thiolated ss-DNA was used as a substrate for BLMs: one terminus was tethered onto an electrode surface, and the other terminus was labelled with the ECL quencher ferrocene to form a hairpin structure. This thiolated ss-DNA self-assembled on to the tris(2,2'-bipyridine)ruthenium-gold nanoparticle composite modified gold electrode. In the presence of Fe(II) · BLM, the ECL DNA biosensing switch undergoes an irreversible cleavage event that can trigger a significant increase in ECL intensity. The relationship of ECL intensity and the concentration of BLMs was found to be linear in the range of 5 fM - 5000 fM with a detection limit of 2 fM. This work demonstrates that the design of a highly sensitive ECL DNA-based biosensing switch that uses the sequence selectivity of DNA cleavage mediated by the antitumor drug BLM in combination with a chemical quencher, such as ferrocene, to quench ECL signal(s), offers a promising approach for the determination of ultratrace amounts of antitumor drugs.

A sensitive graphene oxide-DNA based sensing platform for fluorescence "turn-on" detection of bleomycin.

A sensitive and selective fluorescent sensing platform for bleomycin (BLM) was developed based on BLM-induced DNA strand scission and the difference in affinity of graphene oxide for single-stranded DNA containing different numbers of bases in length.

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.

Sensitive DNA-based electrochemical strategy for trace bleomycin detection.

Bleomycins (BLMs) are widely used in combination with chemotherapy for the treatment of a variety of cancers. The clinical application of BLMs is featured by the occurrence of sometimes fatal side effects, such as renal and lung toxicity, and the potential dose-limiting side effect of pulmonary fibrosis. Therefore, it is highly desirable to develop a sensitive method to quantitatively determine the BLM content in both pharmaceutical analysis and clinical samples, to make full use of therapeutic efficacy and to weaken its toxicity. Here, we proposed a simple, rapid, and convenient electrochemical assay for trace BLM detection. A reported DNA motif, as substrate for BLMs, is prepared to self-assemble onto the gold electrode to fabricate an electrochemical DNA (E-DNA) sensor, with a terminus tethered on the electrode surface and the other terminus labeled with ferrocenyl moiety as a signal reporter to form a stem-loop structure, giving an arise of remarkable faradaic current. In the presence of Fe(II)·BLM, the E-DNA sensor undergoes the irreversible cleavage event, which can be transduced into a significant decrease in current peak. This proposed sensor reveals an impressive sensitivity as low as 100 pM BLMs and exhibits a good performance as well as in serum sample. Considering the high sensitivity and specificity of this proposed sensor, as well as the cost-effective and simple-to-implement features of the electrochemical technique, we believe that this method shows distinct advantages over conventional methods and it is a promising alternative for the determination of trace amounts of BLMs in clinical samples.

Biphasic pulses enhance bleomycin efficacy in a spontaneous canine perianal tumors model.

Perianal tumors (adenoma and carcinoma of the hepatoid glands) are frequently reported in veterinary literature. They are locally aggressive tumors with a low tendency to metastatic spread. An hormonal ethiology has been identified for the development of perianal adenomas in male dogs, while the carcinomas are free from hormonal influence. Standard treatments include surgery, cryotherapy or, in selected cases, radiation therapy. In this article we describe the outcome of a small cohort of canine patients with perianal tumors treated with bleomycin selectively driven by trains of biphasic pulses (electrochemotherapy). Twelve canine patients, eight with adenoma and four with carcinoma of the perianal glands, entered the study and received two sessions of ECT under sedation. The pets had local injection ofbleomycin at the concentration of 1.5 mg/mg and five minutes after the chemotherapy, trains of 8 biphasic electric pulses lasting 50 + 50 micros each, with 1 ms interpulse intervals, were delivered by means of modified caliper and needle array electrodes or, for difficult districts, through paired needle electrode. The overall response rate was 91% with a 83% of complete response (10/12); one dog had a PR that lasted 12 months and another had progressive disease. Electrochemotherapy appears as a safe and efficacious modality for the treatment of perianal tumors and warrants further investigations.

Recent advances in bioinorganic spectroscopy.

Spectroscopic methods covering many energy regions together provide complementary insight into metalloenzyme active sites. These methods probe geometric and electronic structure and define these contributions to reactivity. Two recent advances--determination of the polarizations of electronic transitions in solution using magnetic circular dichroism, electron paramagnetic resonance and quantum chemistry, and experimental estimation of covalency using metal L-edges and ligand K-edges--are particularly important.

Orientation of iron bleomycin and porphyrin complexes on DNA fibers.

Bleomycin (Blm) is an antitumor agent that requires iron and oxygen for strand cleavage of DNA. In this study, ferric bleomycin, Fe(III)Blm, or the nitric oxide adduct of ferrous bleomycin, ON-Fe(II)Blm, were bound to one-dimensionally oriented DNA fibers. Reductive nitrosylation of Fe(III) complexes took place in situ on B-form DNA fibers. Electron paramagnetic resonance (EPR) spectra were obtained as a function of the angle phi between the magnetic field B and the fiber axis Zf. For comparison, EPR spectra were acquired for ON-Fe(II)TMpyP and ON-Fe(II)TMpyP-Im on oriented DNA fibers, where TMpyP is 5,10,15,20-tetrakis(1-methyl-4-pyridino)porphyrin and Im is imidazole. EPR spectra showed both low-spin Fe(III)Blm and ON-Fe(II)Blm bound to B-form DNA in two slightly different binding orientations in the ratio of 1:0.2. With A-form DNA, a fraction of bound Fe(III)Blm was high spin. Specifically, the angle beta between the fiber axis Zf and the g axis, gz, perpendicular to or nearly perpendicular to the equatorial plane of the iron complex was estimated as 20 degrees and 25 degrees for ON-Fe(II)Blm and 30 degrees and 25 degrees for Fe(III)Blm, respectively. The angle gamma that determines the orientation of gx and gy axes was estimated as 90 degrees for the two ON-Fe(II)Blm species and 10 degrees for the two Fe(III)Blm species, respectively. The NO was held rigidly in place as the temperature increased from 123 K to room temperature for ON-Fe(II)Blm but not for ON-Fe(II)TMpyP or ON-Fe(II)TMpyP-Im. It is hypothesized that the NO is structurally oriented by hydrogen bonding like the peroxide is held in HO2(-)-Co(III)Blm (Wu et al. J. Am. Chem. Soc. 1996, 118, 1281-1294). The EPR parameters are consistent with a six-coordinate complex for ON-Fe(II)Blm, although the superhyperfine structure from the trans nitrogen was not detected. The increase in g value anisotropy upon binding ON-Fe(II)Blm to DNA fiber may be caused by an increase in the overlap of d pi and 2p pi* orbitals induced by an interaction of NO with DNA and/or by a perturbation of d orbitals due to the pyrimidine-guanine interaction. It is concluded that the EPR parameters of ON-Fe(II)Blm and Fe(III)Blm bound to oriented DNA support the hypothesis that FeBlm species bind to DNA with adduct structures similar to those formed by related CoBlm species and DNA.

Adsorptive stripping voltammetry of bleomycin.

In 0.05 M H2SO4 solution, two reductive peaks, P1 and P2, of bleomycin were obtained. The peak potentials E(P1) and E(P2) were -0.83 and -1.09 V (versus Ag/AgCl), respectively. The sensitivity of P2 was much higher than that of P1. The peak current of P2 was proportional to the concentration of bleomycin over the range 1.0 x 10(-9)-1.0 x 10(-7) M with a detection limit of 5.0 x 10(-10) M using adsorptive voltammetry at an accumulation time of 120 s (Ei = -0.80 V). The behaviour of the reduction wave was studied and applied to the determination of bleomycin in mouse serum. The reduction process of P1 was irreversible with adsorptive characteristics and the adsorption behavior obeyed the Frumkin adsorptive isotherm. The adsorptive coefficient beta was 8.9 x 10(5), the interaction factor alpha was 0.94 and the Gibbs energy of adsorption delta G degree was -33.93 kJ mol-1. P2 was an irreversible adsorption peak with catalytic hydrogen properties.