Fullerenol-Based Intracellular Delivery of Methotrexate: A Water-Soluble Nanoconjugate for Enhanced Cytotoxicity and Improved Pharmacokinetics

Derivatization of fullerenes to polyhydroxylated fullerenes, i.e., fullerenols (FLU), dramatically decreases their toxicity and has been reported to enhance the solubility as well as cellular permeability. In this paper, we report synthesis of FLU as nanocarrier and subsequent chemical conjugation of Methotrexate (MTX) to FLU with a serum-stable and intracellularly hydrolysable ester bond between FLU and MTX. The conjugate was characterized for physiochemical attributes, micromeritics, drug-loading, and drug-release and evaluated for cancer cell-toxicity, cellular-uptake, hemocompatibility, protein binding, and pharmacokinetics. The developed hemocompatible FL-MTX offered lower protein binding vis-à-vis naïve drug and substantially higher drug loading. The conjugate offered pH-dependent release of 38.20?±?1.19% at systemic pH and 85.67?±?3.39% at the cancer cell pH. FLU-MTX-treated cells showed significant reduction in IC₅₀ value vis-à-vis the cells treated with pure MTX. Analogously, the results from confocal scanning laser microscopy also confirmed the easy access of the dye-tagged FLU-MTX conjugate to the cell interiors. In pharmacokinetics, the AUC of MTX was enhanced by approx. 6.15 times and plasma half-life was enhanced by 2.45 times, after parenteral administration of single equivalent dose in rodents. FLU-MTX offered enhanced availability of drug to the biological system, meanwhile improved the cancer-cell cytotoxicity, sustained the effective plasma drug concentrations, and offered substantial compatibility to erythrocytes.     

Amperometric micro-immunosensor for the detection of tumor biomarker

In this paper, a highly sensitive, reagentless, electrochemical strategy is reported for the detection of a cancer biomarker-Vascular Endothelial Growth Factor (VEGF). Disc shaped carbon fiber microelectrodes were used as the immunosensor platform. Ferrocene monocarboxylic acid labeled anti-VEGF was covalently immobilized on the microelectrode surface using a Jeffamine cross-linker. The formation of immunocomplexes leads to a decrease in the electrochemical signal of ferrocene monocarboxylic acid owing to increased spatial blocking of microelectrode surface. These signal changes enable quantitative detection of VEGF in solution. Voltammetric measurements were conducted to evaluate the interfacial immunoreactions and to quantitatively detect VEGF biomarker. The proposed immunosensing strategy allows a rapid and sensitive means of VEGF analysis with a limit of detection of about 38 pg/mL. This opens up the possibility of employing these electrodes for various single cell analysis and clinical applications. Further, experimental conditions such as concentration of the immobilized antibodies and incubation period were optimized. Following this, the stability and specificity of the immunosensors were also evaluated.     

Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37

In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a "scaffold," recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50(cdc37), whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50(cdc37), like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a "relaxed" ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50(cdc37) binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50(cdc37) is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50(cdc37) forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo.     

Mechanistic Insights into the Neutralization of Cytotoxic Abrin by the Monoclonal Antibody D6F10

Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74–123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin’s toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.     

Plasmid pUPI126-encoded pyrrolnitrin production by Acinetobacter haemolyticus A19 isolated from the rhizosphere of wheat

An Acinetobacter species identified as A. haemolyticus A19 produces an antibiotic and the enzyme chitinase. The antibiotic produced by A. haemolyticus A19 was extracellular and inducible by co-cultivation with Klebsiella pneumoniae in the optimum ratio 2:1, respectively. pH 7, temperature 28 °C, and addition of 2 % (w/v) NaCl are the most suitable environmental conditions for production and activity of the antibiotic. The antibiotic was produced in the early stationary growth phase (48 h) of A. haemolyticus A19. It has a very broad spectrum of antimicrobial activity against plant and human pathogenic bacteria and fungi. The antibiotic was extracted with ethyl acetate and purified by column chromatography with further purification by preparative thin-layer chromatography. Yield of the antibiotic was 15 mg/l. The antibiotic was active at very low concentrations, for example 50 μg/ml, and was water-soluble. It was stable at room temperature for up to 7 days. ¹H NMR analysis revealed the antibiotic was a pyrrolnitrin. It was found that pyrrolnitrin production by A. haemolyticus A19 was encoded by plasmid pUPI126 of molecular weight 25.7 kb. Plasmid pUPI126 was transferred to E. coli HB101 at a frequency of 5 × 10^?5 per μg DNA. It was also conjugally transformed to E. coli HB101 rif ^r mutants at a frequency of 5.9 × 10^?8 per recipient cell. Plasmid pUPI126 was 100 % stable in Acinetobacter and 95 % stable in E. coli HB101. Transconjugants and transformants both produced the antibiotic. This is the first report of plasmid-mediated pyrrolnitrin production by A. haemolyticus A19 isolated from wheat rhizosphere.