Acid-Modulated Peptide Synthesis for Application on Oxide Biosensor Interfaces.

In this paper we report an acid-modulated strategy for novel peptide microarray production on biosensor interfaces. We initially selected a controlled pore glass (CPG) as a support for solid-phase peptide synthesis (SPPS) to implement a chemistry that can be performed at the interface of multiple field effect transistor (FET) sensors, eventually to generate label-free peptide microarrays for protein screening. Our chemistry uses a temporary protection of the N-terminal amino function of each amino acid building block with a tert-butyloxycarbonyl (Boc) group that can be removed after each SPPS cycle, in combination with semi-permanent protection of the side chains of trifunctional amino acid residues. Such a protection scheme with a well-proven record of application in conventional, batchwise SPPS has been fine-tuned for optimal performance on CPG and, from there, translated to SPR chips that allow layer-by-layer monitoring of amino acid coupling. Our results validate this acid-modulated synthesis as a feasible approach for producing peptides in high yields and purity on flat glass surfaces, such as those in bio-FETs.

ZnO Structures with Surface Nanoscale Interfaces Formed by Au, Fe2O3, or Cu2O Modifier Nanoparticles: Characterization and Gas Sensing Properties.

Zinc oxide rod structures are synthetized and subsequently modified with Au, Fe2O3, or Cu2O to form nanoscale interfaces at the rod surface. X-ray photoelectron spectroscopy corroborates the presence of Fe in the form of oxide-Fe2O3; Cu in the form of two oxides-CuO and Cu2O, with the major presence of Cu2O; and Au in three oxidation states-Au3+, Au+, and Au0, with the content of metallic Au being the highest among the other states. These structures are tested towards nitrogen dioxide, ethanol, acetone, carbon monoxide, and toluene, finding a remarkable increase in the response and sensitivity of the Au-modified ZnO films, especially towards nitrogen dioxide and ethanol. The results for the Au-modified ZnO films report about 47 times higher response to 10 ppm of nitrogen dioxide as compared to the non-modified structures with a sensitivity of 39.96% ppm-1 and a limit of detection of 26 ppb to this gas. These results are attributed to the cumulative effects of several factors, such as the presence of oxygen vacancies, the gas-sensing mechanism influenced by the nano-interfaces formed between ZnO and Au, and the catalytic nature of the Au nanoparticles.

Linker Hydrophilicity Modulates the Anticancer Activity of RGD-Cryptophycin Conjugates.

Most anticancer agents are hydrophobic and can easily penetrate the tumor cell membrane by passive diffusion. This may impede the development of highly effective and tumor-selective treatment options. A hydrophilic ß-glucuronidase-cleavable linker was used to connect the highly potent antimitotic agent cryptophycin-55 glycinate with the αv ß3 integrin ligand c(RGDfK). Incorporation of the self-immolative linker containing glucuronic acid results in lower cytotoxicity than that of the free payload, suggesting that hydrophilic sugar linkers can preclude passive cellular uptake. In vitro drug-release studies and cytotoxicity assays demonstrated the potential of this small molecule-drug conjugate, providing guidance for the development of therapeutics containing hydrophobic anticancer drugs.

Solid-Phase Synthesis of Biaryl Cyclic Lipopeptides Derived from Arylomycins.

An efficient approach for the solid-phase synthesis of N-methylated tailed biaryl cyclic lipopeptides based on the structure of arylomycins was established. Each of these analogues incorporates an N-terminal linear lipopeptide attached to a biaryl cyclic tripeptide containing a Phe-Tyr, a Tyr-Tyr, or a His-Tyr linkage. This methodology first involved an intramolecular Suzuki-Miyaura arylation of a linear peptidyl resin incorporating the corresponding halogenated amino acid at the N-terminus and a boronotyrosine at the C-terminus. After N-methylation of the resulting biaryl cyclic peptidyl resin, the N-methylated lipopeptidyl tail was then assembled. The biaryl cyclic lipopeptides were purified and characterized.

Tumor-Targeting Peptides: The Functional Screen of Glioblastoma Homing Peptides to the Target Protein FABP3 (MDGI).

We recently identified the glioblastoma homing peptide CooP (CGLSGLGVA) using in vivo phage display screen. The mammary-derived growth inhibitor (MDGI/FABP3) was identified as its interacting partner. Here, we present an alanine scan of A-CooP to investigate the contribution of each amino acid residue to the binding to FABP3 by microscale thermophoresis (MST) and surface plasmon resonance (SPR). We also tested the binding affinity of the A-CooP-K, KA-CooP, and retro-inverso A-CooP analogues to the recombinant FABP3. According to the MST analysis, A-CooP showed micromolar (KD = 2.18 µM) affinity to FABP3. Alanine replacement of most of the amino acids did not affect peptide affinity to FABP3. The A-CooP-K variant showed superior binding affinity, while A-[Ala5]CooP and A-[Ala7]CooP, both replacing a glycine residue with alanine, showed negligible binding to FABP3. These results were corroborated in vitro and in vivo using glioblastoma models. Both A-CooP-K and A-CooP showed excellent binding in vitro and homing in vivo, while A-[Ala5]CooP and control peptides failed to bind the cells or home to the intracranial glioblastoma xenografts. These results provide insight into the FABP3-A-CooP interaction that may be important for future applications of drug conjugate design and development.

Love Wave Sensors with Silver Modified Polypyrrole Nanoparticles for VOCs Monitoring.

Love wave sensors with silver-modified polypyrrole nanoparticles are developed in this work. These systems prove functional at room temperature with enhanced response, sensitivity and response time, as compared to other state-of-the-art surface acoustic wave (SAW) sensors, towards volatile organic compounds (VOCs). Results demonstrate the monitoring of hundreds of ppb of compounds such as acetone, ethanol and toluene with low estimated limits of detection (~3 ppb for acetone). These results are attributed to the use of silver-modified polypyrrole as a second guiding/sensitive layer in the Love wave sensor structure, which provides further chemically active sites for the gas-solid interactions. The sensing of low VOCs concentrations by micro sensing elements as those presented here could be beneficial in future systems for air quality control, food quality control or disease diagnosis via exhaled breath as the limits of detection obtained are within those required in these applications.

Synthesis and Biological Characterization of Monomeric and Tetrameric RGD-Cryptophycin Conjugates.

The effective delivery of cytotoxic agents to tumor cells is a key challenge in anticancer therapy. Multivalent integrinspecific ligands are considered a promising tool to increase the binding affinity, selectivity, and internalization efficiency of small-molecule drug conjugates. Herein, we report the synthesis and biological evaluation of a multimeric conjugate containing the high-affinity integrin αv ß3 binding ligand RAFT-c(RGDfK)4 , a lysosomally cleavable Val-Cit linker, and cryptophycin-55 glycinate, a potent inhibitor of tubulin polymerization. In vitro cytotoxicity assays verified that the multimeric RGD-cryptophycin conjugate displays improved potency compared to the monomeric analogue in integrin αv ß3 overexpressing tumor cell lines, while significantly reduced activity was observed in the integrin-negative cell line.

Conjugates of Cryptophycin and RGD or isoDGR Peptidomimetics for Targeted Drug Delivery.

RGD-cryptophycin and isoDGR-cryptophycin conjugates were synthetized by combining peptidomimetic integrin ligands and cryptophycin, a highly potent tubulin-binding antimitotic agent across lysosomally cleavable Val-Ala or uncleavable linkers. The conjugates were able to effectively inhibit binding of biotinylated vitronectin to integrin αvß3, showing a binding affinity in the same range as that of the free ligands. The antiproliferative activity of the novel conjugates was evaluated on human melanoma cells M21 and M21-L with different expression levels of integrin αvß3, showing nanomolar potency of all four compounds against both cell lines. Conjugates containing uncleavable linker show reduced activity compared to the corresponding cleavable conjugates, indicating efficient intracellular drug release in the case of cryptophycin-based SMDCs. However, no significant correlation between the in vitro biological activity of the conjugates and the integrin αvß3 expression level was observed, which is presumably due to a non-integrin-mediated uptake. This reveals the complexity of effective and selective αvß3 integrin-mediated drug delivery.

Octreotide Conjugates for Tumor Targeting and Imaging.

Tumor targeting has emerged as an advantageous approach to improving the efficacy and safety of cytotoxic agents or radiolabeled ligands that do not preferentially accumulate in the tumor tissue. The somatostatin receptors (SSTRs) belong to the G-protein-coupled receptor superfamily and they are overexpressed in many neuroendocrine tumors (NETs). SSTRs can be efficiently targeted with octreotide, a cyclic octapeptide that is derived from native somatostatin. The conjugation of cargoes to octreotide represents an attractive approach for effective tumor targeting. In this study, we conjugated octreotide to cryptophycin, which is a highly cytotoxic depsipeptide, through the protease cleavable Val-Cit dipeptide linker using two different self-immolative moieties. The biological activity was investigated in vitro and the self-immolative part largely influenced the stability of the conjugates. Replacement of cryptophycin by the infrared cyanine dye Cy5.5 was exploited to elucidate the tumor targeting properties of the conjugates in vitro and in vivo. The compound efficiently and selectively internalized in cells overexpressing SSTR2 and accumulated in xenografts for a prolonged time. Our results on the in vivo properties indicate that octreotide may serve as an efficient delivery vehicle for tumor targeting.

Synthesis and Biological Evaluation of RGD⁻Cryptophycin Conjugates for Targeted Drug Delivery.

Cryptophycins are potent tubulin polymerization inhibitors with picomolar antiproliferative potency in vitro and activity against multidrug-resistant (MDR) cancer cells. Because of neurotoxic side effects and limited efficacy in vivo, cryptophycin-52 failed as a clinical candidate in cancer treatment. However, this class of compounds has emerged as attractive payloads for tumor-targeting applications. In this study, cryptophycin was conjugated to the cyclopeptide c(RGDfK), targeting integrin αvß3, across the protease-cleavable Val-Cit linker and two different self-immolative spacers. Plasma metabolic stability studies in vitro showed that our selected payload displays an improved stability compared to the parent compound, while the stability of the conjugates is strongly influenced by the self-immolative moiety. Cathepsin B cleavage assays revealed that modifications in the linker lead to different drug release profiles. Antiproliferative effects of Arg-Gly-Asp (RGD)⁻cryptophycin conjugates were evaluated on M21 and M21-L human melanoma cell lines. The low nanomolar in vitro activity of the novel conjugates was associated with inferior selectivity for cell lines with different integrin αvß3 expression levels. To elucidate the drug delivery process, cryptophycin was replaced by an infrared dye and the obtained conjugates were studied by confocal microscopy.

In Vivo Antitumor Activity of a Novel Acetazolamide-Cryptophycin Conjugate for the Treatment of Renal Cell Carcinomas.

Traditional chemotherapeutics used in cancer therapy do not preferentially accumulate in tumor tissues. The conjugation to delivery vehicles like antibodies or small molecules has been proposed as a strategy to increase the tumor uptake and improve the therapeutic window of these drugs. Here, we report the synthesis and the biological evaluation of a novel small molecule-drug conjugate (SMDC) comprising a high-affinity bidentate acetazolamide derivative, targeting carbonic anhydrase IX (CAIX), and cryptophycin, a potent microtubule destabilizer. The biological activity of the novel SMDC was evaluated in vitro, measuring binding to the CAIX antigen by surface plasmon resonance and cytotoxicity against SKRC-52 cells. In vivo studies showed a delayed growth of tumors in nude mice bearing SKRC-52 renal cell carcinomas.

Cerium Oxide-Tungsten Oxide Core-Shell Nanowire-Based Microsensors Sensitive to Acetone.

Gas sensitive cerium oxide-tungsten oxide core-shell nanowires are synthesized and integrated directly into micromachined platforms via aerosol assisted chemical vapor deposition. Tests to various volatile organic compounds (acetone, ethanol, and toluene) involved in early disease diagnosis demonstrate enhanced sensitivity to acetone for the core-shell structures in contrast to the non-modified materials (i.e., only tungsten oxide or cerium oxide). This is attributed to the high density of oxygen vacancy defects at the shell, as well as the formation of heterojunctions at the core-shell interface, which provide the modified nanowires with 'extra' chemical and electronic sensitization as compared to the non-modified materials.

Novel unit B cryptophycin analogues as payloads for targeted therapy.

Cryptophycins are naturally occurring cytotoxins with great potential for chemotherapy. Since targeted therapy provides new perspectives for treatment of cancer, new potent analogues of cytotoxic agents containing functional groups for conjugation to homing devices are required. We describe the design, synthesis and biological evaluation of three new unit B cryptophycin analogues. The O-methyl group of the unit B D-tyrosine analogue was replaced by an O-(allyloxyethyl) moiety, an O-(hydroxyethyl) group, or an O-(((azidoethoxy)ethoxy)ethoyxethyl) substituent. While the former two maintain cytotoxicity in the subnanomolar range, the attachment of the triethylene glycol spacer with a terminal azide results in a complete loss of activity. Docking studies of the novel cryptophycin analogues to ß-tubulin provided a rationale for the observed cytotoxicities.

Erratum: In Vivo Antitumor Activity of a Novel Acetazolamide-Cryptophycin Conjugate for the Treatment of Renal Cell Carcinoma.

[This corrects the article DOI: 10.1021/acsomega.8b02350.].

Cryptophycins: cytotoxic cyclodepsipeptides with potential for tumor targeting.

Cryptophycins are a class of 16-membered highly cytotoxic macrocyclic depsipeptides isolated from cyanobacteria. The biological activity is based on their ability to interact with tubulin. They interfere with microtubule dynamics and prevent microtubules from forming correct mitotic spindles, which causes cell-cycle arrest and apoptosis. Their strong antiproliferative activities with 100-fold to 1000-fold potency compared with those of paclitaxel and vinblastine have been observed. Cryptophycins are highly promising drug candidates, as their biological activity is not negatively affected by P-glycoprotein, a drug efflux system commonly found in multidrug-resistant cancer cell lines and solid tumors. Cryptophycin-52 had been investigated in phase II clinical trials but failed because of its high neurotoxicity. Recently, cryptophycin conjugates with peptides and antibodies have been developed for targeted delivery in tumor therapy. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Design, Preparation, and Characterization of Zn and Cu Metallopeptides Based On Tetradentate Aminopyridine Ligands Showing Enhanced DNA Cleavage Activity.

The conjugation of redox-active complexes that can function as chemical nucleases to cationic tetrapeptides is pursued in this work in order to explore the expected synergistic effect between these two elements in DNA oxidative cleavage. Coordination complexes of biologically relevant first row metal ions, such as Zn(II) or Cu(II), containing the tetradentate ligands 1,4-dimethyl-7-(2-pyridylmethyl)-1,4,7-triazacyclononane ((Me2)PyTACN) and (2S,2S')-1,1'-bis(pyrid-2-ylmethyl)-2,2'-bipyrrolidine ((S,S')-BPBP) have been linked to a cationic LKKL tetrapeptide sequence. Solid-phase synthesis of the peptide-tetradentate ligand conjugates has been developed, and the preparation and characterization of the corresponding metallotetrapeptides is described. The DNA cleavage activity of Cu and Zn metallopeptides has been evaluated and compared to their metal binding conjugates as well as to the parent complexes and ligands. Very interestingly, the oxidative Cu metallopeptides 1Cu and 2Cu show an enhanced activity compared to the parent complexes, [Cu(PyTACN)](2+) and [Cu(BPBP)](2+), respectively. Under optimized conditions, 1Cu displays an apparent pseudo first-order rate constant (kobs) of ∼0.16 min(-1) with a supercoiled DNA half-life time (t1/2) of ∼4.3 min. On the other hand, kobs for 2Cu has been found to be ∼0.11 min(-1) with t1/2 ≈ 6.4 min. Hence, these results point out that the DNA cleavage activities promoted by the metallopeptides 1Cu and 2Cu render ∼4-fold and ∼23 rate accelerations in comparison with their parent Cu complexes. Additional binding assays and mechanistic studies demonstrate that the enhanced cleavage activities are explained by the presence of the cationic LKKL tetrapeptide sequence, which induces an improved binding affinity to the DNA, thus bringing the metal ion, which is responsible for cleavage, in close proximity.

Enzyme-triggered delivery of chlorambucil from conjugates based on the cell-penetrating peptide BP16.

The undecapeptide KKLFKKILKKL-NH2 (BP16) is a non-toxic cell-penetrating peptide (CPP) that is mainly internalized into cancer cells through a clathrin dependent endocytic mechanism and localizes in late endosomes. Moreover, this CPP is able to enhance the cellular uptake of chlorambucil (CLB) improving its cytotoxicity. In this work, we further explored the cell-penetrating properties of BP16 and those of its arginine analogue BP308. We investigated the influence on the cytotoxicity and on the cellular uptake of conjugating CLB at the N- or the C-terminal end of these undecapeptides. The effect of incorporating the cathepsin B-cleavable sequence Gly-Phe-Leu-Gly in CLB-BP16 and CLB-BP308 conjugates was also evaluated. The activity of CLB was significantly improved when conjugated at the N- or the C-terminus of BP16, or at the N-terminus of BP308. While CLB alone was not active (IC50 of 73.7 to >100 µM), the resulting conjugates displayed cytotoxic activity against CAPAN-1, MCF-7, PC-3, 1BR3G and SKMEL-28 cell lines with IC50 values ranging from 8.7 to 25.5 µM. These results were consistent with the internalization properties observed for the corresponding 5(6)-carboxyfluorescein-labeled conjugates. The presence of the tetrapeptide Gly-Phe-Leu-Gly at either the N- or the C-terminus of CLB-BP16 conjugates further increased the efficacy of CLB (IC50 of 3.6 to 16.2 µM), which could be attributed to its selective release in the lysosomal compartment. Enzymatic assays with cathepsin B showed the release of CLB-Gly-OH from these sequences within a short time. Therefore, the combination of BP16 with an enzymatic cleavable sequence can be used as a drug delivery system for the effective uptake and release of drugs in cancer cells.

Improved detection of magnetic signals by a MEMS sensor using stochastic resonance.

We introduce the behavior of the electrical output response of a magnetic field sensor based on microelectromechanical systems (MEMS) technology under different levels of controlled magnetic noise. We explored whether a particular level of magnetic noise applied on the vicinity of the MEMS sensor can improve the detection of subthreshold magnetic fields. We examined the increase in the signal-to-noise ratio (SNR) of such detected magnetic fields as a function of the magnetic noise intensity. The data disclosed an inverted U-like graph between the SNR and the applied magnetic noise. This finding shows that the application of an intermediate level of noise in the environment of a MEMS magnetic field sensor improves its detection capability of subthreshold signals via the stochastic resonance phenomenon.

Digital signal processing by virtual instrumentation of a MEMS magnetic field sensor for biomedical applications.

We present a signal processing system with virtual instrumentation of a MEMS sensor to detect magnetic flux density for biomedical applications. This system consists of a magnetic field sensor, electronic components implemented on a printed circuit board (PCB), a data acquisition (DAQ) card, and a virtual instrument. It allows the development of a semi-portable prototype with the capacity to filter small electromagnetic interference signals through digital signal processing. The virtual instrument includes an algorithm to implement different configurations of infinite impulse response (IIR) filters. The PCB contains a precision instrumentation amplifier, a demodulator, a low-pass filter (LPF) and a buffer with operational amplifier. The proposed prototype is used for real-time non-invasive monitoring of magnetic flux density in the thoracic cage of rats. The response of the rat respiratory magnetogram displays a similar behavior as the rat electromyogram (EMG).

Respiratory magnetogram detected with a MEMS device.

Magnetic fields generated by the brain or the heart are very useful in clinical diagnostics. Therefore, magnetic signals produced by other organs are also of considerable interest. Here we show first evidence that thoracic muscles can produce a strong magnetic flux density during respiratory activity, that we name respiratory magnetogram. We used a small magnetometer based on microelectromechanical systems (MEMS), which was positioned inside the open thoracic cage of anaesthetized and ventilated rats. With this new MEMS sensor of about 20 nT resolution, we recorded a strong and rhythmic respiratory magnetogram of about 600 nT.