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We report herein a Pt(IV) prodrug with metronidazole in axial positions

We report herein the design, synthesis, and biological investigation of a series of novel Pt(IV) prodrugs with non-steroidal anti-inflammatory drugs naproxen, diclofenac, and flurbiprofen, as well as these with stearic acid in the axial position. Six Pt(IV) prodrugs

Alzheimer’s disease (AD) is the most common form of dementia, a progressive neurological disorder characterized by short and long-term memory loss, including cognitive and functional impairment, which is refractory to current therapy. It is suggested that the aggregation of β-amyloid (Aβ) peptide on neuronal cell surface leads to various deviations of its vital function due to myriad pathways defined by internalization of calcium ions, apoptosis promotion, reduction of membrane potential, synaptic activity loss etc. These are associated with structural reorganizations and pathologies of the cell cytoskeleton mainly involving actin filaments and microtubules, and consequently – alterations of cell mechanical properties. Thus, the effect of amyloid oligomers on cells’ Young’s modulus has been observed in a variety of studies. However, the precise connection between the formation of amyloid aggregates on cell membranes and their effects on local mechanical properties of living cells is still unresolved. In this work, we have used correlative scanning ion-conductance microscopy (SICM) to study cell topography, Young’s modulus mapping and confocal imaging of Aβ aggregates formation on living cell surfaces with subsequent assessment of the reactive oxygen species levels inside single cells using platinum nanoelectrodes. We showed that correlative SICM technique, in conjunction with topography mapping and confocal imaging, can be used for Patch-Clamp recordings from living cells with evidently formed FAM-labeled Aβ aggregates on its surface. As we demonstrated, SICM can be successfully applied to studying cytotoxicity mechanisms of Aβ aggregates on living cell surface.

Synthesis, characterization (HRMS, NMR, EPR, UV-Vis spectroscopy, and electrochemistry) and in vitro cytotoxic investigation of a series of new ferrocene-containing derivatives, based on ω-ferrocene carboxylic acids and 2-alkylthioimidazolin-4-ones as well as their copper coordination compounds, have been reported. A series of ferrocene–imidazolone conjugates with different lengths and donor properties of the linker fragments have been synthesized; all the obtained compounds were studied in complexation reactions with copper(I) and copper(II) perchlorates. It is shown that the synthesised conjugates rapidly and efficiently convert coordinating copper ions into the Cu(+1) oxidation state, but a change in the length of the linker in ferrocene–imidazolone ligands fundamentally changes the ratio of products formed as a result of iron oxidation/copper reduction and complexation by various mechanisms. After comparing the data on the qualitative and quantitative composition of the reaction mixtures and the kinetic characteristics of the processes occurring in the mixtures of ferrocene–imidazolones with copper(II) perchlorate, a scheme of the ongoing reactions was proposed which may fully explain all the observed processes and products. It has been shown that the obtained bimetallic Cu/Fe coordination compounds are more toxic than cisplatin and induce apoptosis in micromolar concentrations on Hek-293 and MCF-7 cell lines.

A series of 73 ligands and 73 of their Cu+2 and Cu+1 copper complexes with different geometries, oxidation states of the metal, and redox activities were synthesized and characterized. The aim of the study was to establish the structure-activity relationship within a series of analogues with different substituents at the N(3) position, which govern the redox potentials of the Cu+2/Cu+1 redox couples, ROS generation ability, and intracellular accumulation. Possible cytotoxicity mechanisms, such as DNA damage, DNA intercalation, telomerase inhibition, and apoptosis induction, have been investigated. ROS formation in MCF-7 cells and three-dimensional (3D) spheroids was proven using the Pt-nanoelectrode. Drug accumulation and ROS formation at 40-60 μm spheroid depths were found to be the key factors for the drug efficacy in the 3D tumor model, governed by the Cu+2/Cu+1 redox potential. A nontoxic in vivo single-dose evaluation for two binuclear mixed-valence Cu+1/Cu+2 redox-active coordination compounds, 72k and 61k, was conducted.

In vivo monitoring of reactive oxygen species (ROS) in tumors during treatment with anticancer therapy is important for understanding the mechanism of action and in the design of new anticancer drugs. In this work, a platinized nanoelectrode is placed into a single cell for detection of the ROS signal, and drug-induced ROS production is then recorded. The main advantages of this method are the short incubation time with the drug and its high sensitivity which allows the detection of low intracellular ROS concentrations. We have shown that our new method can measure the ROS response to chemotherapy in tumor-bearing mice in real-time. ROS levels were measured in vivo inside the tumor at different depths in response to doxorubicin. This work provides an effective new approach for the measurement of intracellular ROS by platinized nanoelectrodes.

In search of the compounds that interfere with amyloid transformation of alpha-synuclein, 9 natural and synthetic cinnamic acid derivatives were studied. They are structurally similar to a half of curcumin, which has pronounced anti-aggregatory and anti-amyloid effects. We have shown that some of these derivatives prevent ovine prion protein amyloidization. Subsequently, thioflavin T binding assay showed that 3 out of 9 studied compounds effectively prevented amyloid transformation of alpha-synuclein with IC50 of 13, 50 and 251 μM. Molecular modeling approach revealed possible binding sites of the three selected ligands with alpha-synuclein fibrils, while monomeric alpha-synuclein does not bind to the ligands according to experimental results. This led us to believe that compounds may act by changing the structure of primary aggregates, preventing the formation of full-length fibrils. The inhibiting effect of the ligands on aggregation of alpha-synuclein was further confirmed by monitoring aggregation via turbidimetry, susceptibility to proteolytic cleavage, changes in beta-sheet content, and scanning ion-conductance microscopy. Studied derivatives were not cytotoxic, and, moreover, two studied compounds (ferulic and 3,4-dimethoxycinnamic acid) are found in plant sources and are natural metabolites present in human blood, so they can be promising candidate drugs for synucleinopathies, including Parkinson’s disease.

Prostate cancer is one of the most commonly diagnosed men's cancers and remains one of the leading causes of cancer death. The development of approaches to the treatment of this oncological disease is an ongoing process. In this work, we have carried out the selection of ligands for the creation of conjugates based on the drug docetaxel and synthesized a series of three docetaxel conjugates. In vitro cytotoxicity of these molecules was evaluated using the MTT assay. Based on the assay results, we selected the conjugate which showed cytotoxic potential close to unmodified docetaxel. At the same time, the molar solubility of the resulting compound increased up to 20 times in comparison with the drug itself. In vivo evaluation on 22Rv1 (PSMA+) xenograft model demonstrated a good potency of the synthesized conjugate to inhibit tumor growth: the inhibition turned out to be more than 80% at a dose of 30 mg/kg. Pharmacokinetic parameters of conjugate distribution were analyzed. Also, it was found that PSMA-targeted docetaxel conjugate is less toxic than docetaxel itself, the decrease of molar acute toxicity in comparison with free docetaxel was up to 20%. Obtained conjugate PSMA-DOC is a good candidate for further expanded preclinical trials because of high antitumor activity, fewer side toxic effects and better solubility.

The mechanical properties of PC-3 tumor cells of human prostate cancer before and after exposure to substances acting on the actin cytoskeleton, microtubules, and cell nucleus were measured by scanning ion-conducting microscopy. Changes in local mechanical properties corresponding to the mechanisms of action of these substances were found

Primary physicochemical steps in microwounding of plants were investigated using electrochemical nano- and microprobes, with a focus on the role of oxygen in the wounding responses of individual plant cells. Electrochemical measurements of cell oxygen content were made with carbon-filled quartz micropipettes with platinum-coated tips (oxygen nanosensors). These novel platinum nanoelectrodes are useful for understanding cell oxygen metabolism and can be employed to study the redox biochemistry and biology of cells, tissues and organisms. We show here that microinjury of Chara corallina internodal cells with the tip of a glass micropipette is associated with a drastic decrease in oxygen concentration at the vicinity of the stimulation site. This decrease is reversible and lasts for up to 40 minutes. Membrane stretching, calcium influx, and cytoskeleton rearrangements were found to be essential for the localized oxygen depletion induced by cell wall microwounding. Inhibition of electron transport in chloroplasts or mitochondria did not affect the magnitude or timing of the observed response. In contrast, the inhibition of NADPH oxidase activity caused a significant reduction in the amplitude of the decrease in oxygen concentration. We suggest that the observed creation of localized anoxic conditions in response to cell wall puncture might be mediated by NADPH oxidase.

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is a suitable target for specific delivery of antitumor drugs and diagnostic agents due to its overexpression in prostate cancer cells. In the current work, we describe the design, synthesis, and biological evaluation of novel low-molecular PSMA ligands and conjugates with fluorescent dyes FAM-5, SulfoCy5, and SulfoCy7. In vitro evaluation of synthesized PSMA ligands on the activity of PSMA shows that the addition of aromatic amino acids into a linker structure leads to a significant increase in inhibition. The conjugates of the most potent ligand with FAM-5 as well as SulfoCy5 demonstrated high affinities to PSMA-expressing tumor cells in vitro. In vivo biodistribution in 22Rv1 xenografts in Balb/c nude mice of PSMA-SulfoCy5 and PSMA-SulfoCy7 conjugates with a novel PSMA ligand demonstrated good visualization of PSMA-expressing tumors. Also, the conjugate PSMA-SulfoCy7 demonstrated the absence of any explicit toxicity up to 87.9 mg/kg.

Herein, we describe the design, synthesis, and biological evaluation of novel betulin and N-acetyl-d-galactosamine (GalNAc) glycoconjugates and suggest them as targeted agents against hepatocellular carcinoma. We prepared six conjugates derived via the C-3 and C-28 positions of betulin with one or two saccharide ligands. These molecules demonstrate high affinity to the asialoglycoprotein receptor (ASGPR) of hepatocytes assessed by in silico modeling and surface plasmon resonance tests. Cytotoxicity studies in vitro revealed a bivalent conjugate with moderate activity, selectivity of action, and cytostatic properties against hepatocellular carcinoma cells HepG2. An additional investigation confirmed the specific engagement with HepG2 cells by the enhanced generation of reactive oxygen species. Stability tests demonstrated its lability to acidic media and to intracellular enzymes. Therefore, the selected bivalent conjugate represents a new potential agent targeted against hepatocellular carcinoma. Further extensive studies of the cellular uptake in vitro and the real-time microdistribution in the murine liver in vivo for fluorescent dye-labeled analogue showed its selective internalization into hepatocytes due to the presence of GalNAc ligand in comparison with reference compounds. The betulin and GalNAc glycoconjugates can therefore be considered as a new strategy for developing therapeutic agents based on natural triterpenoids.

In this work, we have developed covalent and low molecular weight docetaxel delivery systems based on conjugation with N-acetyl-d-galactosamine and studied their properties related to hepatocellular carcinoma cells. The resulting glycoconjugates have an excellent affinity to the asialoglycoprotein receptor (ASGPR) in the nanomolar range of concentrations and a high cytotoxicity level comparable to docetaxel. Likewise, we observed the 21-75-fold increase in water solubility in comparison with parent docetaxel and prodrug lability to intracellular conditions with half-life values from 25.5 to 42 h. We also found that the trivalent conjugate possessed selective toxicity against hepatoma cells vs control cell lines (20-35 times). The absence of such selectivity in the case of monovalent conjugates indicates the effect of ligand valency. Specific ASGPR-mediated cellular uptake of conjugates was proved in vitro using fluorescent-labeled analogues. In addition, we showed an enhanced generation of reactive oxygen species in the HepG2 cells, which could be inhibited by the natural ligand of ASGPR. Overall, the obtained results highlight the potential of ASGPR-directed cytostatic taxane drugs for selective therapy of hepatocellular carcinoma.

Reactive oxygen species (ROS) actively participate in many metabolic processes in liver cells, including cell proliferation and apoptosis. Intracellular ROS not only contribute in signaling, but also oxidize lipids, proteins, and DNA. As a result, ROS are involved in the development of many diseases. Lifetime visualization of ROS using sensors that are sensitive to the presence of free radicals is an urgent but difficult task due to short lifetimes of radicals and varied concentrations in different cell compartments. We synthesized lipid nanoparticles (LNP) with average diameters $100 \pm 10$ nm and two different ROS sensitive dyes (L-012 and hydrocyanine 5) for effective detection of ROS in liver cells. We demonstrated that L-012 sodium salt leaks from the nanoparticles, while hydrocy5 labeled LNP are stable and shows an increase of fluorescence in more than 5 times after reaction with hydrogen peroxide. We also validated animal model with increased ROS production by scanning ion-conductance microscopy. Both lipopolysaccharide (LPS) and carbon tetrachloride (CCl4) treatment caused intensive ROS burst in 24 h after injection. Using fluorescent tomography, we analyzed LNP biodistribution in Balb/c mice and observed its preferable accumulation in liver. Thus, incorporation of ROS-sensitive dyes into LNP can improve the stability of the sensors, minimize degradation during blood circulation, and increase its accumulation in the target organ. Creating delivery systems for lifetime ROS imaging can be a useful tool for studying changes in ROS in liver diseases.

Magnetic nanoparticle size is one of the key factors determining its magnetic and biological properties. Herein, we report a synthesis of magnetite uniform-sized cubic nanoparticles with 10, 15 and 20 nm edge lengths for magnetic resonance imaging (MRI). The structure and physicochemical properties of obtained magnetite nanocubes (MNCbs) were studied by multiple methods. MNCbs conjugated with Pluronic F-127 exhibited high colloidal stability in aqueous medium. Synthesized MNCbs demonstrated improved T2-relaxation comparing to commercially available contrast agents proving that these nanocubes can be used for MRI.

In this work, the effect of the new synthesized anticancer conjugates on the HepG2 and PC-3 cell lines was evaluated by determining ROS inside single cells using an electrochemical method.

Melanoma is one of the most aggressive and lethal form of cancer. Photodynamic therapy (PDT) is a clinically approved technique for cancer treatment, including non-melanoma skin cancer. However, the most of conventional photosensitizers are of low efficacy against melanoma due to the possible dark toxicity at high drug concentrations, melanin pigmentation, and induction of anti-oxidant defense mechanisms. In the current research we propose non-toxic flavin mononucleotide (FMN), which is a water-soluble form of riboflavin (vitamin B2) as a promising agent for photodynamic therapy of melanoma. We demonstrated selective accumulation of FMN in melanoma cells in vivo and in vitro in comparison with keratinocytes and fibroblasts. Blue light irradiation with dose 5 J/cm2 of melanoma cells pre-incubated with FMN led to cell death through apoptosis. Thus, the IC50 values of human melanoma A375, Mel IL, and Mel Z cells were in a range of FMN concentration 10–30 µM that can be achieved in tumor tissue under systemic administration. The efficiency of reactive oxygen species (ROS) generation under FMN blue light irradiation was measured in single melanoma cells by a label-free technique using an electrochemical nanoprobe in a real-time control manner. Melanoma xenograft regression in mice was observed as a result of intravenous injection of FMN followed by blue-light irradiation of tumor site. The inhibition of tumor growth was 85–90% within 50 days after PDT treatment.

Iron oxide nanoparticles have attracted a great deal of research interest and have been widely used in bioscience and clinical research including as contrast agents for magnetic resonance imaging, hyperthermia and magnetic field assisted radionuclide therapy. It is therefore important to develop methods, which can provide high-throughput screening of biological responses that can predict toxicity. The use of nanoelectrodes for single cell analysis can play a vital role in this process by providing relatively fast, comprehensive, and cost-effective assessment of cellular responses. We have developed a new method for in vitro study of the toxicity of magnetic nanoparticles (NP) based on the measurement of intracellular reactive oxygen species (ROS) by a novel nanoelectrode. Previous studies have suggested that ROS generation is frequently observed with NP toxicity. We have developed a stable probe for measuring intracellular ROS using platinized carbon nanoelectrodes with a cavity on the tip integrated into a micromanipulator on an upright microscope. Our results show a significant difference for intracellular levels of ROS measured in HEK293 and LNCaP cancer cells before and after exposure to 10 nm size iron oxide NP. These results are markedly different from ROS measured after cell incubation with the same concentration of NP using standard methods where no differences have been detected. In summary we have developed a label-free method for assessing nanoparticle toxicity using the rapid (less than 30 minutes) measurement of ROS with a novel nanoelectrode.

The applications of atomic force microscopy and the methods based on atomic force microscopy that can be useful in medical nanoanalytics have been reviewed. The main fields of possible application of scanning probe microscopy in medicine have been outlined. Among these are studying the resistance of bacterial cells to modern antibiotics and drugs, morphological analysis of blood components, trichology, nanotoxicology, DNA sequencing, and biocompatibility of medicinal materials. Examples of application of atomic force microscopy for studies in these fields have been considered, and prospects for its use in medicine have been demonstrated.

The study of nanomechanical cantilever systems is among the priority directions in the progression of nanotechnologies. Principally new ways of designing biosensors based on nanocantilever transducers are investigated, the effect of orientation of receptor immunoglobulin molecules in the sensor layer on the formation of lateral strain during complementary binding is examined for the first time, and unique techniques for creation of selective receptor transducers based on cantilevers are developed. The unique data of this study make it possible to state that, owing to the presence of 13-thiotridecane-1,1,2-triol molecules in the probe DNA layer, the lateral strain tends to increase during hybridization of complementary molecules. Theoretical predictions and experimental data are compared, and the effect of the formation of lateral strain in polymer layers on the bending of the cantilever transducer is revealed. The nature of lateral strain arising in films of biopolymers (proteins and DNA) during complementary binding (formation of the immune complex for protein molecules and hybridization for DNA) is ascertained.

We have described rapid, label free detection of Influenza A viruses with cantilever transducer modified with synthetic sialylglycopolymer as a receptor layer. Surface stress induced by binding of viruses to receptor layer was investigated as analytical signal. Synthetic sialylglycopolymer receptor layer can be implemented in nanoscale strain-gauge cantilever transducers for high sensitive viruse detection. Strain-gage transducers with such sensor layer demonstrate long lifetime, high sensitivity and regeneration possibility. Nanomechanical cantilever system with optical detector was used for surface stress measurements. We have shown that positive label free detection of Influenza A could be achieved at a concentration less than 106 viruses in ml, which is comparable to the detection sensitivity of polymerase chain reaction (PCR). However, in contrast to PCR, cantilever sensor detection was label free, in situ, and rapid (less than 30 min), potentially requiring minimal or no sample preparation.

We describe the rapid, label-free detection of Influenza A viruses using a cantilever transducer modified with a synthetic sialylglycopolymer receptor layer. Surface stresses induced by viruses binding to the receptor layer were used as the analytical signal. The synthetic sialylglycopolymer receptor layer can be used in nanoscale strain-gauge cantilever transducers for highly sensitive virus detection. Strain-gage transducers using such sensor layers exhibit long lifetimes, high sensitivities, and possible regeneration. Nanomechanical cantilever systems using optical detectors were used for the surface stress measurements. We demonstrated the positive, label-free detection of Influenza A at concentrations below 10(6) viruses per ml. In contrast to hemagglutination assays, cantilever sensors are label free, in situ, and rapid (less than 30 min), and they require minimal or nearly no sample preparation.

International audience; The measurement of key molecules in individual cells with minimal disruption to the biological milieu is the next frontier in single-cell analyses. Nanoscale devices are ideal analytical tools because of their small size and their potential for high spatial and temporal resolution recordings. Here, we report the fabrication of disk-shaped carbon nanoelectrodes whose radius can be precisely tuned within the range 5200 nm. The functionalization of the nanoelectrode with platinum allowed the monitoring of oxygen consumption outside and inside a brain slice. Furthermore, we show that nanoelectrodes of this type can be used to impale individual cells to perform electrochemical measurements within the cell with minimal disruption to cell function. These nanoelectrodes can be fabricated combined with scanning ion conductance microscopy probes, which should allow high resolution electrochemical mapping of species on or in living cells.

A new way to histidine-tag protein immobilization with a new type of terpyridine ligand, 4’-(12-mercaptododecanyloxy)-[2,2’;6’,2”]terpyridine, on gold coated cantilever has been proposed.

Here we describe a new instrument for surface stress measurements in monolayers of self-assembled films–thiol molecules on a cantilever surface. The device is constructed on the basis of an atomic force microscope. Our main interest was the relation between electrostatic forces and hydrogen bonds forces acting in the thiol monolayers. It was shown that sorption of the thiol monomers onto the cantilever surface causes its deflection. In the case when thiols with amino groups were used the cantilever bent towards the golden side of the cantilever, which has been covered by thiols. The monomers, which did not have amino groups, caused the bent in the opposite direction.

Mechanical properties of living cells determined by cytoskeletal elements play a crucial role in a wide range of biological functions. However, low-stress mapping of mechanical properties with nanoscale resolution but with a minimal effect on the fragile structure of cells remains difficult. Scanning Ion-Conductance Microscopy (SICM) for quantitative nanomechanical mapping (QNM) is based on intrinsic force interactions between nanopipettes and samples and has been previously suggested as a promising alternative to conventional techniques. In this work, we have provided an alternative estimation of intrinsic force and stress and demonstrated the possibility to perform qualitative and quantitative analysis of cell nanomechanical properties of a variety of living cells. Force estimation on decane droplets with well-known elastic properties, similar to living cells, revealed that the forces applied using a nanopipette are much smaller than in the case using atomic force microscopy. We have shown that we can perform nanoscale topography and QNM using a scanning procedure with no detectable effect on live cells, allowing long-term QNM as well as detection of nanomechanical properties under drug-induced alterations of actin filaments and microtubulin.