Electrochemical Nanopipette Sensor for In Vitro/In Vivo Detection of Cu2+ Ions.

In vitro/in vivo detection of copper ions is a challenging task but one which is important in the development of new approaches to the diagnosis and treatment of cancer and hereditary diseases such as Alzheimer's, Wilson's, etc. In this paper, we present a nanopipette sensor capable of measuring Cu2+ ions with a linear range from 0.1 to 10 µM in vitro and in vivo. Using the gold-modified nanopipette sensor with a copper chelating ligand, we evaluated the accumulation ability of the liposomal form of an anticancer Cu-containing complex at three levels of biological organization. First, we detected Cu2+ ions in a single cell model of human breast adenocarcinoma MCF-7 and in murine melanoma B16 cells. The insertion of the nanoelectrode did not result in leakage of the cell membrane. We then evaluated the distribution of the Cu-complex in MCF-7 tumor spheroids and found that the diffusion-limited accumulation was a function of the depth, typical for 3D culture. Finally, we demonstrated the use of the sensor for Cu2+ ion detection in the brain of an APP/PS1 transgenic mouse model of Alzheimer's disease and tumor-bearing mice in response to injection (2 mg kg-1) of the liposomal form of the anticancer Cu-containing complex. Enhanced stability and selectivity, as well as distinct copper oxidation peaks, confirmed that the developed sensor is a promising tool for testing various types of biological systems. In summary, this research has demonstrated a minimally invasive electrochemical technique with high temporal resolution that can be used for the study of metabolism of copper or copper-based drugs in vitro and in vivo.

Scanning Ion-Conductance Microscopy for Studying ß-Amyloid Aggregate Formation on Living Cell Surfaces.

ß-Amyloid aggregation on living cell surfaces is described as responsible for the neurotoxicity associated with different neurodegenerative diseases. It is suggested that the aggregation of ß-amyloid (Aß) 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. 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 the 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 Aß aggregate formation on living cell surfaces. However, it is well-known that the cytoskeleton state is highly connected to the intracellular level of reactive oxygen species (ROS). The effect of Aß leads to the induction of oxidative stress, actin polymerization, and stress fiber formation. We measured the reactive oxygen species levels inside single cells using platinum nanoelectrodes to demonstrate the connection of ROS and Young's modulus of cells. SICM can be successfully applied to studying the cytotoxicity mechanisms of Aß aggregates on living cell surfaces.

In Vitro/In Vivo Electrochemical Detection of Pt(II) Species.

The biodistribution of chemotherapy compounds within tumor tissue is one of the main challenges in the development of antineoplastic drugs, and techniques for simple, inexpensive, sensitive, and selective detection of various analytes in tumors are of great importance. In this paper we propose the use of platinized carbon nanoelectrodes (PtNEs) for the electrochemical detection of platinum-based drugs in various biological models, including single cells and tumor spheroids in vitro and inside solid tumors in vivo. We have demonstrated the quantitative direct detection of Pt(II) in breast adenocarcinoma MCF-7 cells treated with cisplatin and a cisplatin-based DNP prodrug. To realize the potential of this technique in advanced tumor models, we measured Pt(II) in 3D tumor spheroids in vitro and in tumor-bearing mice in vivo. The concentration gradient of Pt(II) species correlated with the distance from the sample surface in MCF-7 tumor spheroids. We then performed the detection of Pt(II) species in tumor-bearing mice treated intravenously with cisplatin and DNP. We found that there was deeper penetration of DNP in comparison to cisplatin. This research demonstrates a minimally invasive, real-time electrochemical technique for the study of platinum-based drugs.

Electrochemical Detection of a Novel Pt(IV) Prodrug with the Metronidazole Axial Ligand in the Hypoxic Area.

We report herein a Pt(IV) prodrug with metronidazole in axial positions Pt-Mnz. The nitroaromatic axial ligand was conjugated with a cisplatin scaffold to irreversibly reduce under hypoxic conditions, thereby retaining the Pt(IV) prodrug in the area of hypoxia. X-ray near-edge adsorption spectroscopy (XANES) on dried drug-preincubated tumor cell samples revealed a gradual release of cisplatin from the Pt-Mnz prodrug instead of rapid intracellular degradation. The ability of the prodrug to penetrate into three-dimensional (3D) spheroid cellular cultures was evaluated by a novel electrochemical assay via a platinum-coated carbon nanoelectrode, capable of single-cell measurements. Using a unique technique of electrochemical measurements in single tumor spheroids, we were able to both detect the real-time response of the axial ligand to hypoxia and establish the depth of penetration of the drug into the tumor model.

Synthesis and Preclinical Evaluation of Small-Molecule Prostate-Specific Membrane Antigen-Targeted Abiraterone Conjugate.

Prostate cancer is the second most common type of cancer among men. The main method of its treatment is androgen deprivation therapy, which has a wide range of side effects. One of the solutions to this challenge is the targeted delivery of drugs to prostate cancer cells. In this study, we performed the synthesis of a novel small-molecule PSMA-targeted conjugate based on abiraterone. Cytotoxicity, the induction of intracellular reactive oxygen species, and P450-cytochrome species inhibition were investigated for this conjugate PSMA-abiraterone. The conjugate demonstrated a preferential effect on prostate tumor cells, remaining inactive at up to 100 µM in human fibroblast cells. In addition, it revealed preferential efficacy, specifically on PSMA-expressing lines with a 65% tumor growth inhibition level on 22Rv1 (PSMA+) xenografts after 14-fold oral administration of PSMA-Abi at a single dose of 500 mg/kg (7.0 g/kg total dose) was observed. This compound showed significantly reduced acute toxicity with comparable efficacy compared to AbiAc.

Discovery of Bivalent GalNAc-Conjugated Betulin as a Potent ASGPR-Directed Agent against Hepatocellular Carcinoma.

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.

New Small-Molecule Glycoconjugates of Docetaxel and GalNAc for Targeted Delivery to Hepatocellular Carcinoma.

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.

Dispirooxindoles Based on 2-Selenoxo-Imidazolidin-4-Ones: Synthesis, Cytotoxicity and ROS Generation Ability.

A regio- and diastereoselective synthesis of two types of dispiro derivatives of 2-selenoxoimidazolidin-4-ones, differing in the position of the nitrogen atom in the central pyrrolidine ring of the spiro-fused system-namely, 2-selenoxodispiro[imidazolidine-4,3'-pyrrolidine-2',3″-indoline]-2″,5-diones (5a-h) and 2-senenoxodispiro[imidazolidine-4,3'-pyrrolidine-4',3″-indoline]-2″,5-diones (6a-m)-were developed based on a 1,3-dipolar cycloaddition of azomethine ylides generated from isatin and sarcosine or formaldehyde and sarcosine to 5-arylidene or 5-indolidene-2-selenoxo-tetrahydro-4H-imidazole-4-ones. Selenium-containing dispiro indolinones generally exhibit cytotoxic activity near to the activity of the corresponding oxygen and sulfur-containing derivatives. Compounds 5b, 5c, and 5e demonstrated considerable in vitro cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) test (concentration of compounds that caused 50% death of cells (CC50) 7.6-8.7 µM) against the A549 cancer cell line with the VA13/A549 selectivity index 5.2-6.9; some compounds (5 and 6) increased the level of intracellular reactive oxygen species (ROS) in the experiment on A549 and PC3 cells using platinized carbon nanoelectrode. The tests for p53 activation for compounds 5 and 6 on the transcriptional reporter suggest that the investigated compounds can only have an indirect p53-dependent mechanism of action. For the compounds 5b, 6b, and 6l, the ROS generation may be one of the significant mechanisms of their cytotoxic action.

In Vitro and In Vivo Electrochemical Measurement of Reactive Oxygen Species After Treatment with Anticancer Drugs.

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.

Prolonged oxygen depletion in microwounded cells of Chara corallina detected with novel oxygen nanosensors.

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.

Effect of LDL Extracted from Human Plasma on Membrane Stiffness in Living Endothelial Cells and Macrophages via Scanning Ion Conductance Microscopy.

Mechanical properties of living cells play a crucial role in a wide range of biological functions and pathologies, including atherosclerosis. We used low-stress Scanning Ion-Conductance Microscopy (SICM) correlated with confocal imaging and demonstrated the topographical changes and mechanical properties alterations in EA.hy926 and THP-1 exposed to LDL extracted from CVD patients' blood samples. We show that the cells stiffened in the presence of LDL, which also triggered caveolae formation. Endothelial cells accumulated less cholesterol in the form of lipid droplets in comparison to THP-1 cells based on fluorescence intensity data and biochemical analysis; however, the effect on Young's modulus is higher. The cell stiffness is closely connected to the distribution of lipid droplets along the z-axis. In conclusion, we show that the sensitivity of endothelial cells to LDL is higher compared to that of THP-1, triggering changes in the cytoskeleton and membrane stiffness which may result in the increased permeability of the intima layer due to loss of intercellular connections and adhesion.

Light-Responsive Pt(IV) Prodrugs with Controlled Photoactivation and Low Dark Toxicity.

Light-induced release of cisplatin from Pt(IV) prodrugs represents a promising approach for precise control over the antiproliferative activity of Pt-based chemotherapeutic drugs. This method has the potential to overcome crucial drawbacks of conventional cisplatin therapy, such as high general toxicity toward healthy organs and tissues. Herein, we report two Pt(IV) prodrugs with BODIPY-based photoactive ligands Pt-1 and Pt-2, which were designed using carbamate and triazole linkers, respectively. Both prodrugs demonstrated the ability to release cisplatin under blue light irradiation without the requirement of an external reducing agent. Dicarboxylated Pt-2 prodrug turned out to be more stable in the dark and more sensitive to light than its monocarbamate Pt-1 counterpart; these observations were explained using DFT calculations. The investigation of the photoreduction mechanism of Pt-1 and Pt-2 prodrugs using DFT modeling and ΔG0 PET estimation suggests that the photoinduced electron transfer from the singlet excited state of the BODIPY axial ligand to the Pt(IV) center is the key step in the light-induced release of cisplatin from the complexes. Cytotoxicity studies demonstrated that both prodrugs were nontoxic in the dark and toxic to MCF-7 cells under low-dose irradiation with blue light, and the observed effect was solely due to the cisplatin release from the Pt(IV) prodrugs. Our research presents an elegant synthetic approach to light-activated Pt(IV) prodrugs and presents findings that may contribute to the future rational design of photoactivatable Pt(IV) prodrugs.

Analytical Models for Measuring the Mechanical Properties of Yeast.

The mechanical properties of yeast play an important role in many biological processes, such as cell division and growth, maintenance of internal pressure, and biofilm formation. In addition, the mechanical properties of cells can indicate the degree of damage caused by antifungal drugs, as the mechanical parameters of healthy and damaged cells are different. Over the past decades, atomic force microscopy (AFM) and micromanipulation have become the most widely used methods for evaluating the mechanical characteristics of microorganisms. In this case, the reliability of such an estimate depends on the choice of mathematical model. This review presents various analytical models developed in recent years for studying the mechanical properties of both cells and their individual structures. The main provisions of the applied approaches are described along with their limitations and advantages. Attention is paid to the innovative method of low-invasive nanomechanical mapping with scanning ion-conductance microscopy (SICM), which is currently starting to be successfully used in the discovery of novel drugs acting on the yeast cell wall and plasma membrane.

Recent Advances in Nanopore Technology for Copper Detection and Their Potential Applications.

Recently, nanopore technology has emerged as a promising technique for the rapid, sensitive, and selective detection of various analytes. In particular, the use of nanopores for the detection of copper ions has attracted considerable attention due to their high sensitivity and selectivity. This review discusses the principles of nanopore technology and its advantages over conventional techniques for copper detection. It covers the different types of nanopores used for copper detection, including biological and synthetic nanopores, and the various mechanisms used to detect copper ions. Furthermore, this review provides an overview of the recent advancements in nanopore technology for copper detection, including the development of new nanopore materials, improvements in signal amplification, and the integration of nanopore technology with other analytical methods for enhanced detection sensitivity and accuracy. Finally, we summarize the extensive applications, current challenges, and future perspectives of using nanopore technology for copper detection, highlighting the need for further research in the field to optimize the performance and applicability of the technique.

Scanning Ion-Conductance Microscopy for Studying Mechanical Properties of Neuronal Cells during Local Delivery of Glutamate.

Mechanical properties of neuronal cells have a key role for growth, generation of traction forces, adhesion, migration, etc. Mechanical properties are regulated by chemical signaling, neurotransmitters, and neuronal ion exchange. Disturbance of chemical signaling is accompanied by several diseases such as ischemia, trauma, and neurodegenerative diseases. It is known that the disturbance of chemical signaling, like that caused by glutamate excitotoxicity, leads to the structural reorganization of the cytoskeleton of neuronal cells and the deviation of native mechanical properties. Thus, to investigate the mechanical properties of living neuronal cells in the presence of glutamate, it is crucial to use noncontact and low-stress methods, which are the advantages of scanning ion-conductance microscopy (SICM). Moreover, a nanopipette may be used for the local delivery of small molecules as well as for a probe. In this work, SICM was used as an advanced technique for the simultaneous local delivery of glutamate and investigation of living neuronal cell morphology and mechanical behavior caused by an excitotoxic effect of glutamate.

Single-Cell Analysis with Silver-Coated Pipette by Combined SERS and SICM.

The study of individual cell processes that occur both on their surface and inside is highly interesting for the development of new medical drugs, cytology and cell technologies. This work presents an original technique for fabricating the silver-coated pipette and its use for the cell analysis by combination with surface-enhanced Raman spectroscopy (SERS) and scanning ion-conducting microscopy (SICM). Unlike the majority of other designs, the pipette opening in our case remains uncovered, which is important for SICM. SERS-active Ag nanoparticles on the pipette surface are formed by vacuum-thermal evaporation followed by annealing. An array of nanoparticles had a diameter on the order of 36 nm and spacing of 12 nm. A two-particle model based on Laplace equations is used to calculate a theoretical enhancement factor (EF). The surface morphology of the samples is investigated by scanning electron microscopy while SICM is used to reveal the surface topography, to evaluate Young's modulus of living cells and to control an injection of the SERS-active pipettes into them. A Raman microscope-spectrometer was used to collect characteristic SERS spectra of cells and cell components. Local Raman spectra were obtained from the cytoplasm and nucleus of the same HEK-293 cancer cell. The EF of the SERS-active pipette was 7 × 105. As a result, we demonstrate utilizing the silver-coated pipette for both the SICM study and the molecular composition analysis of cytoplasm and the nucleus of living cells by SERS. The probe localization in cells is successfully achieved.

Scanning ion-conductance microscopy technique for studying the topography and mechanical properties of Candida parapsilosis yeast microorganisms.

Super-resolution microscopy is widely used in the development of novel antimicrobial testing in vitro. In the presented work, a scanning protocol was developed by the method of scanning ion-conducting microscopy (SICM), which makes it possible to study microorganisms without rigid fixation and in saline, obtaining an index map of nanosized structures. The effect of azole and echinocandins drugs on the morphology and mechanical properties of Candida parapsilosis yeast was studied. The findings are consistent with previously proposed drug mechanisms and reports that have examined antifungal agents using AFM, SEM, and TEM. We have shown that the SICM method is capable of scanning and detecting the nanomechanical properties of yeast non-invasively.

ROS Production by a Single Neutrophil Cell and Neutrophil Population upon Bacterial Stimulation.

The reactive oxygen species (ROS) production by a single neutrophil after stimulation with S. aureus and E. coli was estimated by an electrochemical amperometric method with a high time resolution. This showed significant variability in the response of a single neutrophil to bacterial stimulation, from a "silent cell" to a pronounced response manifested by a series of chronoamperometric spikes. The amount of ROS produced by a single neutrophil under the influence of S. aureus was 5.5-fold greater than that produced under the influence of E. coli. The response of a neutrophil granulocyte population to bacterial stimulation was analyzed using luminol-dependent biochemiluminescence (BCL). The stimulation of neutrophils with S. aureus, as compared to stimulation with E. coli, caused a total response in terms of ROS production that was seven-fold greater in terms of the integral value of the light sum and 13-fold greater in terms of the maximum peak value. The method of ROS detection at the level of a single cell indicated the functional heterogeneity of the neutrophil population, but the specificity of the cellular response to different pathogens was the same at the cellular and population levels.

Investigation of the Antifungal and Anticancer Effects of the Novel Synthesized Thiazolidinedione by Ion-Conductance Microscopy.

In connection with the emergence of new pathogenic strains of Candida, the search for more effective antifungal drugs becomes a challenge. Part of the preclinical trials of such drugs can be carried out using the innovative ion-conductance microscopy (ICM) method, whose unique characteristics make it possible to study the biophysical characteristics of biological objects with high accuracy and low invasiveness. We conducted a study of a novel synthesized thiazolidinedione's antimicrobial (for Candida spp.) and anticancer properties (on samples of the human prostate cell line PC3), and its drug toxicity (on a sample of the human kidney cell line HEK293). We used a scanning ion-conductance microscope (SICM) to obtain the topography and mechanical properties of cells and an amperometric method using Pt-nanoelectrodes to register reactive oxygen species (ROS) expression. All data and results are obtained and presented for the first time.

Liposomal form of 2-alkylthioimidazolone-based copper complexes for combined cancer therapy.

Aim: To develop an optimized approach for encapsulating a 2-alkylthioimidazolone-based copper coordination compound within liposomes, which could offer treatment of cancer and bacterial infections by reactive oxygen species generation toxicity mechanisms. Materials & methods: For drug-loaded liposome preparation, lipids and drug mixture in organic solvents was injected into copper salt solution, forming a coordination compound simultaneously embedded in the lipid bilayer. In vitro tests were performed on MCF7 and MDA-MB-231 breast cancer cells. Results: Liposomes had a loading capacity of up to 1.75% (molar drug-to-lipid ratio). In vitro tests showed increased viability and accumulation of the liposomal formulation compared with free drug as well as lack of cytotoxicity in hepatocytes. Conclusion: This optimized technique for encapsulating large copper complexes in liposomes could be used to improve their delivery and better treat cancer and bacterial infections.

This work introduces a new technique for copper-containing drugs encapsulation in a drug-delivery system. The drug, a promising copper compound, is embedded in lipid nanovesicles ­ tiny fat particles ­ for intravenous injection. In addition to chemical characterization of the obtained drug form, tests on cancer cells showed a noticeable effect, whereas healthy cell types were not harmed. Copper possesses not only anticancer effects but also antimicrobial properties, which are also shown by the drug form, and a test of combined suppression of cancer cell lines and bacteria was successful. Hence, the obtained drug form has the potential for dual treatment of cancer and bacterial infections.