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.

Changes in ROS/RNS Levels in Endothelial Cells in Experimental Bacteremia.

A high-precision system was developed for the quantification of biological analytes in single cells (reactive oxygen species (ROS) and reactive nitrogen species (RNS)) based on the electrochemical amperometric method. The efficacy of this system was evaluated using an experimental bacteremia model. Endothelial cells exhibited increased ROS/RNS production when stimulated by Staphylococcus aureus. However, they remained inactive when exposed to either unprimed or primed neutrophils that had been pre-incubated with bacteria. It is noteworthy that the sequential stimulation of endothelial cells with bacteria followed by neutrophils resulted in a significant increase in the ROS/RNS level, which demonstrated a correlation with the number of neutrophils in contact with the endothelial cells. These results highlight the potential of our system to quantitatively assess ROS/RNS dynamics in complex biological systems. They also offer insights into the interplay between various cellular components in experimental bacteremia.

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.

An Open-Source Wireless Electrophysiology System for In Vivo Neuronal Activity Recording in the Rodent Brain: 2.0.

Current trends in neurobiological research focus on analyzing complex interactions within brain structures. To conduct relevant experiments, it is often essential to employ animals with unhampered mobility and utilize electrophysiological equipment capable of wirelessly transmitting data. In prior research, we introduced an open-source wireless electrophysiology system to surmount these challenges. Nonetheless, this prototype exhibited several limitations, such as a hefty weight for the wireless module, redundant system components, a diminished sampling rate, and limited battery longevity. In this study, we unveil an enhanced version of the open-source wireless electrophysiology system, tailored for in vivo monitoring of neural activity in rodent brains. This new system has been successfully tested in real-time recordings of in vivo neural activity. Consequently, our development offers researchers a cost-effective and proficient tool for studying complex brain functions.

Positive Allosteric Modulators of SERCA Pump Restore Dendritic Spines and Rescue Long-Term Potentiation Defects in Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects memory formation and storage processes. Dysregulated neuronal calcium (Ca2+) has been identified as one of the key pathogenic events in AD, and it has been suggested that pharmacological agents that stabilize Ca2+ neuronal signaling can act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+-stabilizing agents and exhibit neuroprotective properties. In the present study, we evaluated effects of a set of novel SERCA PAM agents on the rate of Ca2+ extraction from the cytoplasm of the HEK293T cell line, on morphometric parameters of dendritic spines of primary hippocampal neurons in normal conditions and in conditions of amyloid toxicity, and on long-term potentiation in slices derived from 5xFAD transgenic mice modeling AD. Several SERCA PAM compounds demonstrated neuroprotective properties, and the compound NDC-9009 showed the best results. The findings in this study support the hypothesis that the SERCA pump is a potential therapeutic target for AD treatment and that NDC-9009 is a promising lead molecule to be used in the development of disease-modifying agents for AD.

Recent Advances in 64Cu/67Cu-Based Radiopharmaceuticals.

Copper-64 (T1/2 = 12.7 h) is a positron and beta-emitting isotope, with decay characteristics suitable for both positron emission tomography (PET) imaging and radiotherapy of cancer. Copper-67 (T1/2 = 61.8 h) is a beta and gamma emitter, appropriate for radiotherapy ß-energy and with a half-life suitable for single-photon emission computed tomography (SPECT) imaging. The chemical identities of 64Cu and 67Cu isotopes allow for convenient use of the same chelating molecules for sequential PET imaging and radiotherapy. A recent breakthrough in 67Cu production opened previously unavailable opportunities for a reliable source of 67Cu with high specific activity and purity. These new opportunities have reignited interest in the use of copper-containing radiopharmaceuticals for the therapy, diagnosis, and theranostics of various diseases. Herein, we summarize recent (2018-2023) advances in the use of copper-based radiopharmaceuticals for PET, SPECT imaging, radiotherapy, and radioimmunotherapy.

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.

Intravital electrochemical nanosensor as a tool for the measurement of reactive oxygen/nitrogen species in liver diseases.

Reactive oxygen/nitrogen species (ROS/RNS) are formed during normal cellular metabolism and contribute to its regulation, while many pathological processes are associated with ROS/RNS imbalances. Modern methods for measuring ROS/RNS are mainly based on the use of inducible fluorescent dyes and protein-based sensors, which have several disadvantages for in vivo use. Intravital electrochemical nanosensors can be used to quantify ROS/RNS with high sensitivity without exogenous tracers and allow dynamic ROS/RNS measurements in vivo. Here, we developed a method for quantifying total ROS/RNS levels in the liver and evaluated our setup in live mice using three common models of liver disease associated with ROS activation: acute liver injury with CCl4, partial hepatectomy (HE), and induced hepatocellular carcinoma (HCC). We have demonstrated using intravital electrochemical detection that any exposure to the peritoneum in vivo leads to an increase in total ROS/RNS levels, from a slight increase to an explosion, depending on the procedure. Analysis of the total ROS/RNS level in a partial hepatectomy model revealed oxidative stress, both in mice 24 h after HE and in sham-operated mice. We quantified dose-dependent ROS/RNS production in CCl4-induced injury with underlying neutrophil infiltration and cell death. We expect that in vivo electrochemical measurements of reactive oxygen/nitrogen species in the liver may become a routine approach that provides valuable data in research and preclinical studies.

Astrocyte Activation Markers.

Astrocytes are the most common type of glial cells that provide homeostasis and protection of the central nervous system. Important specific characteristic of astrocytes is manifestation of morphological heterogeneity, which is directly dependent on localization in a particular area of the brain. Astrocytes can integrate into neural networks and keep neurons active in various areas of the brain. Moreover, astrocytes express a variety of receptors, channels, and membrane transporters, which underlie their peculiar metabolic activity, and, hence, determine plasticity of the central nervous system during development and aging. Such complex structural and functional organization of astrocytes requires the use of modern methods for their identification and analysis. Considering the important fact that determining the most appropriate marker for polymorphic and multiple subgroups of astrocytes is of decisive importance for studying their multifunctionality, this review presents markers, modern imaging techniques, and identification of astrocytes, which comprise a valuable resource for studying structural and functional properties of astrocytes, as well as facilitate better understanding of the extent to which astrocytes contribute to neuronal activity.

In Vivo Penetrating Microelectrodes for Brain Electrophysiology.

In recent decades, microelectrodes have been widely used in neuroscience to understand the mechanisms behind brain functions, as well as the relationship between neural activity and behavior, perception and cognition. However, the recording of neuronal activity over a long period of time is limited for various reasons. In this review, we briefly consider the types of penetrating chronic microelectrodes, as well as the conductive and insulating materials for microelectrode manufacturing. Additionally, we consider the effects of penetrating microelectrode implantation on brain tissue. In conclusion, we review recent advances in the field of in vivo microelectrodes.

Aß-Targeting Bifunctional Chelators (BFCs) for Potential Therapeutic and PET Imaging Applications.

Currently, more than 55 million people live with dementia worldwide, and there are nearly 10 million new cases every year. Alzheimer's disease (AD) is the most common neurodegenerative disease resulting in personality changes, cognitive impairment, memory loss, and physical disability. Diagnosis of AD is often missed or delayed in clinical practice due to the fact that cognitive deterioration occurs already in the later stages of the disease. Thus, methods to improve early detection would provide opportunities for early treatment of disease. All FDA-approved PET imaging agents for Aß plaques use short-lived radioisotopes such as 11C (t1/2 = 20.4 min) and 18F (t1/2 = 109.8 min), which limit their widespread use. Thus, a novel metal-based imaging agent for visualization of Aß plaques is of interest, due to the simplicity of its synthesis and the longer lifetimes of its constituent isotopes. We have previously summarized a metal-containing drug for positron emission tomography (PET), magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT) imaging of Alzheimer's disease. In this review, we have summarized a recent advance in design of Aß-targeting bifunctional chelators for potential therapeutic and PET imaging applications, reported after our previous review.

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.

Variation in tumor pH affects pH-triggered delivery of peptide-modified magnetic nanoparticles.

Acidification of the extracellular matrix, an intrinsic characteristic of many solid tumors, is widely exploited for physiologically triggered delivery of contrast agents, drugs, and nanoparticles to tumor. However, pH of tumor microenvironment shows intra- and inter-tumor variation. Herein, we investigate the impact of this variation on pH-triggered delivery of magnetic nanoparticles (MNPs) modified with pH-(low)-insertion peptide (pHLIP). Fluorescent flow cytometry, laser confocal scanning microscopy and transmission electron microscopy data proved that pHLIP-conjugated MNPs interacted with 4T1 cells in two-dimensional culture and in spheroids more effectively at pH 6.4 than at pH 7.2, and entered the cell via clathrin-independent endocytosis. The accumulation efficiency of pHLIP-conjugated MNPs in 4T1 tumors after their intravenous injection, monitored in vivo by magnetic resonance imaging, showed variation. Analysis of the tumor pH profiles recorded with implementation of original nanoprobe pH sensor, revealed obvious correlation between pH measured in the tumor with the amount of accumulated MNPs.

An Open-Source Wireless Electrophysiological Complex for In Vivo Recording Neuronal Activity in the Rodent's Brain.

Multi-electrode arrays (MEAs) are a widely used tool for recording neuronal activity both in vitro/ex vivo and in vivo experiments. In the last decade, researchers have increasingly used MEAs on rodents in vivo. To increase the availability and usability of MEAs, we have created an open-source wireless electrophysiological complex. The complex is scalable, recording the activity of neurons in the brain of rodents during their behavior. Schematic diagrams and a list of necessary components for the fabrication of a wireless electrophysiological complex, consisting of a base charging station and wireless wearable modules, are presented.

Pt(IV) Prodrugs with NSAIDs as Axial Ligands.

A chemo-anti-inflammatory strategy is of interest for the treatment of aggressive cancers. The platinum (IV) prodrug with non-steroidal anti-inflammatory drugs (NSAIDs) as axial ligands is designed to efficiently enter tumor cells due to high lipophilicity and release the cytotoxic metabolite and NSAID intracellularly, thereby reducing side effects and increasing the therapeutic efficacy of platinum chemotherapy. Over the last 7 years, a number of publications have been devoted to the design of such Pt(IV) prodrugs in combination with anti-inflammatory chemotherapy, with high therapeutic efficacy in vitro and In vivo. In this review, we summarize the studies devoted to the development of Pt(IV) prodrugs with NSAIDs as axial ligands, the study of the mechanism of their cytotoxic action and anti-inflammatory activity, the structure-activity ratio, and therapeutic efficacy.

Optogenetic Activation of Astrocytes-Effects on Neuronal Network Function.

Optogenetics approach is used widely in neurobiology as it allows control of cellular activity with high spatial and temporal resolution. In most studies, optogenetics is used to control neuronal activity. In the present study optogenetics was used to stimulate astrocytes with the aim to modulate neuronal activity. To achieve this goal, light stimulation was applied to astrocytes expressing a version of ChR2 (ionotropic opsin) or Opto-α1AR (metabotropic opsin). Optimal optogenetic stimulation parameters were determined using patch-clamp recordings of hippocampal pyramidal neurons' spontaneous activity in brain slices as a readout. It was determined that the greatest increase in the number of spontaneous synaptic currents was observed when astrocytes expressing ChR2(H134R) were activated by 5 s of continuous light. For the astrocytes expressing Opto-α1AR, the greatest response was observed in the pulse stimulation mode (T = 1 s, t = 100 ms). It was also observed that activation of the astrocytic Opto-a1AR but not ChR2 results in an increase of the fEPSP slope in hippocampal neurons. Based on these results, we concluded that Opto-a1AR expressed in hippocampal astrocytes provides an opportunity to modulate the long-term synaptic plasticity optogenetically, and may potentially be used to normalize the synaptic transmission and plasticity defects in a variety of neuropathological conditions, including models of Alzheimer's disease and other neurodegenerative disorders.

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.