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.

Neutrophil as a Carrier for Cancer Nanotherapeutics: A Comparative Study of Liposome, PLGA, and Magnetic Nanoparticles Delivery to Tumors.

Insufficient drug accumulation in tumors is still a major concern for using cancer nanotherapeutics. Here, the neutrophil-based delivery of three nanoparticle types-liposomes, PLGA, and magnetite nanoparticles-was assessed both in vitro and in vivo. Confocal microscopy and a flow cytometry analysis demonstrated that all the studied nanoparticles interacted with neutrophils from the peripheral blood of mice with 4T1 mammary adenocarcinoma without a significant impact on neutrophil viability or activation state. Intravital microscopy of the tumor microenvironment showed that the neutrophils did not engulf the liposomes after intravenous administration, but facilitated nanoparticle extravasation in tumors through micro- and macroleakages. PLGA accumulated along the vessel walls in the form of local clusters. Later, PLGA nanoparticle-loaded neutrophils were found to cross the vascular barrier and migrate towards the tumor core. The magnetite nanoparticles extravasated in tumors both via spontaneous macroleakages and on neutrophils. Overall, the specific type of nanoparticles largely determined their behavior in blood vessels and their neutrophil-mediated delivery to the tumor. Since neutrophils are the first to migrate to the site of inflammation, they can increase nanodrug delivery effectiveness for nanomedicine application.

Synthesis of Prostate-Specific Membrane Antigen-Targeted Bimodal Conjugates of Cytotoxic Agents and Antiandrogens and Their Comparative Assessment with Monoconjugates.

Prostate cancer is the second most common cancer among men. We designed and synthesized new ligands targeting prostate-specific membrane antigen and suitable for bimodal conjugates with diagnostic and therapeutic agents. In vitro studies of the affinity of the synthesized compounds to the protein target have been carried out. Based on these ligands, a series of bimodal conjugates with a combination of different mitosis inhibitors and antiandrogenic drugs were synthesized. The cytotoxicity of the compounds obtained in vitro was investigated on three different cell lines. The efficacy of the two obtained conjugates was evaluated in vivo in xenograft models of prostate cancer. These compounds have been shown to be highly effective in inhibiting the growth of PSMA-expressing tumors.

Pt(IV) Prodrugs with Non-Steroidal Anti-inflammatory Drugs in the Axial Position.

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 5-10 were designed, which showed superior antiproliferative activity compared to cisplatin as well as an ability to overcome tumor cell line resistance to cisplatin. By tuning the drug lipophilicity via variation of the axial ligands, the most potent Pt(IV) prodrug 7 was obtained, with an enhanced cellular accumulation of up to 153-fold that of cisplatin and nanomolar cytotoxicity both in 2D and 3D cell cultures. Pt2+ species were detected at different depths of MCF-7 spheroids after incubation with Pt(IV) prodrugs using a Pt-coated carbon nanoelectrode. Cisplatin accumulation in vivo in the murine mammary EMT6 tumor tissue of BALB/c mice after Pt(IV) prodrug injection was proved electrochemically as well. The drug tolerance study on BALB/c mice showed good tolerance of 7 in doses up to 8 mg/kg.

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.

Room temperature synthesized solid solution AuFe nanoparticles and their transformation into Au/Fe Janus nanocrystals.

Solid solution AuFe nanoparticles were synthesized for the first time under ambient conditions by an adapted method previously established for the Fe3O4-Au core-shell morphology. These AuFe particles preserved the fcc structure of Au incorporated with paramagnetic Fe atoms. The metastable AuFe can be segregated by transformation into Janus Au/Fe particles with bcc Fe and fcc Au upon annealing. The ferromagnetic Fe was epitaxially grown on low index fcc Au planes. This preparation route delivers new perspective materials for magnetoplasmonics and biomedical applications and suggests the reconsideration of existing protocols for magnetite-gold core-shell synthesis.

Synthesis and Biological Evaluation of PSMA Ligands with Aromatic Residues and Fluorescent Conjugates Based on Them.

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.

Neutrophil and Nanoparticles Delivery to Tumor: Is It Going to Carry That Weight?

The application of cell carriers for transporting nanodrugs to the tumor draws much attention as the alternative to the passive drug delivery. In this concept, the neutrophil (NΦ) is of special interest as this cell is able to uptake nanoparticles (NPs) and cross the vascular barrier in response to tumor signaling. There is a growing body of literature describing NP-NΦ interactions in vitro and in vivo that demonstrates the opportunity of using these cells to improve the efficacy of cancer therapy. However, a number of conceptual and technical issues need to be resolved for translating the technology into clinics. The current review summarizes the recent advances and challenges associated with NP-NΦ interactions, with the special focus on the complex interplay between the NP internalization pathways and the modulation of NΦ activity, and its potential consequences for nanodrug delivery.

Cobalt Ferrite Nanoparticles for Tumor Therapy: Effective Heating versus Possible Toxicity.

Magnetic nanoparticles (MNPs) are widely considered for cancer treatment, in particular for magnetic hyperthermia (MHT). Thereby, MNPs are still being optimized for lowest possible toxicity on organisms while the magnetic properties are matched for best heating capabilities. In this study, the biocompatibility of 12 nm cobalt ferrite MNPs, functionalized with citrate ions, in different dosages on mice and rats of both sexes was investigated for 30 days after intraperitoneal injection. The animals' weight, behavior, and blood cells changes, as well as blood biochemical parameters are correlated to histological examination of organs revealing that cobalt ferrite MNPs do not have toxic effects at concentrations close to those used previously for efficient MHT. Moreover, these MNPs demonstrated high specific loss power (SLP) of about 400 W g-1. Importantly the MNPs retained their magnetic properties inside tumor tissue after intratumoral administration for several MHT cycles within three days. Thus, cobalt ferrite MNPs represent a perspective platform for tumor therapy by MHT due to their ability to provide effective heating without exerting a toxic effect on the organism. This opens up new avenues for smaller MNPs sizes while their heating efficiency is maintained.

Intravital imaging of liposome behavior upon repeated administration: A step towards the development of liposomal companion diagnostic for cancer nanotherapy.

Accumulation of liposomal drugs into human tumors has substantial variability influencing the probability of positive response to the therapy. Therefore, it becomes very important to identify the eligibility of patients for various treatment options. The existing strategies of tumor stratification using companion diagnostics are based on the assumption that the initial and subsequent doses of nanoparticles (NP) behave in a sufficiently similar manner to enable a valuable prognosis. Here, we use a combination of in vivo imaging techniques to validate the applicability of magnetic liposomes (ML) as a reliable tool to predict whether or not the tumor would respond to nanomedicine therapy. The results demonstrated that liposome biodistribution, interactions with immune cells, and extravasation behavior in tumors were not affected by the pretreatment with liposomes 24 h prior to the repeat dosing. Co-administration of liposomal doxorubicin (DXR) and liposomes loaded with maghemite NP resulted in a high colocalization rate between two nanomedicines in tumors suggesting that neither contrast agent, nor chemotherapeutics altered biodistribution of liposomes. Based on magnetic resonance imaging of 4T1 tumors performed before and 6 h after ML treatment, animals were classified into high and low accumulation subgroups. Higher ML deposition in tumors was associated with a reduction in lesion size and enhanced survival in animals treated with liposomal DXR, but not with DXR alone. Given that liposomes are the most numerous class of clinically approved nanomedicines the development of safe and cost-effective liposomal companion diagnostic suitable for non-invasive imaging is of paramount importance for improving the efficacy of cancer therapy.

Multifunctional Fe3O4-Au Nanoparticles for the MRI Diagnosis and Potential Treatment of Liver Cancer.

Heterodimeric nanoparticles comprising materials with different functionalities are of great interest for fundamental research and biomedical/industrial applications. In this work, Fe3O4-Au nano-heterostructures were synthesized by a one-step thermal decomposition method. The hybrid nanoparticles comprise a highly crystalline 12 nm magnetite octahedron decorated with a single noble metal sphere of 6 nm diameter. Detailed analysis of the nanoparticles was performed by UV-visible spectroscopy, magnetometry, calorimetry and relaxometry studies. The cytotoxic effect of the nanoparticles in the human hepatic cell line Huh7 and PLC/PRF/5-Alexander was also assessed. These Fe3O4-Au bifunctional nanoparticles showed no significant cytotoxicity in these two cell lines. The nanoparticles showed a good theranostic potential for liver cancer treatment, since the r2 relaxivity (166.5 mM-1·s-1 and 99.5 mM-1·s-1 in water and HepG2 cells, respectively) is higher than the corresponding values for commercial T2 contrast agents and the Specific Absorption Rate (SAR) value obtained (227 W/gFe) is enough to make them suitable as heat mediators for Magnetic Fluid Hyperthermia. The gold counterpart can further allow the conjugation with different biomolecules and the optical sensing.

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.

Synthesis and Evaluation of New Trivalent Ligands for Hepatocyte Targeting via the Asialoglycoprotein Receptor.

Since the asialoglycoprotein receptor (also known as the "Ashwell-Morell receptor" or ASGPR) was discovered as the first cellular mammalian lectin, numerous drug delivery systems have been developed and several gene delivery systems associated with multivalent ligands for liver disease targeting are undergoing clinical trials. The success of these systems has facilitated the further study of new ligands with comparable or higher affinity and less synthetic complexity. Herein, we designed two novel trivalent ligands based on the esterification of tris(hydroxymethyl) aminomethane (TRIS) followed by the azide-alkyne Huisgen cycloaddition with azido N-acetyl-d-galactosamine. The presented triazolyl glycoconjugates exhibited good binding to ASGPR, which was predicted using in silico molecular docking and assessed by a surface plasmon resonance (SPR) technique. Moreover, we demonstrated the low level of in vitro cytotoxicity, as well as the optimal spatial geometry and the required amphiphilic balance, for new, easily accessible ligands. The conjugate of a new ligand with Cy5 dye exhibited selective penetration into HepG2 cells in contrast to the ASGPR-negative PC3 cell line.

Temperature-controlled magnetic nanoparticles hyperthermia inhibits primary tumor growth and metastases dissemination.

Magnetic hyperthermia (MHT) is a promising approach for cancer therapy. However, a systematic MHT characterization as function of temperature on the therapeutic efficiency is barely analyzed. Here, we first perform comparative temperature-dependent analysis of the cobalt ferrite nanoparticles-mediated MHT effectiveness in two murine tumors models - breast (4T1) and colon (CT26) cancer in vitro and in vivo. The overall MHT killing capacity in vitro increased with the temperature and CT26 cells were more sensitive than 4T1 when heated to 43 °C. Well in line with the in vitro data, such heating cured non-metastatic CT26 tumors in vivo, while only inhibiting metastatic 4T1 tumor growth without improving the overall survival. High-temperature MHT (>47 °C) resulted in complete 4T1 primary tumor clearance, 25-40% long-term survival rates, and, importantly, more effective prevention of metastasis comparing to surgical extraction. Thus, the specific MHT temperature must be defined for each tumor individually to ensure a successful antitumor therapy.

Extravasating Neutrophils Open Vascular Barrier and Improve Liposomes Delivery to Tumors.

Liposomes are the most extensively used nanocarriers in cancer therapy. Despite the advantages these vehicles provide over free drugs, there are still limitations with regards to the efficiency of liposomes delivery to tumors and off-target accumulation. A better understanding of nanodrugs extravasation mechanisms in different tumor types and normal vessels is needed to improve their antitumor activity. We used intravital microscopy to track for fluorescent liposomes behavior in xenograft tumor models (murine breast cancer 4T1 and melanoma B16, human prostate cancer 22Rv1) and normal skin and identified two distinct extravasation patterns. Microleakage, a local perivascular nanoparticle deposition, was found both in malignant and healthy tissues. This type of liposomes leakage does not provide access to tumor cells and is presumably responsible for drug deposition in normal tissues. In contrast, macroleakage penetrated deep into tissues and localized predominantly on the tumor-host interface. Although neutrophils did not uptake liposomes, their extravasation appeared to initiate both micro- and macroleakages. Based on neutrophils and liposomes extravasation dynamics, we hypothesized that microleakage and macroleakage are subsequent steps of the extravasation process corresponding to liposomes transport through endothelial and subendothelial barriers. Of note, extravasation spots were detected more often in the proximity of neutrophils, and across studied tumor types, neutrophils counts correlated with leakage frequencies. Reduced liposomes accumulation in 4T1 tumors upon Ly6G depletion further corroborated neutrophils role in nanoparticles delivery. Elucidating liposomes extravasation routes has a potential to help improve existing strategies and develop effective nanodrugs for cancer therapy.

The Centriolar Adjunct⁻Appearance and Disassembly in Spermiogenesis and the Potential Impact on Fertility.

During spermiogenesis, the proximal centriole forms a special microtubular structure: the centriolar adjunct. This structure appears at the spermatid stage, which is characterized by a condensed chromatin nucleus. We showed that the centriolar adjunct disappears completely in mature porcine spermatozoa. In humans, the centriolar adjunct remnants are present in a fraction of mature spermatids. For the first time, the structure of the centriolar adjunct in the cell, and its consequent impact on fertility, were examined. Ultrastructural analysis using transmission electron microscopy was performed on near 2000 spermatozoa per person, in two patients with idiopathic male sterility (IMS) and five healthy fertile donors. We measured the average length of the "proximal centriole + centriolar adjunct" complex in sections, where it had parallel orientation in the section plane, and found that it was significantly longer in the spermatozoa of IMS patients than in the spermatozoa of healthy donors. This difference was independent of chromatin condensation deficiency, which was also observed in the spermatozoa of IMS patients. We suggest that zygote arrest may be related to an incompletely disassembled centriolar adjunct in a mature spermatozoon. Therefore, centriolar adjunct length can be potentially used as a complementary criterion for the immaturity of spermatozoa in the diagnostics of IMS patients.

Size-selected Fe3O4-Au hybrid nanoparticles for improved magnetism-based theranostics.

Size-selected Fe3O4-Au hybrid nanoparticles with diameters of 6-44 nm (Fe3O4) and 3-11 nm (Au) were prepared by high temperature, wet chemical synthesis. High-quality Fe3O4 nanocrystals with bulk-like magnetic behavior were obtained as confirmed by the presence of the Verwey transition. The 25 nm diameter Fe3O4-Au hybrid nanomaterial sample (in aqueous and agarose phantom systems) showed the best characteristics for application as contrast agents in magnetic resonance imaging and for local heating using magnetic particle hyperthermia. Due to the octahedral shape and the large saturation magnetization of the magnetite particles, we obtained an extraordinarily high r 2-relaxivity of 495 mM-1·s-1 along with a specific loss power of 617 W·gFe -1 and 327 W·gFe -1 for hyperthermia in aqueous and agarose systems, respectively. The functional in vitro hyperthermia test for the 4T1 mouse breast cancer cell line demonstrated 80% and 100% cell death for immediate exposure and after precultivation of the cells for 6 h with 25 nm Fe3O4-Au hybrid nanomaterials, respectively. This confirms that the improved magnetic properties of the bifunctional particles present a next step in magnetic-particle-based theranostics.

Magnetite-Gold nanohybrids as ideal all-in-one platforms for theranostics.

High-quality, 25 nm octahedral-shaped Fe3O4 magnetite nanocrystals are epitaxially grown on 9 nm Au seed nanoparticles using a modified wet-chemical synthesis. These Fe3O4-Au Janus nanoparticles exhibit bulk-like magnetic properties. Due to their high magnetization and octahedral shape, the hybrids show superior in vitro and in vivo T2 relaxivity for magnetic resonance imaging as compared to other types of Fe3O4-Au hybrids and commercial contrast agents. The nanoparticles provide two functional surfaces for theranostic applications. For the first time, Fe3O4-Au hybrids are conjugated with two fluorescent dyes or the combination of drug and dye allowing the simultaneous tracking of the nanoparticle vehicle and the drug cargo in vitro and in vivo. The delivery to tumors and payload release are demonstrated in real time by intravital microscopy. Replacing the dyes by cell-specific molecules and drugs makes the Fe3O4-Au hybrids a unique all-in-one platform for theranostics.

Consecutive entosis stages in human substrate-dependent cultured cells.

Entosis, or cell death by invading another cell, is typical for tumor epithelial cells. The formation of cell-in-cell structures is extensively studied in suspension cultures, but remains poorly understood in substrate-dependent cells. Here, we used electron, confocal and time-lapse microscopy in combination with pharmacological inhibition of intracellular components to study the kinetics of entosis using two human substrate-dependent tumor cultures, A431 and MCF7. In total, we identified and characterized five consecutive stages of entosis, which were common for both examined cell lines. We further demonstrated that actin filaments in the entotic as well as invading cells were crucial for entosis. Microtubules and the Golgi apparatus of entotic cells provided membrane expansion required for internalization of the invading cell. Depolymerization of microfilaments and microtubules, and disintegration of the Golgi complex inhibited entosis. We confirmed the presence of adhesive junctions and discovered the formation of desmosomes between the invading and entotic cells. The internalized cell was shown to be degraded due to the lysosomal activation in both cells whereas the disintegration of the Golgi apparatus did not affect the process. Thus, in the substrate-dependent cultures, entosis requires microfilaments, microtubules and the Golgi complex for cell invasion, but not for internalized cell degradation.

Steroid hormones regulate sperm-oviduct interactions in the bovine.

After insemination in the cow, a sperm reservoir is formed within the oviducts, allowing the storage and then progressive release of spermatozoa toward the ovulated oocyte. In order to investigate the hormonal regulation of these events in vitro, the ovarian steroids 17ß-estradiol (E2) and progesterone (P4) were added at various concentrations to monolayers of bovine oviduct epithelial cells (BOEC) before or during co-incubation with spermatozoa. Main findings demonstrate that (1) a 18-h pretreatment of BOEC with 100 pg/mL and 100 ng/mL of E2 decreased by 25% the ability of BOEC to bind spermatozoa after 10 min, and for the highest dose of E2, 60 min of co-incubation; (2) P4 at concentrations of 10, 100 and 1000 ng/mL induced the release within 60 min of 32-47% of bound spermatozoa from BOEC; this sperm-releasing effect was maintained after a 18-h pretreatment of BOEC with 100 pg/mL of E2; (3) E2 in concentrations above 100 pg/mL inhibited the releasing effect of P4 on bound sperm in a dose-dependent manner; (4) spermatozoa bound to BOEC, then released from BOEC by the action of P4-induced higher cleavage and blastocyst rates after in vitro fertilization than the control group. These results support the hypothesis that the dynamic changes in steroid hormones around the time of ovulation regulate the formation of the sperm reservoir and the timed delivery of capacitated spermatozoa to the site of fertilization.