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.

Using ELP Repeats as a Scaffold for De Novo Construction of Gadolinium-Binding Domains within Multifunctional Recombinant Proteins for Targeted Delivery of Gadolinium to Tumour Cells.

Three artificial proteins that bind the gadolinium ion (Gd3+) with tumour-specific ligands were de novo engineered and tested as candidate drugs for binary radiotherapy (BRT) and contrast agents for magnetic resonance imaging (MRI). Gd3+-binding modules were derived from calmodulin. They were joined with elastin-like polypeptide (ELP) repeats from human elastin to form the four-centre Gd3+-binding domain (4MBS-domain) that further was combined with F3 peptide (a ligand of nucleolin, a tumour marker) to form the F3-W4 block. The F3-W4 block was taken alone (E2-13W4 protein), as two repeats (E1-W8) and as three repeats (E1-W12). Each protein was supplemented with three copies of the RGD motif (a ligand of integrin αvß3) and green fluorescent protein (GFP). In contrast to Magnevist (a Gd-containing contrast agent), the proteins exhibited three to four times higher accumulation in U87MG glioma and A375 melanoma cell lines than in normal fibroblasts. The proteins remained for >24 h in tumours induced by Ca755 adenocarcinoma in C57BL/6 mice. They exhibited stability towards blood proteases and only accumulated in the liver and kidney. The technological advantages of using the engineered proteins as a basis for developing efficient and non-toxic agents for early diagnosis of tumours by MRI as well as part of BRT were demonstrated.

Magneto-Mechanical Approach in Biomedicine: Benefits, Challenges, and Future Perspectives.

The magneto-mechanical approach is a powerful technique used in many different applications in biomedicine, including remote control enzyme activity, cell receptors, cancer-selective treatments, mechanically-activated drug releases, etc. This approach is based on the use of a combination of magnetic nanoparticles and external magnetic fields that have led to the movement of such nanoparticles with torques and forces (enough to change the conformation of biomolecules or even break weak chemical bonds). However, despite many theoretical and experimental works on this topic, it is difficult to predict the magneto-mechanical effects in each particular case, while the important results are scattered and often cannot be translated to other experiments. The main reason is that the magneto-mechanical effect is extremely sensitive to changes in any parameter of magnetic nanoparticles and the environment and changes in the parameters of the applied magnetic field. Thus, in this review, we (1) summarize and propose a simplified theoretical explanation of the main factors affecting the efficiency of the magneto-mechanical approach; (2) discuss the nature of the MNP-mediated mechanical forces and their order of magnitude; (3) show some of the main applications of the magneto-mechanical approach in the control over the properties of biological systems.

Myxococcus xanthus Encapsulin as a Promising Platform for Intracellular Protein Delivery.

Introducing a new genetically encoded material containing a photoactivatable label as a model cargo protein, based on Myxococcus xanthus (Mx) encapsulin system stably expressed in human 293T cells. Encapsulin from Mx is known to be a protein-based container for a ferritin-like cargo in its shell which could be replaced with an exogenous cargo protein, resulting in a modified encapsulin system. We replaced Mx natural cargo with a foreign photoactivatable mCherry (PAmCherry) fluorescent protein and isolated encapsulins, containing PAmCherry, from 293T cells. Isolated Mx encapsulin shells containing photoactivatable label can be internalized by macrophages, wherein the PAmCherry fluorescent signal remains clearly visible. We believe that a genetically encoded nanocarrier system obtained in this study, can be used as a platform for controllable delivery of protein/peptide therapeutics in vitro.

CT and MRI Imaging of Theranostic Bimodal Fe3O4@Au NanoParticles in Tumor Bearing Mice.

Gold-containing nanoparticles are proven to be an effective radiosensitizer in the radiotherapy of tumors. Reliable imaging of nanoparticles in a tumor and surrounding normal tissues is crucial both for diagnostics and for nanoparticle application as radiosensitizers. The Fe3O4 core was introduced into gold nanoparticles to form a core/shell structure suitable for MRI imaging. The aim of this study was to assess the in vivo bimodal CT and MRI enhancement ability of novel core/shell Fe3O4@Au theranostic nanoparticles. Core/shell Fe3O4@Au nanoparticles were synthesized and coated with PEG and glucose. C57Bl/6 mice bearing Ca755 mammary adenocarcinoma tumors received intravenous injections of the nanoparticles. CT and MRI were performed at several timepoints between 5 and 102 min, and on day 17 post-injection. Core/shell Fe3O4@Au nanoparticles provided significant enhancement of the tumor and tumor blood vessels. Nanoparticles also accumulated in the liver and spleen and were retained in these organs for 17 days. Mice did not show any signs of toxicity over the study duration. These results indicate that theranostic bimodal Fe3O4@Au nanoparticles are non-toxic and serve as effective contrast agents both for CT and MRI diagnostics. These nanoparticles have potential for future biomedical applications in cancer diagnostics and beyond.

Toxicity of iron oxide nanoparticles: Size and coating effects.

Toxicological research of novel nanomaterials is a major developmental step of their clinical approval. Since iron oxide magnetic nanoparticles have a great potential in cancer treatment and diagnostics, the investigation of their toxic properties is very topical. In this paper we synthesized bovine serum albumin-coated iron oxide nanoparticles with different sizes and their polyethylene glycol derivative. To prove high biocompatibility of obtained nanoparticles the number of in vitro toxicological tests on human fibroblasts and U251 glioblastoma cells was performed. It was shown that albumin nanoparticles' coating provides a stable and biocompatible shell and prevents cytotoxicity of magnetite core. On long exposure times (48 hours), cytotoxicity of iron oxide nanoparticles takes place due to free radical production, but this toxic effect may be neutralized by using polyethylene glycol modification.

Multimodal doxorubicin loaded magnetic nanoparticles for VEGF targeted theranostics of breast cancer.

In presented paper we have developed new system for cancer theranostics based on vascular endothelial growth factor (VEGF) targeted magnetic nanoparticles. Conjugation of anti-VEGF antibodies with bovine serum albumin coated PEGylated magnetic nanoparticles allows for improved binding with murine breast adenocarcinoma 4T1 cell line and facilitates doxorubicin delivery to tumor cells. It was shown that intravenous injection of doxorubicin loaded VEGF targeted nanoparticles increases median survival rate of mice bearing 4T1 tumors up to 50%. On the other hand magnetic resonance imaging (MRI) of 4T1 tumors 24 h after intravenous injection showed accumulation of nanoparticles in tumors, thus allowing simultaneous cancer therapy and diagnostics.

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.

Magnetic and Fluorescent Dual-Labeled Genetically Encoded Targeted Nanoparticles for Malignant Glioma Cell Tracking and Drug Delivery.

Human glioblastoma multiforme (GBM) is a primary malignant brain tumor, a radically incurable disease characterized by rapid growth resistance to classical therapies, with a median patient survival of about 15 months. For decades, a plethora of approaches have been developed to make GBM therapy more precise and improve the diagnosis of this pathology. Targeted delivery mediated by the use of various molecules (monoclonal antibodies, ligands to overexpressed tumor receptors) is one of the promising methods to achieve this goal. Here we present a novel genetically encoded nanoscale dual-labeled system based on Quasibacillus thermotolerans (Qt) encapsulins exploiting biologically inspired designs with iron-containing nanoparticles as a cargo, conjugated with human fluorescent labeled transferrin (Tf) acting as a vector. It is known that the expression of transferrin receptors (TfR) in glioma cells is significantly higher compared to non-tumor cells, which enables the targeting of the resulting nanocarrier. The selectivity of binding of the obtained nanosystem to glioma cells was studied by qualitative and quantitative assessment of the accumulation of intracellular iron, as well as by magnetic particle quantification method and laser scanning confocal microscopy. Used approaches unambiguously demonstrated that transferrin-conjugated encapsulins were captured by glioma cells much more efficiently than by benign cells. The resulting bioinspired nanoplatform can be supplemented with a chemotherapeutic drug or genotherapeutic agent and used for targeted delivery of a therapeutic agent to malignant glioma cells. Additionally, the observed cell-assisted biosynthesis of magnetic nanoparticles could be an attractive way to achieve a narrow size distribution of particles for various applications.

Oncolytic therapy with recombinant vaccinia viruses targeting the interleukin-15 pathway elicits a synergistic response.

We developed recombinant variants of oncolytic vaccinia virus LIVP strain expressing interleukin-15 (IL-15) or its receptor subunit alpha (IL-15Rα) to stimulate IL-15-dependent immune cells. We evaluated their oncolytic activity either alone or in combination with each other in vitro and in vivo using the murine CT26 colon carcinoma and 4T1 breast carcinoma models. We demonstrated that the admixture of these recombinant variants could promote the generation of the IL-15/IL-15Rα complex. In vitro studies indicated that 4T1 breast cancer cells were more susceptible to the developed recombinant viruses. In vivo studies showed significant survival benefits and tumor regression in 4T1 breast cancer syngeneic mice that received a combination of LIVP-IL15-RFP with LIVP-IL15Ra-RFP. Histological analysis showed recruited lymphocytes at the tumor region, while no harmful effects to the liver or spleen of the animals were detected. Evaluating tumor-infiltrated lymphocytes represented profound activation of cytotoxic T cells and macrophages in mice receiving combination therapy. Thus, our experiments showed superior oncolytic effectiveness of simultaneous injection of LIVP-IL15-RFP and LIVP-IL15Ra-RFP in breast cancer-bearing mice. The combined therapy by these recombinant variants represents a potent and versatile approach for developing new immunotherapies for breast cancer.

Oncolytic Efficacy of a Recombinant Vaccinia Virus Strain Expressing Bacterial Flagellin in Solid Tumor Models.

Oncolytic viral therapy is a promising novel approach to cancer treatment. Oncolytic viruses cause tumor regression through direct cytolysis on the one hand and recruiting and activating immune cells on the other. In this study, to enhance the antitumor efficacy of the thymidine kinase-deficient vaccinia virus (VV, Lister strain), recombinant variants encoding bacterial flagellin (subunit B) of Vibrio vulnificus (LIVP-FlaB-RFP), firefly luciferase (LIVP-Fluc-RFP) or red fluorescent protein (LIVP-RFP) were developed. The LIVP-FLuc-RFP strain demonstrated exceptional onco-specificity in tumor-bearing mice, detected by the in vivo imaging system (IVIS). The antitumor efficacy of these variants was explored in syngeneic murine tumor models (B16 melanoma, CT26 colon cancer and 4T1 breast cancer). After intravenous treatment with LIVP-FlaB-RFP or LIVP-RFP, all mice tumor models exhibited tumor regression, with a prolonged survival rate in comparison with the control mice. However, superior oncolytic activity was observed in the B16 melanoma models treated with LIVP-FlaB-RFP. Tumor-infiltrated lymphocytes and the cytokine analysis of the serum and tumor samples from the melanoma-xenografted mice treated with these virus variants demonstrated activation of the host's immune response. Thus, the expression of bacterial flagellin by VV can enhance its oncolytic efficacy against immunosuppressive solid tumors.

Photoinduced Reduction of Novel Dual-Action Riboplatin Pt(IV) Prodrug.

Controlled photoreduction of Pt(IV) prodrugs is a challenging task due to the possibility of targeted light-controlled activation of anticancer agents without affecting healthy tissues. Also, a conjugation of photosensitizers and clinically used platinum drugs into one Pt(IV) prodrug allows combining photodynamic therapy and chemotherapy approaches into one molecule. Herein, we designed the cisplatin-based Pt(IV) prodrug Riboplatin with tetraacetylriboflavin in the axial position. A novel Pt(IV) prodrug is able to act both as a photodynamic therapy (PDT) agent through the conversion of ground-state 3O2 to excited-state 1O2 and as an agent of photoactivated chemotherapy (PACT) through releasing of cisplatin under gentle blue light irradiation, without the requirement of a reducing agent. The light-induced behavior of Riboplatin was investigated using an electrochemical sensor in MCF-7 tumor spheroids. Photocontrolled cisplatin release and ROS generation were detected electrochemically in real time. This appears to be the first confirmation of simultaneous photoactivated release of anticancer drug cisplatin and ROS from a dual-action Pt(IV) prodrug observed from the inside of living tumor spheroids.

New Approach to Non-Invasive Tumor Model Monitoring via Self-Assemble Iron Containing Protein Nanocompartments.

According to the World Health Organization, breast cancer is the most common oncological disease worldwide. There are multiple animal models for different types of breast carcinoma, allowing the research of tumor growth, metastasis, and angiogenesis. When studying these processes, it is crucial to visualize cancer cells for a prolonged time via a non-invasive method, for example, magnetic resonance imaging (MRI). In this study, we establish a new genetically encoded material based on Quasibacillus thermotolerans (Q.thermotolerans, Qt) encapsulin, stably expressed in mouse 4T1 breast carcinoma cells. The label consists of a protein shell containing an enzyme called ferroxidase. When adding Fe2+, a ferroxidase oxidizes Fe2+ to Fe3+, followed by iron oxide nanoparticles formation. Additionally, genes encoding mZip14 metal transporter, enhancing the iron transport, were inserted into the cells via lentiviral transduction. The expression of transgenic sequences does not affect cell viability, and the presence of magnetic nanoparticles formed inside encapsulins results in an increase in T2 relaxivity.

Study of the Therapeutic Efficiency of Transduced Olfactory Ensheathing Cells in Spinal Cord Cysts.

Posttraumatic spinal cord cysts are difficult to treat with medication and surgery. Gene-cell therapy is a promising area of treatment for such patients. However, optimal gene-cell construct for this therapy has not been developed. We investigated the therapeutic efficiency of human olfactory ensheathing cells (OECs) transduced by adenoviral vector encoding the mature form of brain-derived neurotrophic factor (mBDNF) in spinal cord cysts. The adenoviral vectors Ad5/35-CAG-mBDNF and Ad5/35-CAG-Fluc were constructed. Spinal cysts were modeled in female Wistar rats. We selected animals at the early and intermediate stages of recovery with scores to 13 according to the Basso, Beattie and Bresnahan (BBB) scale. The efficiency of therapy was evaluated by BBB tests. No cytotoxicity was detected using the Resazurin/AlamarBlue assay for both vectors at multiplicity of infection (MOIs) of 1, 5, and 25. There was an increase in the proliferation of cells treated with Ad5/35-CAG-mBDNF at MOIs of 5 and 25. The hind limb mobility after the transplantation of Ad5/35-CAG-mBDNF- and Ad5/35-CAG-Fluc-transduced human OECs and nontransduced OECs had approximately the same tendency to improve. Cyst reduction was observed with the transplantation of all the samples. Although Ad5/35-CAG-mBDNF-transduced OECs had high BDNF expression levels in vitro, these cells lacked positive effect in vivo because they did not exhibit significant effect concerning functional test when comparing the groups that received the same numbers of OECs. The therapeutic efficiency of transduced OECs appears to be due to the cell component. The autological and tissue-specific human OECs are promising for the personalized cell therapy. It is extremely important to test new gene-cell constructs based on these cells for further clinical use.

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.

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.

Photodiagnosis and photodynamic effects of bacteriochlorin-naphthalimide conjugates on tumor cells and mouse model.

Photo-induced cytotoxicity and antitumor activity of a series of dual function agents for photodynamic therapy (PDT) and fluorescent imaging based on bacteriochlorin photosensitizer conjugated with various naphthalimide fluorophores was studied in vitro using murine tumor cells of S37 sarcoma and in vivo on mice bearing murine S37 sarcoma. Upon irradiation at the absorption maximum of the photosensitizer, the activity of conjugates was as high as in the case of individual bacteriochlorin, while an additional excitation of the naphthalimide fragment led to an increase in the PDT efficacy due to resonance energy transfer from the fluorophore to photosensitizer. The fluorescence contrast and specific cytotoxic activity measurements indicate that the conjugate of bacteriochlorin with 3,4-dimethoxestyrene-substituted naphthalimide is the most promising agent for the application as theranostic in PDT.

Intra-Arterial Stem Cell Transplantation in Experimental Stroke in Rats: Real-Time MR Visualization of Transplanted Cells Starting With Their First Pass Through the Brain With Regard to the Therapeutic Action.

Cell therapy is an emerging approach to stroke treatment with a potential to limit brain damage and enhance its restoration after the acute phase of the disease. In this study we tested directly reprogrammed neural precursor cells (drNPC) derived from adult human bone marrow cells in the rat middle cerebral artery occlusion (MCAO) model of acute ischemic stroke using human placenta mesenchymal stem cells (pMSC) as a positive control with previously confirmed efficacy. Cells were infused into the ipsilateral (right) internal carotid artery of male Wistar rats 24 h after MCAO. The main goal of this work was to evaluate real-time distribution and subsequent homing of transplanted cells in the brain. This was achieved by performing intra-arterial infusion directly inside the MRI scanner and allowed transplanted cells tracing starting from their first pass through the brain vessels. Immediately after transplantation, cells were observed in the periphery of the infarct zone and in the brain stem, 15 min later small numbers of cells could be discovered deep in the infarct core and in the contralateral hemisphere, where drNPC were seen earlier and in greater numbers than pMSC. Transplanted cells in both groups could no longer be detected in the rat brain 48-72 h after infusion. Histological and histochemical analysis demonstrated that both the drNPC and pMSC were localized inside blood vessels in close contact with the vascular wall. No passage of labeled cells through the blood brain barrier was observed. Additionally, the therapeutic effects of drNPC and pMSC were compared. Both drNPC and pMSC induced substantial attenuation of neurological deficits evaluated at the 7th and 14th day after transplantation using the modified neurological severity score (mNSS). Some of the effects of drNPC and pMSC, such as the influence on the infarct volume and the survival rate of animals, differed. The results suggest a paracrine mechanism of the positive therapeutic effects of IA drNPC and pMSC infusion, potentially enhanced by the cell-cell interactions. Our data also indicate that the long-term homing of transplanted cells in the brain is not necessary for the brain's functional recovery.

Encapsulins-Bacterial Protein Nanocompartments: Structure, Properties, and Application.

Recently, a new class of prokaryotic compartments, collectively called encapsulins or protein nanocompartments, has been discovered. The shell proteins of these structures self-organize to form icosahedral compartments with a diameter of 25-42 nm, while one or more cargo proteins with various functions can be encapsulated in the nanocompartment. Non-native cargo proteins can be loaded into nanocompartments and the surface of the shells can be further functionalized, which allows for developing targeted drug delivery systems or using encapsulins as contrast agents for magnetic resonance imaging. Since the genes encoding encapsulins can be integrated into the cell genome, encapsulins are attractive for investigation in various scientific fields, including biomedicine and nanotechnology.

Novel Copper-Containing Cytotoxic Agents Based on 2-Thioxoimidazolones.

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.