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.

Encapsulins: Structure, Properties, and Biotechnological Applications.

In 1994 a new class of prokaryotic compartments was discovered, collectively called "encapsulins" or "nanocompartments". Encapsulin shell protomer proteins self-assemble to form icosahedral structures of various diameters (24-42 nm). Inside of nanocompartments shells, one or several cargo proteins, diverse in their functions, can be encapsulated. In addition, non-native cargo proteins can be loaded into nanocompartments, and shell surfaces can be modified via various compounds, which makes it possible to create targeted drug delivery systems, labels for optical and MRI imaging, and to use encapsulins as bioreactors. This review describes a number of strategies of encapsulins application in various fields of science, including biomedicine and nanobiotechnologies.

Synthesis and Functional Characterization of CoxFe3-xO4-BaTiO3 Magnetoelectric Nanocomposites for Biomedical Applications.

Nowadays, magnetoelectric nanomaterials are on their way to finding wide applications in biomedicine for various cancer and neurological disease treatment, which is mainly restricted by their relatively high toxicity and complex synthesis. This study for the first time reports novel magnetoelectric nanocomposites of CoxFe3-xO4-BaTiO3 series with tuned magnetic phase structures, which were synthesized via a two-step chemical approach in polyol media. The magnetic CoxFe3-xO4 phases with x = 0.0, 0.5, and 1.0 were obtained by thermal decomposition in triethylene glycol media. The magnetoelectric nanocomposites were synthesized by the decomposition of barium titanate precursors in the presence of a magnetic phase under solvothermal conditions and subsequent annealing at 700 °C. X-ray diffraction revealed the presence of both spinel and perovskite phases after annealing with average crystallite sizes in the range of 9.0-14.5 nm. Transmission electron microscopy data showed two-phase composite nanostructures consisting of ferrites and barium titanate. The presence of interfacial connections between magnetic and ferroelectric phases was confirmed by high-resolution transmission electron microscopy. Magnetization data showed expected ferrimagnetic behavior and σs decrease after the nanocomposite formation. Magnetoelectric coefficient measurements after the annealing showed non-linear change with a maximum of 89 mV/cm*Oe with x = 0.5, 74 mV/cm*Oe with x = 0, and a minimum of 50 mV/cm*Oe with x = 0.0 core composition, that corresponds with the coercive force of the nanocomposites: 240 Oe, 89 Oe and 36 Oe, respectively. The obtained nanocomposites show low toxicity in the whole studied concentration range of 25-400 µg/mL on CT-26 cancer cells. The synthesized nanocomposites show low cytotoxicity and high magnetoelectric effects, therefore they can find wide applications in biomedicine.

Transgenic HA-1-Specific CD8+ T-Lymphocytes Selectively Target Leukemic Cells.

A significant share of allogeneic hematopoietic stem cell transplantations (allo-HSCT) results in the relapse of malignant disease. The T cell immune response to minor histocompatibility antigens (MiHAs) promotes a favorable graft-versus-leukemia response. The immunogenic MiHA HA-1 is a promising target for leukemia immunotherapy, as it is predominantly expressed in hematopoietic tissues and presented by the common HLA A*02:01 allele. Adoptive transfer of HA-1-specific modified CD8+ T cells could complement allo-HSCT from HA-1- donors to HA-1+ recipients. Using bioinformatic analysis and a reporter T cell line, we discovered 13 T cell receptors (TCRs) specific for HA-1. Their affinities were measured by the response of the TCR-transduced reporter cell lines to HA-1+ cells. The studied TCRs showed no cross-reactivity to the panel of donor peripheral mononuclear blood cells with 28 common HLA alleles. CD8+ T cells after endogenous TCR knock out and introduction of transgenic HA-1-specific TCR were able to lyse hematopoietic cells from HA-1+ patients with acute myeloid, T-, and B-cell lymphocytic leukemia (n = 15). No cytotoxic effect was observed on cells from HA-1- or HLA-A*02-negative donors (n = 10). The results support the use of HA-1 as a target for post-transplant T cell therapy.

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.

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.

Encapsulin Based Self-Assembling Iron-Containing Protein Nanoparticles for Stem Cells MRI Visualization.

Over the past decade, cell therapy has found many applications in the treatment of different diseases. Some of the cells already used in clinical practice include stem cells and CAR-T cells. Compared with traditional drugs, living cells are much more complicated systems that must be strictly controlled to avoid undesirable migration, differentiation, or proliferation. One of the approaches used to prevent such side effects involves monitoring cell distribution in the human body by any noninvasive technique, such as magnetic resonance imaging (MRI). Long-term tracking of stem cells with artificial magnetic labels, such as magnetic nanoparticles, is quite problematic because such labels can affect the metabolic process and cell viability. Additionally, the concentration of exogenous labels will decrease during cell division, leading to a corresponding decrease in signal intensity. In the current work, we present a new type of genetically encoded label based on encapsulin from Myxococcus xanthus bacteria, stably expressed in human mesenchymal stem cells (MSCs) and coexpressed with ferroxidase as a cargo protein for nanoparticles' synthesis inside encapsulin shells. mZip14 protein was expressed for the enhancement of iron transport into the cell. Together, these three proteins led to the synthesis of iron-containing nanoparticles in mesenchymal stem cells-without affecting cell viability-and increased contrast properties of MSCs in MRI.

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.