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.

Localized Increased Permeability of Blood-Brain Barrier for Antibody Conjugates in the Cuprizone Model of Demyelination.

The development of new neurotherapeutics depends on appropriate animal models being chosen in preclinical studies. The cuprizone model is an effective tool for studying demyelination and remyelination processes in the brain, but blood-brain barrier (BBB) integrity in the cuprizone model is still a topic for debate. Several publications claim that the BBB remains intact during cuprizone-induced demyelination; others demonstrate results that could explain the increased BBB permeability. In this study, we aim to analyze the permeability of the BBB for different macromolecules, particularly antibody conjugates, in a cuprizone-induced model of demyelination. We compared the traditional approach using Evans blue injection with subsequent dye extraction and detection of antibody conjugates using magnetic resonance imaging (MRI) and confocal microscopy to analyze BBB permeability in the cuprizone model. First, we validated our model of demyelination by performing T2-weighted MRI, diffusion tensor imaging, quantitative rt-PCR to detect changes in mRNA expression of myelin basic protein and proteolipid protein, and Luxol fast blue histological staining of myelin. Intraperitoneal injection of Evans blue did not result in any differences between the fluorescent signal in the brain of healthy and cuprizone-treated mice (IVIS analysis with subsequent dye extraction). In contrast, intravenous injection of antibody conjugates (anti-GFAP or non-specific IgG) after 4 weeks of a cuprizone diet demonstrated accumulation in the corpus callosum of cuprizone-treated mice both by contrast-enhanced MRI (for gadolinium-labeled antibodies) and by fluorescence microscopy (for Alexa488-labeled antibodies). Our results suggest that the methods with better sensitivity could detect the accumulation of macromolecules (such as fluorescent-labeled or gadolinium-labeled antibody conjugates) in the brain, suggesting a local BBB disruption in the demyelinating area. These findings support previous investigations that questioned BBB integrity in the cuprizone model and demonstrate the possibility of delivering antibody conjugates to the corpus callosum of cuprizone-treated mice.

Glioma Stem Cells: Novel Data Obtained by Single-Cell Sequencing.

Glioma is the most common type of primary CNS tumor, composed of cells that resemble normal glial cells. Recent genetic studies have provided insight into the inter-tumoral heterogeneity of gliomas, resulting in the updated 2021 WHO classification of gliomas. Thorough understanding of inter-tumoral heterogeneity has already improved the prognosis and treatment outcomes of some types of gliomas. Currently, the challenge for researchers is to study the intratumoral cell heterogeneity of newly defined glioma subtypes. Cancer stem cells (CSCs) present in gliomas and many other tumors are an example of intratumoral heterogeneity of great importance. In this review, we discuss the modern concept of glioma stem cells and recent single-cell sequencing-driven progress in the research of intratumoral glioma cell heterogeneity. The particular emphasis was placed on the recently revealed variations of the cell composition of the subtypes of the adult-type diffuse gliomas, including astrocytoma, oligodendroglioma and glioblastoma. The novel data explain the inconsistencies in earlier glioma stem cell research and also provide insight into the development of more effective targeted therapy and the cell-based immunotherapy of gliomas. Separate sections are devoted to the description of single-cell sequencing approach and its role in the development of cell-based immunotherapies for glioma.

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.

Supermagnetic Human Serum Albumin (HSA) Nanoparticles and PLGA-Based Doxorubicin Nanoformulation: A Duet for Selective Nanotherapy.

Predicting the ability of nanoparticles (NP) to access the tumor is key to the success of chemotherapy using nanotherapeutics. In the present study, the ability of the dual NP-based theranostic system to accumulate in the tumor was evaluated in vivo using intravital microscopy (IVM) and MRI. The system consisted of model therapeutic doxorubicin-loaded poly(lactide-co-glycolide) NP (Dox-PLGA NP) and novel hybrid Ce3/4+-doped maghemite NP encapsulated within the HSA matrix (hMNP) as a supermagnetic MRI contrasting agent. Both NP types had similar sizes of ~100 nm and negative surface potentials. The level of the hMNP and PLGA NP co-distribution in the same regions of interest (ROI, ~2500 µm2) was assessed by IVM in mice bearing the 4T1-mScarlet murine mammary carcinoma at different intervals between the NP injections. In all cases, both NP types penetrated into the same tumoral/peritumoral regions by neutrophil-assisted extravasation through vascular micro- and macroleakages. The maximum tumor contrasting in MRI scans was obtained 5 h after hMNP injection/1 h after PLGA NP injection; the co-distribution level at this time reached 78%. Together with high contrasting properties of the hMNP, these data indicate that the hMNP and PLGA NPs are suitable theranostic companions. Thus, analysis of the co-distribution level appears to be a useful tool for evaluation of the dual nanoparticle theranostics, whereas assessment of the leakage areas helps to reveal the tumors potentially responsive to nanotherapeutics.

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.

Photochemical synthesis, intercalation with DNA and antitumor evaluation in vitro of benzo[d]thiazolo[3,2-a]quinolin-10-ium derivatives.

A new anticancer benzo[d]thiazolo[3,2-a]quinolin-10-ium derivatives were synthesized and characterized. Anticancer evaluation in vitro against four cancer cell lines including adenocarcinomic human alveolar basal epithelial cells (A549), hepatocellular carcinoma (HepG2), prostate cancer (PC3) and breast cancer (MCF7) indicated that some of prepared compounds shows higher selectivity in comparison with doxorubicin. DNA interaction studies by optical, CD, NMR spectroscopies showed the high affinity of benzothiazole ligands towards the dsDNA. The ligand-DNA interaction occurs through the intercalation of benzo[d]thiazolo[3,2-a]quinolin-10-ium derivatives with nucleic acid. The investigation of formed ligand - DNA complexes by docking and molecular dynamic calculations was applied for analysis of the relationship between structure and anticancer activity. The results suggested that benzo[d]thiazolo[3,2-a]quinolin-10-ium derivatives might serve as a novel scaffold for the future development to new antitumor agents.

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.

Genetically encodable bioluminescent system from fungi.

Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.

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.

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.

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.

Evaluation of cyclic luciferin as a substrate for luminescence measurements in in vitro and in vivo applications.

Bioluminescence imaging (BLI) is a powerful tool for cell tracking, monitoring of gene delivery and expression in small laboratory animals. An alternative luciferase (Luc) substrate cyclic luciferin (Cycluc) was recently advanced for BLI applications as providing a stronger, more stable signal at significantly lower doses than the classical substrate D-luciferin (D-Luc) increasing sensitivity of Luc detection 10 to 100 times. We evaluated benefits of using Cycluc in in vivo studies in mice injected with murine adenocarcinoma 4T1 cells expressing Luc, and in single-cell organisms, the oocytes of Xenopus laevis. No significant increase in the efficacy of detection of the luminescent signal was recorded in either of the systems. Kinetic studies demonstrated that Km for Cycluc was 10000 higher, whereas Vmax was 100 lower than that of D-Luc. Cycluc efficiently bound to the active center of luciferase, but its turnover was extremely low, leading to actual inhibition of bioluminescence. This compromises Cycluc as a substrate for measurement of the activity of the wild-type luciferases, still widely used as reporters for in vivo monitoring microorganisms and tumor cells. It may find better applications with the development of in vivo imaging based on the genetically engineered mutant luciferases with different substrate requirements.

Magnetic and Optical Properties of Gold-Coated Iron Oxide Nanoparticles.

In this work, magnetic and optical properties of magnetic nanoparticles were investigated, where the particles of iron oxide were prepared with a co-precipitation route and the component of gold was built up by reduction of AuCl4- on the surface of iron oxide to assemble nanocomposite structures in the form of an electrostatic stabilized suspension. The size of the particles obtained with TEM increased from of 8.9 ± 2.7 to 16 ± 6 nm after the procedure of hybridisation. In order to distinguish the impact of the gold on the optical properties, UV-Vis and Raman spectroscopy techniques were used. Magnetic properties were studied in the temperature range of 5-300 K and the superparamagnetic state of MNPs at room temperature was confirmed for both systems.

Synthesis of Iron Oxide Nanoclusters by Thermal Decomposition.

Herein, we report a novel one-step solvothermal synthesis of magnetite nanoclusters (MNCs). In this report, we discuss the synthesis, structure, and properties of MNCs and contrast enhancement in T2-weighted MR images using magnetite nanoclusters. The effect of different organic acids, used as surfactants, on the size and shape of MNCs was investigated. The structure and properties of samples were determined by magnetic measurements, TGA, TEM, HRTEM, XRD, FTIR, and MRI. Magnetic measurements show that obtained MNCs have relatively high saturation magnetization values (65.1-81.5 emu/g) and dependence of the coercive force on the average size of MNCs was established. MNCs were transferred into an aqueous medium by Pluronic F-127, and T2-relaxivity values were determined. T2-Weighted MR phantom images clearly demonstrated that such magnetite nanoclusters can be used as contrast agents for MRI.

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.

Diffusion Tensor Imaging in Diagnosis of Post-Traumatic Syringomyelia in Spinal Cord Injury in Rats.

BACKGROUND Post-traumatic syringomyelia (PTS) is a common disease after spinal cord injury (SCI). The present study was performed to evaluate the advantages of diffusion tensor imaging (DTI) in estimating SCI and prognosing PTS in SCI rats. MATERIAL AND METHODS Forty rats were divided into 3 groups based on the extent of the individual SCI and PTS: a control group (n=10), a PTS group (n=8), and an SCI group (n=22). BBB tests were performed preoperatively and postoperatively at (1 d, 3 d, 5 d, 1 w, 2 w, 1 w, 2 w, 3 w, 4 w, 5 w, and 6 w). MRI T2 scanning was conducted postoperatively at (1 w, 2 w, 3 w, 4 w, 5 w, 6 w). DTI and diffusion tensor tractography were used for analyzing neuro-fiber changes after SCI. RESULTS BBB scoring showed no differences between the PTS group and SCI group (P<0.05). PTS was found in 8 rats after SCI. MRI showed PTS formation in 3 rats at 2 w after SCI, and 5 rats showed PTS formation at postoperative 3w after SCI. Compared with the control group, ADC showed significant increase in both the PTS group (P<0.05) and the SCI group (P<0.05), FA showed significant decreases in the PTS (P<0.05) and SCI (P<0.05) groups. Compared with the SCI group, the PTS group showed an increase in ADC, but no statistical difference was found in ADC (P>0.05). The PTS group showed a significant increase in FA (P<0.05). CONCLUSIONS The combination of diffusion tensor imaging and diffusion tensor tractography has characteristics of high-sensitivity and quantitation for PTS prognosis. FA is predictive in the prognosis of PTS formation after SCI.

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.

Precise Delivery Into Chronic Spinal Cord Injury Syringomyelic Cysts with Magnetic Nanoparticles MRI Visualization.

BACKGROUND: Traumatic spinal cord injury (SCI) often results in the deficiency of glia and neurons in cystic cavities. These syringomyelic cysts can prevent axonal regeneration and sprouting. Details of the mechanism of syringomyelic cyst formation are unknown and an effective treatment for overcoming syringomyelic cysts is not available. MATERIAL AND METHODS: Ten adult female Wistar rats underwent contusion SCI modeling resulting in syringomyelic cyst formation. A novel method for locating the cysts was developed and employed. MRI safe silver needles were inserted through the erector spinae of anesthetized rats to create a stable reference point. MRI images of the rodent spine were taken with the needles in situ. This information was used to accurately locate the cyst and determine the 3-dimensional entry point coordinates for nanoparticle delivery. Nanoparticles were injected into the cyst during a primary injection of 8 ul and a secondary injection of 8 ul, to prove the procedure can be accurately repeated. RESULTS: None of the rats died intra- or post-operatively. The syringomyelic cysts were accurately located with the 3-dimensional entry point coordinates. After nanoparticle delivery twice into each rat, the visualized syringomyelic cyst volume significantly decreased from 5.71±0.21 mm3 to 3.23±0.364 mm3 and to 1.48±0.722 mm3. CONCLUSIONS: The present study describes a novel strategy for precise nanoparticle delivery into a syringomyelic cyst, using measurements obtained from MRI images. This strategy may aid in developing a new method for studying chronic spinal cord injury and a novel treatment for syringomyelic cysts.