Fullerene C60 Conjugate with Folic Acid and Polyvinylpyrrolidone for Targeted Delivery to Tumor Cells.

The use of targeted drug delivery systems, including those based on selective absorption by certain receptors on the surface of the target cell, can lead to a decrease in the minimum effective dose and the accompanying toxicity of the drug, as well as an increase in therapeutic efficacy. A fullerene C60 conjugate (FA-PVP-C60) with polyvinylpyrrolidone (PVP) as a biocompatible spacer and folic acid (FA) as a targeting ligand for tumor cells with increased expression of folate receptors (FR) was obtained. Using 13C NMR spectroscopy, FT-IR, UV-Vis spectrometry, fluorometry and thermal analysis, the formation of the conjugate was confirmed and the nature of the binding of its components was established. The average particle sizes of the conjugate in aqueous solutions and cell culture medium were determined using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The FA-PVP-C60 showed antiradical activity against •DPPH, •OH and O2•-, but at the same time, it was shown to generate 1O2. It was found that the conjugate in the studied concentration range (up to 200 µg/mL) is non-toxic in vitro and does not affect the cell cycle. To confirm the ability of the conjugate to selectively accumulate through folate-mediated endocytosis, its uptake into cells was analyzed by flow cytometry and confocal microscopy. It was shown that the conjugate is less absorbed by A549 cells with low FR expression than by HeLa, which has a high level of expression of this receptor.

Tumor alkalization therapy: misconception or good therapeutics perspective? - the case of malignant ascites.

Tumor acidity has been identified as a key factor in promoting cancer progression, metastasis, and resistance. Tumor alkalization therapy has emerged as a potential strategy for cancer treatment. This article provides preclinical and clinical evidence for tumor alkalization therapy as a promising cancer treatment strategy. The potential of tumor alkalization therapy using sodium bicarbonate in the treatment of malignant ascites was studied. The concept of intraperitoneal perfusion with an alkalizing solution to increase the extracellular pH and its antitumor effect were explored. The significant extension in the overall survival of the Ehrlich ascites carcinoma mice treated with sodium bicarbonate solution compared to those treated with a sodium chloride solution was observed. In the sodium bicarbonate group, mice had a median survival of 30 days after tumor cell injection, which was significantly (p<0.05) different from the median survival of 18 days in the sodium chloride group and 14 days in the intact group. We also performed a case study of a patient with ovarian cancer malignant ascites resistant to previous lines of chemotherapy who underwent intraperitoneal perfusions with a sodium bicarbonate solution, resulting in a significant drop of CA-125 levels from 5600 U/mL to 2200 U/mL in and disappearance of ascites, indicating the potential effectiveness of the treatment. The preclinical and clinical results obtained using sodium bicarbonate perfusion in the treatment of malignant ascites represent a small yet significant contribution to the evolving field of tumor alkalization as a cancer therapy. They unequivocally affirm the good prospects of this concept.

OCT4 Expression in Gliomas Is Dependent on Cell Metabolism.

The OCT4 transcription factor is necessary to maintain cell stemness in the early stages of embryogenesis and is involved in the formation of induced pluripotent stem cells, but its role in oncogenesis is not yet entirely clear. In this work, OCT4 expression was investigated in malignant gliomas. Twenty glioma cell lines and a sample of normal adult brain tissue were used. OCT4 expression was found in all studied glioma cell lines but was not detected in normal adult brain tissue. For one of these lines, OCT4 knockdown caused tumor cell death. By varying the culture conditions of these cells, we unexpectedly found that OCT4 expression increased when cells were incubated in serum-free medium, and this effect was significantly enhanced in serum-free and L-glutamine-free medium. L-glutamine and the Krebs cycle, which is slowed down in serum-free medium according to our NMR data, are sources of α-KG. Thus, our data indicate that OCT4 expression in gliomas may be regulated by the α-KG-dependent metabolic reprogramming of cells.

Radiosensitizing Effect of Dextran-Coated Iron Oxide Nanoparticles on Malignant Glioma Cells.

The potential of standard methods of radiation therapy is limited by the dose that can be safely delivered to the tumor, which could be too low for radical treatment. The dose efficiency can be increased by using radiosensitizers. In this study, we evaluated the sensitizing potential of biocompatible iron oxide nanoparticles coated with a dextran shell in A172 and Gl-Tr glioblastoma cells in vitro. The cells preincubated with nanoparticles for 24 h were exposed to ionizing radiation (X-ray, gamma, or proton) at doses of 0.5-6 Gy, and their viability was assessed by the Resazurin assay and by staining of the surviving cells with crystal violet. A statistically significant effect of radiosensitization by nanoparticles was observed in both cell lines when cells were exposed to 35 keV X-rays. A weak radiosensitizing effect was found only in the Gl-Tr line for the 1.2 MeV gamma irradiation and there was no radiosensitizing effect in both lines for the 200 MeV proton irradiation at the Bragg peak. A slight (ca. 10%) increase in the formation of additional reactive oxygen species after X-ray irradiation was found when nanoparticles were present. These results suggest that the nanoparticles absorbed by glioma cells can produce a significant radiosensitizing effect, probably due to the action of secondary electrons generated by the magnetite core, whereas the dextran shell of the nanoparticles used in these experiments appears to be rather stable under radiation exposure.

Fullerenes on a Nanodiamond Platform Demonstrate Antibacterial Activity with Low Cytotoxicity.

Carbon nanoparticles with antimicrobial properties, such as fullerenes, can be distinguished among the promising means of combating pathogens characterized by resistance to commercial antibiotics. However, they have a number of limitations for their use in medicine. In particular, the insolubility of carbon nanoparticles in water leads to a low biocompatibility and especially strong aggregation when transferred to liquid media. To overcome the negative factors and enhance the action of fullerenes in an extended range of applications, for example, in antimicrobial photodynamic therapy, we created new water-soluble complexes containing, in addition to C60 fullerene, purified detonation nanodiamonds (AC960) and/or polyvinylpyrrolidone (PVP). The in vitro antibacterial activity and toxicity to human cells of the three-component complex C60+AC960+PVP were analyzed in comparison with binary C60+PVP and C60+AC960. All complexes showed a low toxicity to cultured human skin fibroblasts and ECV lines, as well as significant antimicrobial activity, which depend on the type of microorganisms exposed, the chemical composition of the complex, its dosage and exposure time. Complex C60+PVP+AC960 at a concentration of 175 µg/mL showed the most stable and pronounced inhibitory microbicidal/microbiostatic effect.

Experimental validation of proton boron capture therapy for glioma cells.

Proton boron capture therapy (PBCT) has emerged from particle acceleration research for enhancing the biological effectiveness of proton therapy. The mechanism responsible for the dose increase was supposed to be related to proton-boron fusion reactions (11B + p → 3α + 8.7 MeV). There has been some experimental evidence that the biological efficiency of protons is significantly higher for boron-11-containing prostate or breast cancer cells. The aim of this study was to evaluate the sensitizing potential of sodium borocaptate (BSH) under proton irradiation at the Bragg peak of cultured glioma cells. To address this problem, cells of two glioma lines were preincubated with 80 or 160 ppm boron-11, irradiated both at the middle of 200 MeV beam Spread-Out Bragg Peak (SOBP) and at the distal end of the 89.7 MeV beam SOBP and assessed for the viability, as well as their ability to form colonies. Our results clearly show that BSH provides for only a slight, if any, enhancement of the effect of proton radiation on the glioma cells in vitro. In addition, we repeated the experiments using the Du145 prostate cancer cell line, for which an increase in the biological efficiency of proton irradiation in the presence of sodium borocaptate was demonstrated previously. The data presented add new argument against the efficiency of proton boron capture therapy when based solely on direct dose-enhancement effect by the proton capture nuclear reaction, underlining the need to investigate the indirect effects of the secondary alpha irradiation depending on the state and treatment conditions of the irradiated tissue.

Exosomes Released by Influenza-Virus-Infected Cells Carry Factors Capable of Suppressing Immune Defense Genes in Naïve Cells.

Background: Exosomes are involved in intercellular communication and can transfer regulatory molecules between cells. Consequently, they can participate in host immune response regulation. For the influenza A virus (IAV), there is very limited information on changes in exosome composition during cell infection shedding light on the potential role of these extracellular membrane vesicles. Thus, the aim of our work was to study changes in exosomal composition following IAV infection of cells, as well as to evaluate their effect on uninfected cells. Methods: To characterize changes in the composition of cellular miRNAs and mRNAs of exosomes during IAV infection of A549 cells, NGS was used, as well as PCR to identify viral genes. Naïve A549 cells were stimulated with infected-cell-secreted exosomes for studying their activity. Changes in the expression of genes associated with the cell's immune response were shown using PCR. The effect of exosomes on IAV replication was shown in MDCK cells using In-Cell ELISA and PCR of the supernatants. Results: A change in the miRNA composition (miR-21-3p, miR-26a-5p, miR-23a-5p, miR-548c-5p) and mRNA composition (RPL13A, MKNK2, TRIB3) of exosomes under the influence of the IAV was shown. Many RNAs were involved in the regulation of the immune response of the cell, mainly by suppressing it. After exosome stimulation of naïve cells, a significant decrease in the expression of genes involved in the immune response was shown (RIG1, IFIT1, MDA5, COX2, NFκB, AnxA1, PKR, IL6, IL18). When infecting MDCK cells, a significant decrease in nucleoprotein levels was observed in the presence of exosomes secreted by mock-infected cells. Viral levels in supernatants also decreased. Conclusions: Exosomes secreted by IAV-infected cells could reduce the immune response of neighboring intact cells, leading to more effective IAV replication. This may be associated both with regulatory functions of cellular miRNAs and mRNAs carried by exosomes, or with the presence of viral mRNAs encoding proteins with an immunosuppressive function.

Extracellular Particles as Carriers of Cholesterol Not Associated with Lipoproteins.

Exosomes and exomeres are the smallest microparticles ranging from 20 to 130 nm in diameter. They are found in almost all biological fluids. Exosomes and exomeres are of considerable interest since they can be involved in intercellular signaling and are biological markers of the state of cells, which can be used for diagnostics. The nomenclature of exosomes remains poorly developed. Most researchers try to classify them based on the mode of formation, physicochemical characteristics, and the presence of tetrasporin markers CD9, CD63, and CD81. The data presented in this work show that although exomeres carry tetrasporin biomarkers, they differ from exosomes strongly in lipid composition, especially in cholesterol content. The production of exomeres by cells is associated with the synthesis of cholesterol in cells and is expressed or suppressed by regulators of the synthesis of mevalonate, an intermediate product of cholesterol metabolism. In addition, the work shows that the concentration of extracellular particles in the body correlates with the concentration of cholesterol in the plasma, but weakly correlates with the concentration of cholesterol in lipoproteins. This suggests that not all plasma cholesterol is associated with lipoproteins, as previously thought.

Nanomechanical characterization of exosomes and concomitant nanoparticles from blood plasma by PeakForce AFM in liquid.

BACKGROUND: To date, EVs characterization techniques are extremely diverse. The contribution of AFM, in particular, is often confined to size distribution. While AFM provides a unique possibility to carry out measurements in situ, nanomechanical characterization of EVs is still missing. METHODS: Blood plasma EVs were isolated by ultracentrifugation, analyzed by flow cytometry and NTA. Followed by cryo-EM, we applied PeakForce AFM to assess morphological and nanomechanical properties of EVs in liquid. RESULTS: Nanoparticles were subdivided by their size estimated for their suspended state into sub-sets of small S1-EVs (< 30 nm), S2-EVs (30-50 nm), and sub-set of large ones L-EVs (50-170 nm). Non-membranous S1-EVs were distinguished by higher Young's modulus (10.33(7.36;15.25) MPa) and were less deformed by AFM tip (3.6(2.8;4.4) nm) compared to membrane exosomes S2-EVs (6.25(4.52;8.24) MPa and 4.8(4.3;5.9) nm). L-EVs were identified as large membrane exosomes, heterogeneous by their nanomechanical properties (22.43(8.26;53.11) MPa and 3.57(2.07;7.89) nm). Nanomechanical mapping revealed a few non-deformed L-EVs, of which Young's modulus rose up to 300 MPa. Taken together with cryo-EM, these results lead us to the suggestion that two or more vesicles could be contained inside a large one being a multilayer vesicle. CONCLUSIONS: We identified particles similar in morphology and showed differences in nanomechanical properties that could be attributed to the features of their inner structure. GENERAL SIGNIFICANCE: Our results further elucidate the identification of EVs and concomitant nanoparticles based on their nanomechanical properties.

Delivery of functional exogenous proteins by plant-derived vesicles to human cells in vitro.

Plant-derived extracellular vesicles (EVs) gain more and more attention as promising carriers of exogenous bioactive molecules to the human cells. Derived from various edible sources, these EVs are remarkably biocompatible, biodegradable and highly abundant from plants. In this work, EVs from grapefruit juice were isolated by differential centrifugation followed by characterization of their size, quantity and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments, we visualized grapefruit EVs with the average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Further, using cell culture models, we have successfully demonstrated that native grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa Fluor 647 labeled bovine serum albumin (BSA) and heat shock protein 70 (HSP70) into both human peripheral blood mononuclear cells and colon cancer cells. Interestingly, loading to plant EVs significantly ameliorated the uptake of exogenous proteins by human cells compared to the same proteins without EVs. Most importantly, we have confirmed the functional activity of human recombinant HSP70 in the colon cancer cell culture upon delivery by GF-EVs. Analysis of the biodistribution of GF-EVs loaded with 125I-labeled BSA in mice demonstrated a significant uptake of the grapefruit-derived extracellular vesicles by the majority of organs. The results of our study indicate that native plant EVs might be safe and effective carriers of exogenous proteins into human cells.

Biomechanical Properties of Blood Plasma Extracellular Vesicles Revealed by Atomic Force Microscopy.

While extracellular vesicles (EVs) are extensively studied by various practical applications in biomedicine, there is still little information on their biomechanical properties due to their nanoscale size. We identified isolated blood plasma vesicles that carried on biomarkers associated with exosomes and exomeres and applied atomic force microscopy (AFM) to study them at single particle level in air and in liquid. Air measurements of exosomes revealed a mechanically indented internal cavity in which highly adhesive sites were located. In contrast, the highly adhesive sites of exomeres were located at the periphery and the observed diameter of the particles was ~35 nm. In liquid, the reversible deformation of the internal cavity of exosomes was observed and a slightly deformed lipid bi-layer was identified. In contrast, exomeres were not deformed and their observed diameter was ~16 nm. The difference in diameters might be associated with a higher sorption of water film in air. The parameters we revealed correlated with the well-known structure and function for exosomes and were observed for exomeres for the first time. Our data provide a new insight into the biomechanical properties of nanoparticles and positioned AFM as an exclusive source of in situ information about their biophysical characteristics.

Evaluation of immune and chemical precipitation methods for plasma exosome isolation.

Exosomes are a type of extracellular vesicles (EVs) secreted by multiple mammalian cell types and involved in intercellular communication. Numerous studies have explored the diagnostic and therapeutic potential of exosomes. The key challenge is the lack of efficient and standard techniques for isolation and downstream analysis of nanovesicles. Conventional isolation methods, such as ultracentrifugation, precipitation, filtration, chromatography, and immune-affinity-based approaches, rely on specific physical properties or on surface biomarkers. However, any of the existing methods has its limitations. Various parameters, such as efficacy, specificity, labor input, cost and scalability, and standardization options, must be considered for the correct choice of appropriate approach. The isolation of exosomes from biological fluids is especially challenged by the complex nature and variability of these liquids. Here, we present a comparison of five protocols for exosome isolation from human plasma: two chemical affinity precipitation methods (lectin-based purification and SubX™ technology), immunoaffinity precipitation, and reference ultracentrifugation-based exosome isolation method in two modifications. An approach for the isolation of exosomes based on the phenomenon of binding and aggregation of these particles via clusters of outer membrane phosphate groups in the presence of SubX™ molecules has been put forward in the present study. The isolated EVs were characterized based upon size, quantity, and protein content.

Crystal and Supramolecular Structure of Bacterial Cellulose Hydrolyzed by Cellobiohydrolase from Scytalidium Candidum 3C: A Basis for Development of Biodegradable Wound Dressings.

The crystal and supramolecular structure of the bacterial cellulose (BC) has been studied at different stages of cellobiohydrolase hydrolysis using various physical and microscopic methods. Enzymatic hydrolysis significantly affected the crystal and supramolecular structure of native BC, in which the 3D polymer network consisted of nanoribbons with a thickness T ≈ 8 nm and a width W ≈ 50 nm, and with a developed specific surface SBET ≈ 260 m2·g-1. Biodegradation for 24 h led to a ten percent decrease in the mean crystal size Dhkl of BC, to two-fold increase in the sizes of nanoribbons, and in the specific surface area SBET up to ≈ 100 m2·g-1. Atomic force and scanning electron microscopy images showed BC microstructure "loosening"after enzymatic treatment, as well as the formation and accumulation of submicron particles in the cells of the 3D polymer network. Experiments in vitro and in vivo did not reveal cytotoxic effect by the enzyme addition to BC dressings and showed a generally positive influence on the treatment of extensive III-degree burns, significantly accelerating wound healing in rats. Thus, in our opinion, the results obtained can serve as a basis for further development of effective biodegradable dressings for wound healing.

Abnormal activity of transcription factors gli in high-grade gliomas.

Malignant transformation is associated with loss of cell differentiation, anaplasia. Transcription factors gli, required for embryonic development, may be involved in this process. We studied the activity of transcription factors gli in high-grade gliomas and their role in maintenance of stem cell state and glioma cell survival. 20 glioma cell lines and a sample of a normal adult brain tissue were used in the present study. We found the expression of gli target genes, including GLI1 and FOXM1, in all tested glioma cell lines, but not in the normal tissue. Interestingly, the expression of gli target genes in some glioma cell lines was observed together with a high level of their transcriptional repressor, Gli3R. Knockdown of GLI3 in one of these lines resulted in decrease of gli target gene expression. These data suggest that Gli3R does not prevent the gli target genes transcription, and gli3 acts in glioma cells more as an activator, than a repressor of transcription. We observed that gli regulated the expression of such genes, as SOX2 or OCT4 that maintain stem cell state, and TET1, involving in DNA demethylation. Treatment with GANT61 or siRNA against GLI1, GLI2, or GLI3 could result in complete glioma cell death, while cyclopamine had a weaker and line-specific effect on glioma cell survival. Thus, the gli transcription factors are abnormally active in high-grade gliomas, regulate expression of genes, maintaining the stem cell state, and contribute to glioma cell survival.

Plasma exosomal miR-21 and miR-181a differentiates follicular from papillary thyroid cancer.

Thyroid cancer (TC) is the most common endocrine malignancy and its incidence has increased over the last few decades. As has been revealed by a number of studies, TC tissue's micro-RNA (miRNA) profile may reflect histological features and the clinical behavior of tumor. However, alteration of the miRNA profile of plasma exosomes associated with TC development has to date not been explored. We isolated exosomes from plasma and assayed their characteristics using laser diffraction particle size analysis, atomic force microscopy, and western blotting. Next, we profiled cancer-associated miRNAs in plasma exosomes obtained from papillary TC patients, before and after surgical removal of the tumor. The diagnostic value of selected miRNAs was evaluated in a large cohort of patients displaying different statuses of thyroid nodule disease. MiRNA assessment was performed by RT-qPCR. In total, 60 patients with different types of thyroid nodal pathology were included in the study. Our results revealed that the development of papillary TC is associated with specific changes in exosomal miRNA profiles; this phenomenon can be used for differential diagnostics. MiRNA-31 was found to be over-represented in the plasma exosomes of patients with papillary TC vs. benign tumors, while miRNA-21 helped to distinguish between benign tumors and follicular TC. MiRNA-21 and MiRNA-181a-5p were found to be expressed reciprocally in the exosomes of patients with papillary and follicular TC, and their comparative assessment may help to distinguish between these types of TC with 100 % sensitivity and 77 % specificity.

Lectin-induced agglutination method of urinary exosomes isolation followed by mi-RNA analysis: Application for prostate cancer diagnostic.

BACKGROUND: Prostate cancer is the most common cancer in men. Prostate-specific antigen has, however, insufficient diagnostic specificity. Novel complementary diagnostic approaches are greatly needed. MiRNAs are small regulatory RNAs which play an important role in tumorogenesis and are being investigated as a cancer biomarker. In addition to their intracellular regulatory functions, miRNAs are secreted into the extracellular space and can be found in various body fluids, including urine. The stability of extracellular miRNAs is defined by association with proteins, lipoprotein particles, and membrane vesicles. Among the known forms of miRNA packaging, tumour-derived exosome-enclosed miRNAs is thought to reflect the vital activity of cancer cells. The assessment of the exosomal fraction of urinary miRNA may present a new and highly specific method for prostate cancer diagnostics; however, this is challenged by the absence of reliable and inexpensive methods for isolation of exosomes. METHODS: Prostate cancer (PC) cell lines and urine samples collected from 35 PC patients and 35 healthy donors were used in the study. Lectins, phytohemagglutinin, and concanavalin A were used to induce agglutination of exosomes. The efficiency of isolation process was evaluated by AFM and DLS assays. The protein content of isolated exosomes was analysed by western blotting. Exosomal RNA was assayed by automated electrophoresis and expression level of selected miRNAs was evaluated by RT-qPCR. The diagnostic potency of the urinary exosomal miRNA assessment was estimated by the ROC method. RESULTS: The formation of multi-vesicular agglutinates in urine can be induced by incubation with lectin at a final concentration of 2 mg/ml. These agglutinates contain urinary exosomes and may be pelleted by centrifugation with a relatively low G-force. The analysis of PC-related miRNA in urinary exosomes revealed significant up-regulation of miR-574-3p, miR-141-5p, and miR-21-5p associated with PC. CONCLUSIONS: Lectin-induced aggregation is a low-cost and easily performed method for isolation of exosomes from urine. Isolated exosomes can be further analysed in terms of miRNA content. The miRNA profile of urinary exosomes reflects development of prostate cancer and may present a promising diagnostic tool.