Assessment of the effects of amphiphilic poly (N­vinylpyrrolidone) nanoparticles loaded with bortezomib on glioblastoma cell lines and zebrafish embryos.

Proteasome inhibitor bortezomib is an anticancer agent approved for treatment of multiple myeloma and mantle cell lymphoma. However, its application in other types of cancer, primarily in solid tumors, is limited due to poor pharmacokinetics, inefficient tissue penetration, low stability and frequent adverse effects. In the present study, a novel micellar nano-scaled delivery system was manufactured, composed of amphiphilic poly(N-vinylpyrrolidone) nanoparticles loaded with bortezomib. Similar nanoparticles loaded with prothionamide, a drug without anticancer effect, were used as control. The size and zeta potential of the obtained polymeric micelles were measured by dynamic light scattering. Bortezomib-loaded micelles exhibited significant cytotoxic activity in vitro in monolayer tumor cell cultures (IC50 ~6.5 µg/ml) and in 3D multicellular tumor spheroids (IC50 ~8.5 µg/ml) of human glioblastoma cell lines U87 and T98G. Additionally, the toxic effects in vivo were studied in zebrafish Danio rerio embryos, with an estimated 50% lethal concentration of 0.1 mg/ml. Considering that bortezomib and other molecules from the class of proteasome inhibitors are potent antitumor agents, nanodelivery approach can help reduce adverse effects and expand the range of its applications for treatment of various oncological diseases.

Retrovirus-like gag protein Arc/Arg3.1 is involved in extracellular-vesicle-mediated mRNA transfer between glioma cells.

BACKGROUND: Activity-regulated cytoskeleton-associated (Arc) protein is predominantly expressed in excitatory glutamatergic neurons of vertebrates, where it plays a pivotal role in regulation of synaptic plasticity. Arc protein forms capsid-like particles, which can encapsulate and transfer mRNA in extracellular vesicles (EVs) between hippocampal neurons. Once glioma cell networks actively interact with neurons via paracrine signaling and formation of neurogliomal glutamatergic synapses, we predicted the involvement of Arc in a process of EV-mediated mRNA transfer between glioma cells. MATERIALS AND METHODS: Arc expression in three human glioma cell lines was evaluated by WB and immunocytochemistry. The properties of Arc protein/mRNA-containing EVs produced by glioma cells were analyzed by RT-PCR, TEM, and WB. Flow cytometry, RT-PCR, and fluorescent microscopy were used to show the involvement of Arc in EV-mediated mRNA transfer between glioma cells. RESULTS: It was found that human glioma cells can produce EVs containing Arc/Arg3.1 protein and Arc mRNA (or "Arc EVs"). Arc EVs from U87 glioma cells internalize and deliver Arc mRNA to recipient U87 cells, where it is translated into a protein. Arc overexpression significantly increases EV production, alters EV morphology, and enhances intercellular transfer of highly expressed mRNA in glioma cell culture. CONCLUSION: These findings indicate involvement of Arc EVs into mRNA transfer between glioma cells that could contribute to tumor progression and affect synaptic plasticity in cancer patients.

Toxicity Evaluation and Controlled-Release of Curcumin-Loaded Amphiphilic Poly-N-vinylpyrrolidone Nanoparticles: In Vitro and In Vivo Models.

Curcumin attracts huge attention because of its biological properties: it is antiproliferative, antioxidant, anti-inflammatory, immunomodulatory and so on. However, its usage has been limited by poor water solubility and low bioavailability. Herein, to solve these problems, we developed curcumin-loaded nanoparticles based on end-capped amphiphilic poly(N-vinylpyrrolidone). Nanoparticles were obtained using the solvent evaporation method and were characterized by dynamic and electrophoretic light scattering, transmission electron (TEM) and atomic force (AFM) microscopy. The average particle size was 200 nm, and the ζ-potential was -4 mV. Curcumin-release studies showed that nanoparticles are stable in aqueous solutions. An in vitro release study showed prolonged action in gastric, intestinal and colonic fluids, consistently, and in PBS. In vitro studies on epidermoid carcinoma and human embryonic kidney cells showed that the cells absorbed more curcumin in nanoparticles compared to free curcumin. Nanoparticles are safe for healthy cells and show high cytotoxicity for glioblastoma cells in cytotoxicity studies in vitro. The median lethal dose was determined in an acute toxicity assay on zebrafish and was 23 µM. Overall, the curcumin-loaded nanoparticles seem promising for cancer treatment.

Comparison of Methods for MicroRNA Isolation from Extracellular Vesicles Obtained from Ascitic Fluids.

Secreted extracellular vesicles (EVs) contain active biomolecules, including miRNAs, composition of which reflects epigenetic changes occurring in cells during pathological processes, in particular, malignant transformation. The accumulated pool of data on the role of EVs in carcinogenesis has stimulated investigations of the EV-derived cancer markers. The most important factor limiting development of this scientific direction is lack of "gold standards" both for methods of EV isolation from biological fluids and for analyzing their molecular content, including composition of miRNAs. Here we first examined efficacy of various methods for small RNA isolation from EVs contained in ascitic fluid for subsequent miRNA analysis. Comparison of different commercial kits showed advantages of the methods based on phenol-chloroform extraction: Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit. Analysis of the small RNA transcriptome showed presence of various classes of molecules in the EVs, among which proportion of miRNAs averaged 6% and reaching 10% with the Total Exosome RNA & Protein Isolation Kit. The PureLink miRNA Isolation Kit demonstrated the lowest efficiency. The miRNeasy Advanced Serum/Plasma Kit showed the highest concentration of the small RNA fraction, miRNA proportion of which, however, did not exceed that obtained with the miRNeasy Serum/Plasma Kit and Total Exosome RNA & Protein Isolation Kit. Moreover, RT-PCR analysis of the individual molecules showed lower levels of each of investigated miRNAs (miR-1246, miR-200b-5p, miR-200c-3p, and miR-23a-3p) when using the miRNeasy Advanced Serum/Plasma Kit. In conclusion, Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit can be considered as optimal kits in terms of performance based on combination of the studied characteristics, including small RNA concentration, percentage of microRNA according to bioanalyzer and sequencing results, and levels of individual miRNAs detected by RT-PCR.

Nanoaggregates of Biphilic Carboxyl-Containing Copolymers as Carriers for Ionically Bound Doxorubicin.

Application of nanocarriers for drug delivery brings numerous advantages, allowing both minimization of side effects common in systemic drug delivery and improvement in targeting, which has made it the focal point of nanoscience for a number of years. While most of the studies are focused on encapsulation of hydrophobic drugs, delivery of hydrophilic compounds is typically performed via covalent attachment, which often requires chemical modification of the drug and limits the release kinetics. In this paper, we report synthesis of biphilic copolymers of various compositions capable of self-assembly in water with the formation of nanoparticles and suitable for ionic binding of the common anticancer drug doxorubicin. The copolymers are synthesized by radical copolymerization of N-vinyl-2-pyrrolidone and acrylic acid using n-octadecyl-mercaptan as a chain transfer agent. With an increase of the carboxyl group's share in the chain, the role of the electrostatic stabilization factor of the nanoparticles increased as well as the ability of doxorubicin as an ion binder. A mathematical description of the kinetics of doxorubicin binding and release is given and thermodynamic functions for the equilibrium ionic binding of doxorubicin are calculated.

Highly Sensitive Nanomagnetic Quantification of Extracellular Vesicles by Immunochromatographic Strips: A Tool for Liquid Biopsy.

Extracellular vesicles (EVs) are promising agents for liquid biopsy-a non-invasive approach for the diagnosis of cancer and evaluation of therapy response. However, EV potential is limited by the lack of sufficiently sensitive, time-, and cost-efficient methods for their registration. This research aimed at developing a highly sensitive and easy-to-use immunochromatographic tool based on magnetic nanoparticles for EV quantification. The tool is demonstrated by detection of EVs isolated from cell culture supernatants and various body fluids using characteristic biomarkers, CD9 and CD81, and a tumor-associated marker-epithelial cell adhesion molecules. The detection limit of 3.7 × 105 EV/µL is one to two orders better than the most sensitive traditional lateral flow system and commercial ELISA kits. The detection specificity is ensured by an isotype control line on the test strip. The tool's advantages are due to the spatial quantification of EV-bound magnetic nanolabels within the strip volume by an original electronic technique. The inexpensive tool, promising for liquid biopsy in daily clinical routines, can be extended to other relevant biomarkers.

Extracellular Vesicles from Uterine Aspirates Represent a Promising Source for Screening Markers of Gynecologic Cancers.

Extracellular vesicles (EVs), including exosomes, are key factors of intercellular communication, performing both local and distant transfers of bioactive molecules. The increasingly obvious role of EVs in carcinogenesis, similarity of molecular signatures with parental cells, precise selection and high stability of cargo molecules make exosomes a promising source of liquid biopsy markers for cancer diagnosis. The uterine cavity fluid, unlike blood, urine and other body fluids commonly used to study EVs, is of local origin and therefore enriched in EVs secreted by cells of the female reproductive tract. Here, we show that EVs, including those corresponding to exosomes, could be isolated from individual samples of uterine aspirates (UA) obtained from epithelial ovarian cancer (EOC) patients and healthy donors using the ultracentrifugation technique. First, the conducted profiling of small RNAs (small RNA-seq) from UA-derived EVs demonstrated the presence of non-coding RNA molecules belonging to various classes. The analysis of the miRNA content in EVs from UA performed on a pilot sample revealed significant differences in the expression levels of a number of miRNAs in EVs obtained from EOC patients compared to healthy individuals. The results open up prospects for using UA-derived EVs as a source of markers for the diagnostics of gynecological cancers, including EOC.

Antigen-Specific Stimulation and Expansion of CAR-T Cells Using Membrane Vesicles as Target Cell Surrogates.

Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low-efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. To develop a specific and improved protocol for CAR-T cell expansion, cell-derived membrane vesicles are taken advantage of, and the simple structural demands of the CAR-antigen interaction. This novel approach is to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens, and then use them for stimulation and expansion of CAR-T cells. The data presented in this article clearly demonstrate that this protocol produced antigen-specific vesicles with the capacity to induce stronger stimulation, proliferation, and functional activity of CAR-T cells than is possible with existing protocols. It is predicted that this new methodology will significantly advance the ability to obtain improved populations of functional CAR-T cells for therapy.

Amphiphilic Poly(N-vinylpyrrolidone) Nanoparticles Conjugated with DR5-Specific Antitumor Cytokine DR5-B for Targeted Delivery to Cancer Cells.

Nanoparticles based on the biocompatible amphiphilic poly(N-vinylpyrrolidone) (Amph-PVP) derivatives are promising for drug delivery. Amph-PVPs self-aggregate in aqueous solutions with the formation of micellar nanoscaled structures. Amph-PVP nanoparticles are able to immobilize therapeutic molecules under mild conditions. As is well known, many efforts have been made to exploit the DR5-dependent apoptosis induction for cancer treatment. The aim of the study was to fabricate Amph-PVP-based nanoparticles covalently conjugated with antitumor DR5-specific TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) variant DR5-B and to evaluate their in vitro cytotoxicity in 3D tumor spheroids. The Amph-PVP nanoparticles were obtained from a 1:1 mixture of unmodified and maleimide-modified polymeric chains, while DR5-B protein was modified by cysteine residue at the N-end for covalent conjugation with Amph-PVP. The nanoparticles were found to enhance cytotoxicity effects compared to those of free DR5-B in both 2D (monolayer culture) and 3D (tumor spheroids) in vitro models. The cytotoxicity of the nanoparticles was investigated in human cell lines, namely breast adenocarcinoma MCF-7 and colorectal carcinomas HCT116 and HT29. Notably, DR5-B conjugation with Amph-PVP nanoparticles sensitized resistant multicellular tumor spheroids from MCF-7 and HT29 cells. Taking into account the nanoparticles loading ability with a wide range of low-molecular-weight antitumor chemotherapeutics into hydrophobic core and feasibility of conjugation with hydrophilic therapeutic molecules by click chemistry, we suggest further development to obtain a versatile system for targeted drug delivery into tumor cells.

Secretion of Mutant DNA and mRNA by the Exosomes of Breast Cancer Cells.

Exosomes are the small vesicles that are secreted by different types of normal and tumour cells and can incorporate and transfer their cargo to the recipient cells. The main goal of the present work was to study the tumour exosomes' ability to accumulate the parent mutant DNA or RNA transcripts with their following transfer to the surrounding cells. The experiments were performed on the MCF7 breast cancer cells that are characterized by the unique coding mutation in the PIK3CA gene. Using two independent methods, Sanger sequencing and allele-specific real-time PCR, we revealed the presence of the fragments of the mutant DNA and RNA transcripts in the exosomes secreted by the MCF7 cells. Furthermore, we demonstrated the MCF7 exosomes' ability to incorporate into the heterologous MDA-MB-231 breast cancer cells supporting the possible transferring of the exosomal cargo into the recipient cells. Sanger sequencing of the DNA from MDA-MB-231 cells (originally bearing a wild type of PIK3CA) treated with MCF7 exosomes showed no detectable amount of mutant DNA or RNA; however, using allele-specific real-time PCR, we revealed a minor signal from amplification of a mutant allele, showing a slight increase of mutant DNA in the exosome-treated MDA-MB-231 cells. The results demonstrate the exosome-mediated secretion of the fragments of mutant DNA and mRNA by the cancer cells and the exosomes' ability to transfer their cargo into the heterologous cells.

Stomatin is highly expressed in exosomes of different origin and is a promising candidate as an exosomal marker.

Proteins involved in the organizing of lipid rafts can be found in exosomes, as shown for caveolin-1, and they could contribute to exosomal cargo sorting, as shown for flotillins. Stomatin belongs to the same stomatin/prohibitin/flotillin/HflK/C family of lipid rafts proteins, but it has never been studied in exosomes except for extracellular vesicles (EVs) originating from blood cells. Here we first show the presence of stomatin in exosomes produced by epithelial cancer cells (non-small cell lung cancer, breast, and ovarian cancer cells) as well as in EVs from biological fluids, including blood plasma, ascitic fluids, and uterine flushings. A high abundance of stomatin in EVs of various origins and its enrichment in exosomes make stomatin a promising exosomal marker. Comparison with other lipid raft proteins and exosomal markers showed that the level of stomatin protein in exosomes from different sources corresponds well to that of CD9, while it differs essentially from flotillin-1 and flotillin-2 homologs, which in turn are present in exosomes in nearly equal proportions. In contrast, the level of vesicular caveolin-1 as well as its EV-to-cellular ratio vary drastically depending on cell type.

Adsorption of extracellular vesicles onto the tube walls during storage in solution.

Short term storage of extracellular vesicle (EV) solutions at +4°C is a common practice, but the stability of EVs during this procedure has not been fully understood yet. Using nanoparticle tracking analysis, we have shown that EVs isolated from the conditioned medium of HT-29 cells exhibit a pronounced concentration decrease when stored in PBS in ordinary polypropylene tubes within the range of (0.5-2.1) × 1010 particles/ml. EV losses reach 51±3% for 0.5 ml of EVs in Eppendorf 2 ml tube at 48 hours of storage at +4°C. Around 2/3 of the observed losses have been attributed to the adsorption of vesicles onto tube walls. This result shows that the lower part (up to at least 2 × 1010 particles/ml) of the practically relevant concentration range for purified EVs is prone to adsorption losses at +4°C. Total particle losses could be reduced to 18-21% at 48 hours by using either Eppendorf Protein LoBind tubes or ordinary tubes with the surface blocked with bovine serum albumin or EVs. Reduction of losses to 15% has been shown for isolated EVs dissolved in the supernatant after 100 000 g centrifugation as a model of conditioned medium. Also, a previously unknown feature of diffusion-controlled adsorption was revealed for EVs. In addition to the decrease in particle count, this process causes the predominant losses of smaller particles.

Protein nanoparticles with ligand-binding and enzymatic activities.

PURPOSE: To develop a general method for NP fabrication from various proteins with maintenance of biological activity. METHODS: A novel general approach for producing protein nanoparticles (NP) by nanoprecipitation of the protein solutions in 1,1,1,3,3,3-hexafluoroisopropanol is described. Protein NP sizes and shapes were analyzed by dynamic light scattering, scanning electron and atomic force microscopy (SEM and AFM). Chemical composition of the NP was confirmed using ultraviolet (UV) spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and circular dichroism (CD). Biological properties of the NP were analyzed in ELISA, immunofluorescent analysis and lysozyme activity assay. RESULTS: Water-insoluble NP were constructed from globular (bovine serum albumin (BSA), lysozyme, immunoglobulins), fibrillar (fibrinogen) proteins and linear polylysines by means of nanoprecipitation of protein solutions in fluoroalcohols. AFM and SEM revealed NP sizes of 20-250 nm. The NP chemical structure was confirmed by UV spectroscopy, protease digestion and EDX spectroscopy. CD spectra revealed a stable secondary structure of proteins in NP. The UV spectra, microscopy and SDS-PAA gel electrophoresis (PAGE) proved the NP stability at +4°C for 7 months. Co-precipitation of proteins with fluorophores or nanoprecipitation of pre-labeled BSA resulted in fluorescent NP that retained antigenic structures as shown by their binding with specific antibodies. Moreover, NP from monoclonal antibodies could bind with the hepatitis B virus antigen S. Besides that, lysozyme NP could digest bacterial cellular walls. CONCLUSION: Thus, the water-insoluble, stable protein NP were produced by nanoprecipitation without cross-linking and retained ligand-binding and enzymatic activities.

Exosome-Mediated Transfer of Cancer Cell Resistance to Antiestrogen Drugs.

Exosomes are small vesicles which are produced by the cells and released into the surrounding space. They can transfer biomolecules into recipient cells. The main goal of the work was to study the exosome involvement in the cell transfer of hormonal resistance. The experiments were performed on in vitro cultured estrogen-dependent MCF-7 breast cancer cells and MCF-7 sublines resistant to SERM tamoxifen and/or biguanide metformin, which exerts its anti-proliferative effect, at least in a part, via the suppression of estrogen machinery. The exosomes were purified by differential ultracentrifugation, cell response to tamoxifen was determined by MTT test, and the level and activity of signaling proteins were determined by Western blot and reporter analysis. We found that the treatment of the parent MCF-7 cells with exosomes from the resistant cells within 14 days lead to the partial resistance of the MCF-7 cells to antiestrogen drugs. The primary resistant cells and the cells with the exosome-induced resistance were characterized with these common features: decrease in ERα activity and parallel activation of Akt and AP-1, NF-κB, and SNAIL1 transcriptional factors. In general, we evaluate the established results as the evidence of the possible exosome involvement in the transferring of the hormone/metformin resistance in breast cancer cells.

Application of vasoactive and matrix-modifying drugs can improve polyplex delivery to tumors upon intravenous administration.

Low efficacy of cationic polymer-based formulations (polyplexes) for systemic gene delivery to tumors remains the crucial concern for their clinical translation. Here we show that modulating the physiological state of a tumor using clinically approved pharmaceuticals can improve delivery of intravenously injected polyplexes to murine melanoma tumors with different characteristics. Direct comparison of drugs with different mechanisms of action has shown that application of nitroglycerin or losartan improved extravasation and tumor uptake of polyplex nanoparticles, whereas angiotensin II had almost no effect on polyplex accumulation and microdistribution in the tumor tissue. Application of nitroglycerin and losartan caused from 2- to 6-fold enhanced efficacy of polyplex-mediated gene delivery depending on the tumor model. The results obtained on polyplex behavior in tumor tissues depending on physiological state of the tumor can be relevant to optimize delivery of polyplexes and other nanomedicines with similar physicochemical properties.

Morphology and aggregation of RADA-16-I peptide Studied by AFM, NMR and molecular dynamics simulations.

RADA-16-I is a self-assembling peptide which forms biocompatible fibrils and hydrogels. We used molecular dynamics simulations, atomic-force microscopy, NMR spectroscopy, and thioflavin T binding assay to examine size, structure, and morphology of RADA-16-I aggregates. We used the native form of RADA-16-I (H-(ArgAlaAspAla)4 -OH) rather than the acetylated one commonly used in the previous studies. At neutral pH, RADA-16-I is mainly in the fibrillar form, the fibrils consist of an even number of stacked ß-sheets. At acidic pH, RADA-16-I fibrils disassemble into monomers, which form an amorphous monolayer on graphite and monolayer lamellae on mica. RADA-16-I fibrils were compared with the fibrils of a similar peptide RLDL-16-I. Thickness of ß-sheets measured by AFM was in excellent agreement with the molecular dynamics simulations. A pair of RLDL-16-I ß-sheets was thicker (2.3 ± 0.4 nm) than a pair of RADA-16-I ß-sheets (1.9 ± 0.1 nm) due to the volume difference between alanine and leucine residues.