In recent years, there has been a growing interest in plant extracellular vesicles (pEVs) due to their immense potential for medical applications, particularly as carriers for drug delivery. To use the benefits of pEVs in the future, it is necessary to identify methods that facilitate their production in sufficient quantities while maintaining high quality. In this study, a comparative analysis of yields of tobacco pEV derived from apoplastic fluid, sterile calli, and suspension cultures, was performed to identify the most suitable plant material for vesicle isolation. Subsequent experiments focused on assessing the efficiency of small interfering RNA (siRNA) loading into callus-derived vesicles, employing various methods such as sonication, incubation, incubation supplemented with saponin, lipofection, and electroporation. Differences in loading efficiency among vesicles derived from apoplastic fluid, calli, and suspension cultures were observed. Moreover, our investigation extended to the presence of tobacco secondary metabolites, specifically anabasine and nicotine, within vesicles originating from three distinct tobacco sources. The outcomes of our study highlight variations not only in vesicle yields based on their source but also in their loadability and the presence of nicotine and anabasine. These findings contribute valuable insights into optimizing the production and application of pEVs for future medicinal purposes.
- MeSH
- buněčné kultury metody MeSH
- extracelulární vezikuly * metabolismus MeSH
- malá interferující RNA metabolismus genetika MeSH
- nikotin MeSH
- tabák * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- malá interferující RNA MeSH
- nikotin MeSH
The aim of this study was to develop multifunctional magnetic poly(ε-caprolactone) (PCL) mats with antibacterial properties for bone tissue engineering and osteosarcoma prevention. To provide good dispersion of magnetic iron oxide nanoparticles (IONs), they were first grafted with PCL using a novel three-step approach. Then, a series of PCL-based mats containing a fixed amount of ION@PCL particles and an increasing content of ascorbic acid (AA) was prepared by electrospinning. AA is known for increasing osteoblast activity and suppressing osteosarcoma cells. Composites were characterized in terms of morphology, mechanical properties, hydrolytic stability, antibacterial performance, and biocompatibility. AA affected both the fiber diameter and the mechanical properties of the nanocomposites. All produced mats were nontoxic to rat bone marrow-derived mesenchymal cells; however, a composite with 5 wt.% of AA suppressed the initial proliferation of SAOS-2 osteoblast-like cells. Moreover, AA improved antibacterial properties against Staphylococcus aureus and Escherichia coli compared to PCL. Overall, these magnetic composites, reported for the very first time, can be used as scaffolds for both tissue regeneration and osteosarcoma prevention.
- Klíčová slova
- L‐ascorbic acid, iron oxide nanoparticles, nanocomposites, poly(ε‐caprolactone),
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- Escherichia coli účinky léků MeSH
- kosti a kostní tkáň MeSH
- krysa rodu Rattus MeSH
- kyselina askorbová * chemie farmakologie MeSH
- lidé MeSH
- magnetické nanočástice chemie MeSH
- nádorové buněčné linie MeSH
- nanokompozity chemie MeSH
- osteoblasty metabolismus cytologie MeSH
- osteosarkom patologie MeSH
- polyestery * chemie MeSH
- Staphylococcus aureus * účinky léků růst a vývoj MeSH
- testování materiálů MeSH
- tkáňové inženýrství * MeSH
- tkáňové podpůrné struktury chemie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky MeSH
- kyselina askorbová * MeSH
- magnetické nanočástice MeSH
- polycaprolactone MeSH Prohlížeč
- polyestery * MeSH
BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. METHODS: An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. RESULTS: We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. CONCLUSION: We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.
- Klíčová slova
- Cancer cell migration, GAMG glioblastoma, HEK-Blue IL-6, IL-6, IL-6R blockers, Malignant melanoma, Migrastatics, Pancreatic carcinoma, Protein engineering,
- MeSH
- HEK293 buňky MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- pohyb buněk * účinky léků MeSH
- proliferace buněk * účinky léků MeSH
- receptory interleukinu-6 * metabolismus MeSH
- vazba proteinů účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- IL6R protein, human MeSH Prohlížeč
- receptory interleukinu-6 * MeSH
This study develops and characterizes novel biodegradable soft hydrogels with dual porosity based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers cross-linked by hydrolytically degradable linkers. The structure and properties of the hydrogels are designed as scaffolds for tissue engineering and they are tested in vitro with model mesenchymal stem cells (rMSCs). Detailed morphological characterization confirms dual porosity suitable for cell growth and nutrient transport. The dual porosity of hydrogels slightly improves rMSCs proliferation compared to the hydrogel with uniform pores. In addition, the laminin coating supports the adhesion of rMSCs to the hydrogel surface. However, hydrogels modified by heptapeptide RGDSGGY significantly stimulate cell adhesion and growth. Moreover, the RGDS-modified hydrogels also affect the topology of proliferating rMSCs, ranging from single-cell to multicellular clusters. The 3D reconstruction of the hydrogels with cells obtained by laser scanning confocal microscopy (LSCM) confirms cell penetration into the inner structure of the hydrogel and its corresponding microstructure. The prepared biodegradable oligopeptide-modified hydrogels with dual porosity are suitable candidates for further in vivo evaluation in soft tissue regeneration.
- Klíčová slova
- N-(2-hydroxypropyl)methacrylamide, double porosity hydrogels, hydrogels, mesenchymal stem cells, scaffolds for tissue engineering,
- MeSH
- buněčná adheze MeSH
- hydrogely * chemie MeSH
- mezenchymální kmenové buňky * MeSH
- poréznost MeSH
- tkáňové inženýrství MeSH
- tkáňové podpůrné struktury chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- hydrogely * MeSH
Extracellular vesicles (EVs) are lipid-enclosed structures that facilitate intercellular communication by transferring cargo between cells. Although predominantly studied in mammals, extracellular vesicles are ubiquitous across metazoans, and thus research in non-mammalian models is critical for fully elucidating extracellular vesicles biology. Recent advances demonstrate that extracellular vesicles mediate diverse physiological processes in non-mammalian vertebrates, including fish, amphibians, and reptiles. Piscine extracellular vesicles promote fin regeneration in zebrafish and carry heat shock proteins regulated by stress. Frog extracellular vesicles containing microRNAs modulate angiogenesis, while turtle extracellular vesicles coordinate reproductive functions. Venom from snakes contains extracellular vesicles that mirror the whole venom composition and interact with mammalian cells. Invertebrates also possess extracellular vesicles involved in immunity, development, and pathogenesis. Molluscan extracellular vesicles participate in shell formation and host interactions. Arthropod models, including Drosophila, genetically dissect conserved pathways controlling extracellular vesicles biogenesis and signalling. Nematode extracellular vesicles regulate larval development, animal communication, and ageing via conserved extracellular vesicles proteins. Ancient metazoan lineages utilise extracellular vesicles as well, with cnidarian extracellular vesicles regulating immunity and regeneration. Ultimately, expanding extracellular vesicles research beyond typical biomedical models to encompass phylogenetic diversity provides an unparalleled perspective on the conserved versus specialised aspects of metazoan extracellular vesicles roles over ∼500 million years. With a primary focus on the literature from the past 5 years, this review aims to reveal fundamental insights into EV-mediated intercellular communication mechanisms shaping animal physiology.
- Klíčová slova
- cargo, development, exosomes, extracellular vesicles, non-mammalian animal models, regeneration, signalling,
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The slug Arion vulgaris has attracted major attention as one of the worst invasive herbivore pests in Europe and is renowned for the stiff mucus it secretes for locomotion. In this study we focused on the isolation and characterisation of extracellular vesicles, specifically exosomes and exosome-like vesicles, from Arion secretions. We developed a method for slug mucus collection and subsequent vesicle isolation by ultracentrifugation. The isolated vesicles with an average diameter of ~ 100 nm carry abundant proteins and short RNAs, as well as adhesion molecules similar to mammalian galectins. We demonstrated that the slug extracellular vesicles are internalised by plant cells and human cancer cells in in vitro assays and are loadable by bioactive compounds, which makes them an interesting tool for utilisation in biotechnology.
- MeSH
- biotechnologie MeSH
- exozómy * metabolismus MeSH
- hlen MeSH
- lidé MeSH
- plži * MeSH
- savci MeSH
- zavlečené druhy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plant extracellular vesicles (pEVs) derived from numerous edible sources gain a lot of attention in recent years, mainly due to the potential to efficiently carry bioactive molecules into mammalian cells. In the present study, we focus on isolation of PDNVs (plant-derived nanovesicles) and pEVs from callus culture and from BY-2 culture of Nicotiana tabacum (tobacco). Tobacco was selected as a source of plant vesicles, as it is commonly used by human, moreover it is a model organism with established techniques for cultivation of explant cultures in vitro. Explant cultures are suitable for the isolation of pEVs in large quantities, due to their fast growth in sterile conditions. As the efficiency of isolation methods varies, we were comparing two methods of isolation. We evaluated biophysical and biochemical properties of plant vesicles, as well as differences between isolates. We encountered difficulties in the form of vesicles aggregation, which is often described in publications focused on mammalian nanovesicles. In an effort to prevent vesicle aggregation, we used trehalose in different stages of isolation. We show tobacco-derived vesicles successfully enter tobacco and mesenchymal cell lines. We observed that tobacco-nanovesicles isolated by different methods incorporated fluorescent dye with different efficiency. The results of our study show tobacco-derived vesicles isolated by various isolation methods are able to enter plant, as well as mammalian cells.
- MeSH
- biologický transport MeSH
- extracelulární vezikuly * metabolismus MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- mezenchymální kmenové buňky * MeSH
- rostliny metabolismus MeSH
- savci MeSH
- tabák MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Layer-by-layer (LbL) polyelectrolyte coatings are intensively studied as reservoirs of bioactive proteins for modulating interactions between biomaterial surfaces and cells. Mild conditions for the incorporation of growth factors into delivery systems are required to maintain protein bioactivity. Here, we present LbL films composed of water-soluble N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC), heparin (Hep), and tannic acid (TA) fabricated under physiological conditions with the ability to release heparin-binding proteins. Surface plasmon resonance analysis showed that the films formed on an anchoring HTCC/TA bilayer, with TA serving as a physical crosslinker, were more stable during their assembly, leading to increased film thickness and increased protein release. X-ray reflectivity measurements confirmed intermixing of the deposited layers. Protein release also increased when the proteins were present as an integral part of the Hep layers rather than as individual protein layers. The 4-week release pattern depended on the protein type; VEGF, CXCL12, and TGF-β1 exhibited a typical high initial release, whereas FGF-2 was sustainably released over 4 weeks. Notably, the films were nontoxic, and the released proteins retained their bioactivity, as demonstrated by the intensive chemotaxis of T-lymphocytes in response to the released CXCL12. Therefore, the proposed LbL films are promising biomaterial coating candidates for stimulating cellular responses.
Silver nanoparticles are versatile platforms with a variety of applications in the biomedical field. In this framework, their presence in biological media inevitably leads to the interaction with proteins thus conducting to the formation of biomolecular coronas. This feature alters the identity of the nanomaterial and may affect many biological events. These considerations motivated the investigation of protein adsorption onto the surface of polymer-stabilized AgNPs. The metallic colloids were coated by polyethyleneimine (PEI), polyvinylpyrrolidone (PVP), and poly(2-vinyl pyridine)-b-poly(ethylene oxide) (PEO-b-P2VP), and nanoparticle-protein interaction was probed by using a library of analytical techniques. The experimental data revealed a higher extent of protein adsorption at the surface of AgNPs@PVP whereas PEO-b-P2VP coating conducted to the least amount. The main component of the protein coronas was evidenced to be bovine serum albumin (BSA), which is indeed the protein at the highest abundancy in the model biological media. We have further demonstrated reduced cytotoxicity of the silver colloids coated by biomolecular coronas as compared to the pristine counterparts. Nevertheless, the protein coatings did not notably reduce the antimicrobial performance of the polymer-stabilized AgNPs. Accordingly, although the protein-repelling property is frequently targeted towards longer in vivo circulation of nanoparticles, we herein underline that protein coatings, which are commonly treated as artifacts to be avoided, may indeed enhance the biological performance of nanomaterials. These findings are expected to be highly relevant in the design of polymer-stabilized metallic colloids intended to be used in healthcare.
- Klíčová slova
- Antimicrobial properties, Cytotoxicity, Nano-bio interface, Protein corona, Silver colloids,
- MeSH
- antibakteriální látky farmakologie MeSH
- ethylenoxid MeSH
- koloidy MeSH
- kovové nanočástice * MeSH
- polyethylenimin farmakologie MeSH
- polymery farmakologie MeSH
- povidon farmakologie MeSH
- proteinová korona * metabolismus MeSH
- pyridiny MeSH
- sérový albumin hovězí MeSH
- stříbro farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky MeSH
- ethylenoxid MeSH
- koloidy MeSH
- polyethylenimin MeSH
- polymery MeSH
- povidon MeSH
- proteinová korona * MeSH
- pyridiny MeSH
- sérový albumin hovězí MeSH
- stříbro MeSH
Background: Exosomes are extracellular vesicles with the ability to encapsulate bioactive molecules, such as therapeutics. This study identified a new exosome mediated route of doxorubicin and poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA)-bound doxorubicin trafficking in the tumor mass. Materials & methods: Exosome loading was achieved via incubation of the therapeutics with an adherent human breast adenocarcinoma cell line and its derived spheroids. Exosomes were characterized using HPLC, nanoparticle tracking analysis (NTA) and western blotting. Results: The therapeutics were successfully loaded into exosomes. Spheroids secreted significantly more exosomes than adherent cells and showed decreased viability after treatment with therapeutic-loaded exosomes, which confirmed successful transmission. Conclusion: To the best of our knowledge, this study provides the first evidence of pHPMA-drug conjugate secretion by extracellular vesicles.
Background: In cancer treatment, low-molecular-weight drugs (e.g., doxorubicin [DOX]) with a broad spectrum of side effects are commonly used. Through their conjugation with hydrophilic polymers – N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers – for example, most of the side effects can be reduced. These drug–polymer conjugates are delivered via bloodstream into the tumor. This study aimed to identify a new exosome-mediated route of DOX and polyHPMA(pHPMA)–DOX conjugates trafficking inside the tumor mass. Exosomes are small lipid membrane vesicles constitutively released from most of the cell types, including the tumor cells. Exosomes are able to encapsulate low-molecular-weight drugs. Methods: Exosomes were loaded with DOX and pHPMA-DOX in vitro via coincubation with cancer cells. Exosomes were isolated from the conditioned-cultivation medium after their release from cells and characterized (size, numbers, protein marker profiles). Results: The therapeutics were successfully loaded into exosomes and transmitted to the tumor cells. To the best of our knowledge, this is the first evidence of the pHPMA–drug conjugate secretion by exosomes.
- Klíčová slova
- cytotoxicity, drug delivery, extracellular vesicles, polymer, tumor,
- MeSH
- adenokarcinom * farmakoterapie MeSH
- doxorubicin farmakologie terapeutické užití MeSH
- exozómy * MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- polymery MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- doxorubicin MeSH
- polymery MeSH
