In recent years, fluorescent nanodiamond (fND) particles containing nitrogen-vacancy (NV) centers gained recognition as an attractive probe for nanoscale cellular imaging and quantum sensing. For these applications, precise localization of fNDs inside of a living cell is essential. Here we propose such a method by simultaneous detection of the signal from the NV centers and the spectroscopic Raman signal from the cells to visualize the nucleus of living cells. However, we show that the commonly used Raman cell signal from the fingerprint region is not suitable for organelle imaging in this case. Therefore, we develop a method for nucleus visualization exploiting the region-specific shape of C-H stretching mode and further use k-means cluster analysis to chemically distinguish the vicinity of fNDs. Our technique enables, within a single scan, to detect fNDs, distinguish by chemical localization whether they have been internalized into cell and simultaneously visualize cell nucleus without any labeling or cell-fixation. We show for the first time spectral colocalization of unmodified high-pressure high-temperature fND probes with the cell nucleus. Our methodology can be, in principle, extended to any red- and near-infrared-luminescent cell-probes and is fully compatible with quantum sensing measurements in living cells.
- MeSH
- buněčné jádro ultrastruktura MeSH
- cytologické techniky MeSH
- fluorescenční barviva MeSH
- kultivované buňky MeSH
- lidé MeSH
- molekulární zobrazování metody MeSH
- nádorové buněčné linie MeSH
- nanodiamanty * MeSH
- Ramanova spektroskopie MeSH
- zubní dřeň cytologie diagnostické zobrazování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein corona formation has been regarded as an obstacle to developing diagnostic and therapeutic nanoparticles for in vivo applications. Serum proteins that assemble around nanoparticles can hinder their targeting efficiency. Virus-based nanoparticles should be naturally predisposed to evade such barriers in host organisms. Here, we demonstrate that virus-like particles derived from mouse polyomavirus do not form a rich protein corona. These particles can be efficiently targeted to cells that overproduce transferrin receptors, e.g., cancer cells, by conjugating transferrin to the particle surface. In this study, we provide evidence that the interaction of virus-like particles with their newly assigned target receptor is not obstructed by serum proteins. The particles enter target cells via a clathrin-dependent endocytic pathway that is not naturally used by the virus. Our results support the notion that the natural properties of virus-like particles make them well-suited for development of nanosized theranostic tools resistant to detargeting by protein coronas.
- MeSH
- krevní proteiny chemie metabolismus MeSH
- lidé MeSH
- myši MeSH
- nanočástice chemie MeSH
- Polyomavirus chemie MeSH
- proteinová korona chemie metabolismus MeSH
- receptory transferinu metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Biohydrogels, composed of naturally occurring biopolymers are typically preferred over their synthetic analogues in bioapplications thanks to their biocompatibility, bioactivity, mechanical or degradation properties. Shaping biohydrogels on the single-cell length scales (micrometers) is a key ability needed to create bioequivalent artificial cell/tissue constructs and cannot be achieved with current methods. This work introduces a method for photolithographic synthesis of arbitrarily shaped microgels composed purely of a biopolymer of choice. The biopolymer is mixed with a sacrificial photocrosslinkable polymer, and the mixture is photocrosslinked in a lithographic process, yielding anisotropic microgels with the biopolymer entrapped in the network. Subsequent ionic or covalent biopolymer crosslinking followed by template cleavage yields a microgel composed purely of a biopolymer with the 3D shape dictated by the photocrosslinking process. Method feasibility is demonstrated with two model polysaccharide biopolymers (alginate, chitosan) using suitable crosslinking methods. Next, alginate microgels were used as microtaggants on a pharmaceutical oral solid dose formulation to prevent its counterfeiting. Since the alginate is approved as an additive in the food and pharmaceutical industries, the presented tagging system can be implemented in practical use much easier than systems comprising synthetic polymers.
Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.
- MeSH
- kovové nanočástice chemie MeSH
- lidé MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
Energetic ions represent an important tool for the creation of controlled structural defects in solid nanomaterials. However, the current preparative irradiation techniques in accelerators show significant limitations in scaling-up, because only very thin layers of nanoparticles can be efficiently and homogeneously irradiated. Here, we show an easily scalable method for rapid irradiation of nanomaterials by light ions formed homogeneously in situ by a nuclear reaction. The target nanoparticles are embedded in B2O3 and placed in a neutron flux. Neutrons captured by 10B generate an isotropic flux of energetic α particles and 7Li+ ions that uniformly irradiates the surrounding nanoparticles. We produced 70 g of fluorescent nanodiamonds in an approximately 30-minute irradiation session, as well as fluorescent silicon carbide nanoparticles. Our method thus increased current preparative yields by a factor of 102-103. We envision that our technique will increase the production of ion-irradiated nanoparticles, facilitating their use in various applications.
- MeSH
- ionty účinky záření MeSH
- nanočástice * účinky záření MeSH
- záření * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The blocking of specific protein-protein interactions using nanoparticles is an emerging alternative to small molecule-based therapeutic interventions. However, the nanoparticles designed as "artificial proteins" generally require modification of their surface with (bio)organic molecules and/or polymers to ensure their selectivity and specificity of action. Here, we show that nanosized diamond crystals (nanodiamonds, NDs) without any synthetically installed (bio)organic interface enable the specific and efficient targeting of the family of extracellular signalling molecules known as fibroblast growth factors (FGFs). We found that low nanomolar solutions of detonation NDs with positive ζ-potential strongly associate with multiple FGF ligands present at sub-nanomolar concentrations and effectively neutralize the effects of FGF signalling in cells without interfering with other growth factor systems and serum proteins unrelated to FGFs. We identified an evolutionarily conserved FGF recognition motif, ∼17 amino acids long, that contributes to the selectivity of the ND-FGF interaction. In addition, we inserted this motif into a de novo constructed chimeric protein, which significantly improved its interaction with NDs. We demonstrated that the interaction of NDs, as purely inorganic nanoparticles, with proteins can mitigate pathological FGF signalling and promote the restoration of cartilage growth in a mouse limb explant model. Based on our observations, we foresee that NDs may potentially be applied as nanotherapeutics to neutralize disease-related activities of FGFs in vivo.
- MeSH
- aminokyselinové motivy MeSH
- buněčné linie MeSH
- chrupavka fyziologie MeSH
- embryo savčí MeSH
- fibroblastové růstové faktory metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- myši MeSH
- nanodiamanty chemie MeSH
- proliferace buněk MeSH
- receptory fibroblastových růstových faktorů metabolismus MeSH
- signální transdukce MeSH
- techniky tkáňových kultur MeSH
- tibie fyziologie MeSH
- vazba proteinů MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Glutamate carboxypeptidase II (GCPII) is a membrane protease overexpressed by prostate cancer cells and detected in the neovasculature of most solid tumors. Targeting GCPII with inhibitor-bearing nanoparticles can enable recognition, imaging, and delivery of treatments to cancer cells. Compared to methods based on antibodies and other large biomolecules, inhibitor-mediated targeting benefits from the low molecular weight of the inhibitor molecules, which are typically stable, easy-to-handle, and able to bind the enzyme with very high affinity. Although GCPII is established as a molecular target, comparing previously reported results is difficult due to the different methodological approaches used. In this work, we investigate the robustness and limitations of GCPII targeting with a diverse range of inhibitor-bearing nanoparticles (various structures, sizes, bionanointerfaces, conjugation chemistry, and surface densities of attached inhibitors). Polymer-coated nanodiamonds, virus-like particles based on bacteriophage Qβ and mouse polyomavirus, and polymeric poly(HPMA) nanoparticles with inhibitors attached by different means were synthesized and characterized. We evaluated their ability to bind GCPII and interact with cancer cells using surface plasmon resonance, inhibition assay, flow cytometry, and confocal microscopy. Regardless of the diversity of the investigated nanosystems, they all strongly interact with GCPII (most with low picomolar Ki values) and effectively target GCPII-expressing cells. The robustness of this approach was limited only by the quality of the nanoparticle bionanointerface, which must be properly designed by adding a sufficient density of hydrophilic protective polymers. We conclude that the targeting of cancer cells overexpressing GCPII is a viable approach transferable to a broad diversity of nanosystems.
- MeSH
- antigeny povrchové metabolismus MeSH
- antitumorózní látky aplikace a dávkování MeSH
- chemie farmaceutická MeSH
- glutamátkarboxypeptidasa II antagonisté a inhibitory metabolismus MeSH
- hydrofobní a hydrofilní interakce MeSH
- inhibitory enzymů aplikace a dávkování MeSH
- lidé MeSH
- ligandy MeSH
- nádorové buněčné linie MeSH
- nádory farmakoterapie patologie MeSH
- nanokonjugáty chemie MeSH
- rekombinantní proteiny metabolismus MeSH
- syntetická chemie okamžité shody MeSH
- thiazolidiny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nanoparticle-cell interactions begin with the cellular uptake of the nanoparticles, a process that eventually determines their cellular fate. In the present work, we show that the morphological features of nanodiamonds (NDs) affect both the anchoring and internalization stages of their endocytosis. While a prickly ND (with sharp edges/corners) has no trouble of anchoring onto the plasma membrane, it suffers from difficult internalization afterwards. In comparison, the internalization of a round ND (obtained by selective etching of the prickly ND) is not limited by its lower anchoring amount and presents a much higher endocytosis amount. Molecular dynamics simulation and continuum modelling results suggest that the observed difference in the anchoring of round and prickly NDs likely results from the reduced contact surface area with the cell membrane of the former, while the energy penalty associated with membrane curvature generation, which is lower for a round ND, may explain its higher probability of the subsequent internalization.
Development of multifunctional nanoscale sensors working under physiological conditions enables monitoring of intracellular processes that are important for various biological and medical applications. By attaching paramagnetic gadolinium complexes to nanodiamonds (NDs) with nitrogen-vacancy (NV) centres through surface engineering, we developed a hybrid nanoscale sensor that can be adjusted to directly monitor physiological species through a proposed sensing scheme based on NV spin relaxometry. We adopt a single-step method to measure spin relaxation rates enabling time-dependent measurements on changes in pH or redox potential at a submicrometre-length scale in a microfluidic channel that mimics cellular environments. Our experimental data are reproduced by numerical simulations of the NV spin interaction with gadolinium complexes covering the NDs. Considering the versatile engineering options provided by polymer chemistry, the underlying mechanism can be expanded to detect a variety of physiologically relevant species and variables.
- MeSH
- biosenzitivní techniky metody MeSH
- časové faktory MeSH
- koncentrace vodíkových iontů MeSH
- konfokální mikroskopie MeSH
- kvantová teorie MeSH
- nanodiamanty chemie ultrastruktura MeSH
- nanotechnologie metody MeSH
- optické zobrazování metody MeSH
- oxidace-redukce MeSH
- reprodukovatelnost výsledků MeSH
- transmisní elektronová mikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Virus-like particles based on polyomaviruses (PVLPs) are promising delivery devices for various cargoes, including nucleic acids, imaging probes, and therapeutic agents. In biological environments, the major coat protein VP1 interacts with ubiquitously distributed sialic acid residues, and therefore PVLPs show a broad tropism. For selective targeting, appropriate engineering of the PVLP surface is needed. Here, we describe a chemical approach to retarget PVLPs to cancer cells displaying abnormally high levels of transferrin receptor. We created an array of transferrin molecules on the surface of PVLPs by combining a high-yielding bioconjugation approach with specific point modification of transferrin. This artificial surface protein architecture enables (i) suppression of natural VP1-specific interactions by blocking the surface conformational epitope on the VP1 protein, (ii) unusually high cellular uptake efficiency, and (iii) selective retargeting of PVLPs to osteosarcoma (U2OS) and lymphoblastoid leukemia (CCRF-CEM) cells.
- MeSH
- biologický transport MeSH
- kapsida chemie metabolismus MeSH
- lidé MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- nádorové buněčné linie MeSH
- nosiče léků chemie metabolismus MeSH
- Polyomavirus chemie MeSH
- povrchové vlastnosti MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
