surface plasmon resonance
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To address the challenge of drug accumulation and penetration at the tumor site(s), herein we describe a first-in-class nanocarrier containing 24 copies each of two bioactive peptides (BAPs) genetically fused in frame to the 24 N-termini of a human ferritin H-type construct, named THE-10. The two BAPs are specific for PD-L1 and integrin αVβ3/αVβ5 plus Neuropilin (iRGD) respectively, conferring immune checkpoint blockade and drug-internalization properties. In turn, the THE-10 backbone brings 48 BAPs contiguous for synergism, prolonged blood half-life, and release into the tumor microenvironment upon conditional cleavage of a metalloprotease-sensitive site. Predicted THE-10 multitasking activity was experimentally supported as follows. Size-exclusion chromatography and surface plasmon resonance demonstrated BAP cleavage/release and receptor binding (nanomolar KD). Live-cell/time-lapse imaging demonstrated 4-fold-increased internalization of naked therapeutic antibodies, mirrored by enhanced cytotoxicity of the corresponding Antibody-Drug Conjugate. Slight antitumor effects were observed in vivo by treating immune checkpoint-sensitive syngeneic mouse colorectal model with THE-10 alone. Drug boosting was instead considerable on colorectal and pancreatic tumor allografts when THE-10 was co-administered with both small and large chemotherapeutic agents, outperforming the original iRGD cyclic peptide. Thus, THE-10 may enhance target therapy, chemotherapy and immunotherapy altogether, e.g. it candidates as a multitasking, all-round, antineoplastic therapy booster.
- MeSH
- ferritiny * chemie genetika farmakologie MeSH
- imunoterapie * MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nanočástice * chemie MeSH
- nosiče léků * chemie MeSH
- protinádorové látky farmakologie chemie MeSH
- rekombinantní proteiny chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The PB2 subunit of the influenza virus polymerase complex is essential for viral replication, primarily through a mechanism known as cap-snatching. In this process, PB2 binds to the 5' cap structure of host pre-mRNAs, enabling the viral polymerase to hijack the host transcriptional machinery. This binding facilitates the cleavage and integration of the capped RNA fragment into viral mRNA, thereby promoting efficient viral replication. Inhibiting the PB2-cap interaction is therefore crucial, as it directly disrupts the viral replication cycle. Consequently, targeting PB2 with specific inhibitors is a promising strategy for antiviral drug development against influenza. However, there are currently no available methods for the high-throughput screening of potential inhibitors. The development of new inhibitor screening methods of potential PB2 binders is the focus of this study. In this study, we present two novel methods, DIANA and AlphaScreen, for screening influenza PB2 cap-binding inhibitors and evaluate their effectiveness compared to the established differential scanning fluorimetry (DSF) technique. Using a diverse set of substrates and compounds based on the previously described PB2 binder pimodivir, we thoroughly assessed the capabilities of these new methods. Our findings demonstrate that both DIANA and AlphaScreen are highly effective for PB2 inhibitor screening, offering distinct advantages over traditional techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). These advantages include improved scalability, reduced sample requirements, and the capacity for label-free detection. Notably, DIANA's ability to determine Ki values from a single-well measurement significantly enhances its practicality and efficiency in inhibitor screening. This research represents a significant step forward in the development of more efficient and scalable screening strategies, helping advance efforts in the discovery of antiviral drugs against influenza.
- MeSH
- antivirové látky * farmakologie chemie MeSH
- fluorometrie metody MeSH
- lidé MeSH
- piperidiny farmakologie MeSH
- pyridiny MeSH
- pyrimidiny MeSH
- pyrroly MeSH
- RNA čepičky metabolismus MeSH
- RNA-dependentní RNA-polymerasa antagonisté a inhibitory metabolismus MeSH
- rychlé screeningové testy * metody MeSH
- virové proteiny * antagonisté a inhibitory metabolismus MeSH
- virus chřipky A účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Glioblastoma multiforme je mozkový nádor s obecně špatnou prognózou a incidencí 2-3/100000 obyvatel. V této studii chceme využít nový přístup k léčbě nádorů prostřednictvím fototermální terapie za použití zlatých nanotyčinek s potenciálem neinvazivního odstranění nádoru včetně radio- a chemo-rezistentních buněk. Lokalizovaná povrchová plazmonová rezonance nanotyčinek umožňuje přeměnit světelnou energii na teplo, jehož vhodná intenzita usmrtí nádorové buňky apoptózou a dodatečně stimuluje protinádorovou imunitní odpověď. Zlaté nanotyčinky mohou být současně naladěny na požadovanou vlnovou délku zdroje záření vhodného pro klinické použití. V rámci projektu vyvineme techniku pro cílení glioblastomu nanotyčkami za použití mesenchymálních a neurálních kmenových buněk jako jejich nosičů. Lečebná strategie bude otestována in vivo na myších pomocí techniky kranialního okna, kdy bude možné sledovat růst nádoru, distribuci nanotyčinek a efekt terapie v reálném čase.; Glioblastoma multiforme is a brain tumor with incidence of 2-3/100000 persons and generally poor prognosis. In this study we want to employ a new approach of photothermal cancer therapy using gold nanorods (GNRs) with potential to eradicate also the radio- and chemo-resistant cells. The localized surface plasmon resonance of GNRs allows effective transformation of light energy to heat that can be optimized to kill the cancer cells by apoptosis with additional stimulation of anti-tumor immune response. GNRs can be optically tuned to desired wavelength of the irradiation source suitable for clinical use. Mesenchymal or neural stem cells will be used as a vehicle for targeting of GNRs to brain tumors in mouse. Cranial window technique will be adapted for life imaging of tumor growth, GNRs distribution and detection of thermal effects of GNRs in one step.
- Klíčová slova
- kmenové buňky, stem cells, glioblastoma, glioblastom, zlaté nanotyčky, fototermální terapie, kraniální okno, gold nanorods, photothermal therapy, cranial window,
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability. Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.
Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.
- MeSH
- exozómy * chemie MeSH
- karcinom * metabolismus patologie MeSH
- lektiny analýza metabolismus MeSH
- lidé MeSH
- polysacharidy analýza metabolismus MeSH
- tekutá biopsie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The modification of biomaterial surfaces has become increasingly relevant in the context of ongoing advancements in tissue engineering applications and the development of tissue-mimicking polymer materials. In this study, we investigated the layer-by-layer (LbL) deposition of polyelectrolyte multilayer protein reservoirs consisting of poly-l-lysine (PLL) and hyaluronic acid (HA) on the hydrophobic surface of poly(glycerol sebacate) (PGS) elastomer. Using the methods of isothermal titration calorimetry and surface plasmon resonance, we systematically investigated the interactions between the polyelectrolytes and evaluated the deposition process in real time, providing insight into the phenomena associated with film assembly. PLL/HA LbL films deposited on PGS showed an exceptional ability to incorporate bone morphogenetic protein-2 (BMP-2) compared to other growth factors tested, thus highlighting the potential of PLL/HA LbL films for osteoregenerative applications. The concentration of HA solution used for film assembly did not affect the thickness and topography of the (PLL/HA)10 films, but had a notable impact on the hydrophilicity of the PGS surface and the BMP-2 release kinetics. The release kinetics were successfully described using the Weibull model and hyperbolic tangent function, underscoring the potential of these less frequently used models to compare the protein release from LbL protein reservoirs.
Aberrant glycosylation of glycoproteins has been linked with various pathologies. Therefore, understanding the relationship between aberrant glycosylation patterns and the onset and progression of the disease is an important research goal that may provide insights into cancer diagnosis and new therapy development. In this study, we use a surface plasmon resonance imaging biosensor and a lectin array to investigate aberrant glycosylation patterns associated with oncohematological disease-myelodysplastic syndromes (MDS). In particular, we detected the interaction between the lectins and glycoproteins present in the blood plasma of patients (three MDS subgroups with different risks of progression to acute myeloid leukemia (AML) and AML patients) and healthy controls. The interaction with lectins from Aleuria aurantia (AAL) and Erythrina cristagalli was more pronounced for plasma samples of the MDS and AML patients, and there was a significant difference between the sensor response to the interaction of AAL with blood plasma from low and medium-risk MDS patients and healthy controls. Our data also suggest that progression from MDS to AML is accompanied by sialylation of glycoproteins and increased levels of truncated O-glycans and that the number of lectins that allow discriminating different stages of disease increases as the disease progresses.
- MeSH
- akutní myeloidní leukemie * MeSH
- biosenzitivní techniky * MeSH
- glykoproteiny metabolismus MeSH
- glykosylace MeSH
- krevní plazma metabolismus MeSH
- lektiny MeSH
- lidé MeSH
- myelodysplastické syndromy * terapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Salivary glands from blood-feeding arthropods secrete several molecules that inhibit mammalian hemostasis and facilitate blood feeding and pathogen transmission. The salivary functions from Simulium guianense, the main vector of Onchocerciasis in South America, remain largely understudied. Here, we have characterized a salivary protease inhibitor (Guianensin) from the blackfly Simulium guianense. MATERIALS AND METHODS: A combination of bioinformatic and biophysical analyses, recombinant protein production, in vitro and in vivo experiments were utilized to characterize the molecula mechanism of action of Guianensin. Kinetics of Guianensin interaction with proteases involved in vertebrate inflammation and coagulation were carried out by surface plasmon resonance and isothermal titration calorimetry. Plasma recalcification and coagulometry and tail bleeding assays were performed to understand the role of Guianensin in coagulation. RESULTS: Guianensin was identified in the sialotranscriptome of adult S. guianense flies and belongs to the Kunitz domain of protease inhibitors. It targets various serine proteases involved in hemostasis and inflammation. Binding to these enzymes is highly specific to the catalytic site and is not detectable for their zymogens, the catalytic site-blocked human coagulation factor Xa (FXa), or thrombin. Accordingly, Guianensin significantly increased both PT (Prothrombin time) and aPTT (Activated partial thromboplastin time) in human plasma and consequently increased blood clotting time ex vivo. Guianensin also inhibited prothrombinase activity on endothelial cells. We show that Guianensin acts as a potent anti-inflammatory molecule on FXa-induced paw edema formation in mice. CONCLUSION: The information generated by this work highlights the biological functionality of Guianensin as an antithrombotic and anti-inflammatory protein that may play significant roles in blood feeding and pathogen transmission.
- MeSH
- antiflogistika farmakologie MeSH
- endoteliální buňky MeSH
- hemostatika * MeSH
- hemostáza MeSH
- lidé MeSH
- myši MeSH
- savci MeSH
- Simuliidae * MeSH
- slinné proteiny a peptidy farmakologie MeSH
- zánět MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Intramural MeSH
MXenes and their related nanocomposites with superior physicochemical properties such as high surface area, ease of synthesis and functionalization, high drug loading capacity, collective therapy potentials, pH-triggered drug release behavior, high photothermal conversion, and excellent photodynamic efficiency have been explored as alluring materials in photomedicine; the application of photons in medicine is facilitated for imaging and various disease treatment methods such as photothermal cancer/tumor ablation. Non-invasive theranostic strategies with synergistic activities have been developed using photothermal, photodynamic, and magnetic therapies together with remotely controlled drug/gene delivery for the diagnosis and treatment of various malignant diseases. Photothermal/photodynamic therapy and photoacoustic imaging using MXene-based structures have shown great promise in cancer phototherapy. However, hybridization and surface functionalization should be further explored to obtain biocompatible MXene-based composites/platforms with unique properties, high stability, and improved functionality in photomedicine. Toxicological and long-term biosafety assessments as well as clinical translation evaluations ought to be given high priority in research. Although some limited studies have revealed the excellent potentials of MXenes and their derivatives in photomedicine, further steps should be taken towards extensive research and detailed analysis in the field of optimizing the properties and improving the performance of these materials with a clinical and industrial outlook. Optical biosensing platforms have been developed along with electrochemical sensors and wearable sensors constructed from MXenes and their derivatives; future studies warrant the comprehensive analysis of optical transduction aspects such as colorimetry, electrochemiluminescence, photoluminescence, surface-enhanced Raman scattering, and surface plasmon resonance. Herein, the potentials of MXenes in photomedicine are deliberated encompassing important challenges and future research directions.
- MeSH
- fotochemoterapie * MeSH
- fototerapie metody MeSH
- indukovaná hypertermie * metody MeSH
- lidé MeSH
- nádory * diagnostické zobrazování farmakoterapie MeSH
- nanokompozity * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Base excision repair is one of the important DNA repair mechanisms in cells. The fundamental role in this complex process is played by DNA glycosylases. Here, we present a novel approach for the real-time measurement of uracil DNA glycosylase activity, which employs selected oligonucleotides immobilized on the surface of magnetic nanoparticles and Förster resonance energy transfer. We also show that the approach can be performed by surface plasmon resonance sensor technology. We demonstrate that the immobilization of oligonucleotides provides much more reliable data than the free oligonucleotides including molecular beacons. Moreover, our results show that the method provides the possibility to address the relationship between the efficiency of uracil DNA glycosylase activity and the arrangement of the used oligonucleotide probes. For instance, the introduction of the nick into oligonucleotide containing the target base (uracil) resulted in the substantial decrease of uracil DNA glycosylase activity of both the bacterial glycosylase and glycosylases naturally present in nuclear lysates.
- MeSH
- buněčné jádro metabolismus MeSH
- lidé MeSH
- magnetické nanočástice oxidů železa MeSH
- oligonukleotidové sondy chemie metabolismus MeSH
- oprava DNA MeSH
- rezonanční přenos fluorescenční energie MeSH
- uracil-DNA-glykosidasa metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
