RESUMO
We have developed an Ir(PPy)3 photoredox-catalyzed cross-coupling reaction that allows installation of quinoxalinones at the C7 position of thiazolino ring-fused 2-pyridones (TRPs) under mild conditions. The methodology tolerates various substituted quinoxalinones and biologically relevant substituents on the C8 position of the TRP. The TRP scaffold has large potential in the development of lead compounds, and while the coupled products are interesting from a drug-development perspective, the methodology will be useful for developing more potent and drug-like TRP-based candidates.
RESUMO
Antibodies, disruptive potent therapeutic agents against pharmacological targets, face a barrier in crossing immune systems and cellular membranes. To overcome these, various strategies have been explored including shuttling via liposomes or biocamouflaged nanoparticles. Here, we demonstrate the feasibility of loading antibodies into exosome-mimetic nanovesicles derived from human red-blood-cell membranes, which can act as nanocarriers for intracellular delivery. Goat-antichicken antibodies are loaded into erythrocyte-derived nanovesicles, and their loading yields are characterized and compared with smaller dUTP-cargo molecules. Applying dual-color coincident fluorescence burst analyses, the loading yield of nanocarriers is rigorously profiled at the single-vesicle level, overcoming challenges due to size-heterogeneity and demonstrating a maximum antibody-loading yield of 38-41% at the optimal vesicle radius of 52 nm. The achieved average loading yields, amounting to 14% across the entire nanovesicle population, with more than two antibodies per loaded vesicle, are fully comparable to those obtained for the much smaller dUTP molecules loaded in the nanovesicles after additional exosome-spin-column purification. The results suggest a promising new avenue for therapeutic delivery of antibodies, potentially encompassing also intracellular targets and suitable for large-scale pharmacological applications, which relies on the exosome-mimetic properties, biocompatibility, and low-immunogenicity of bioengineered nanocarriers synthesized from human erythrocyte membranes.
RESUMO
The amino acids tryptophan, tyrosine, and phenylalanine have been extensively used for different label-free protein studies, based on the intensity, lifetime, wavelength and/or polarization of their emitted fluorescence. Similar to most fluorescent organic molecules, these amino acids can undergo transitions into dark meta-stable states, such as triplet and photo-radical states. On the one hand, these transitions limit the fluorescence signal, but they are also highly environment-sensitive and can offer an additional set of parameters, reflecting interactions, folding states, and immediate environments around the proteins. In this work, by analyzing the average intensity of tyrosine emission under different excitation modulations with the transient state monitoring (TRAST) technique, we explored the photo physics of tyrosine as a basis for such environment-sensitive readouts. From how the dark state transitions of tyrosine varied with excitation intensity and solvent conditions we first established a photophysical model for tyrosine. Next, we studied Calmodulin (containing two tyrosines), and how its conformation is changed upon calcium binding. From these TRAST experiments, performed with 280 nm time-modulated excitation, we show that tyrosine dark state transitions clearly change with the calmodulin conformation, and may thus represent a useful source of information for (label-free) analyses of protein conformations and interactions.
Assuntos
Calmodulina , Tirosina , Tirosina/química , Calmodulina/metabolismo , Espectrometria de Fluorescência/métodos , Conformação Proteica , Triptofano/química , CorantesRESUMO
This work details the synthesis of paramagnetic upconversion nanoparticles doped with Fe3+ in various morphologies via the thermal decomposition method, followed by comprehensive characterization of their structures, optical properties and magnetism using diverse analytical techniques. Our findings demonstrate that by precisely modulating the ratio of oleic acid to octadecene in the solvent, one can successfully obtain hexagonal nanodiscs with a consistent and well-defined morphology. Further adjustments in the oleic acid to octadecene ratio, coupled with fine-tuning of the Na+/F- ratio, led to the production of small-sized nanorods with uniform morphology. Significantly, all Fe3+-doped nanoparticles displayed pronounced paramagnetism, with magnetic susceptibility measurements at 1 T and room temperature of 0.15 emu g-1 and 0.14 emu g-1 for the nanodiscs and nanorods, respectively. To further enhance their magnetic properties, we replaced the Y-matrix with a Gd-matrix, and by fine-tuning the oleic acid/octadecene and Na+/F- ratios, we achieved nanoparticles with uniform morphology. The magnetic susceptibility was 0.82 emu g-1 at 1 T and room temperature. Simultaneously, we could control the nanoparticle size by altering the synthesis temperature. These upconversion nanostructures, characterized by both paramagnetic properties and regular morphology, represent promising dual-mode nanoprobe candidates for optical biological imaging and magnetic resonance imaging.
RESUMO
Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, are a necessary basis for fluorescence-based super-resolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans-cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time resolution and is thus broadly applicable to different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional useful strategy for fluorescence-based multiplexing that can be implemented on a broad range of both stationary and moving samples.
Assuntos
Corantes Fluorescentes , Microfluídica , Espectrometria de Fluorescência/métodos , Carbocianinas/química , Rodaminas , Corantes Fluorescentes/químicaRESUMO
Photodynamic therapy (PDT) fundamentally relies on local generation of PDT precursor states in added photosensitizers (PS), particularly triplet and photo-radical states. Monitoring these states in situ can provide important feedback but is difficult in practice. The states are strongly influenced by local oxygenation, pH and redox conditions, often varying significantly at PDT treatment sites. To overcome this problem, we followed local PDT precursor state populations of PS compounds, via their fluorescence intensity response to systematically varied excitation light modulation. Thereby, we could demonstrate local monitoring of PDT precursor states of methylene blue (MB) and IRdye700DX (IR700), and determined their transitions rates under different oxygenation, pH and redox conditions. By fiber-optics, using one fiber for both excitation and fluorescence detection, the triplet and photo-radical state kinetics of locally applied MB and IR700 could then be monitored in a tissue sample. Finally, potassium iodide and ascorbate were added as possible PDT adjuvants, enhancing intersystem crossing and photoreduction, respectively, and their effects on the PDT precursor states of MB and IR700 could be locally monitored. Taken together, the presented procedure overcomes current methodological limitations and can offer feedback, guiding both excitation and PDT adjuvant application, and thereby more efficient and targeted PDT treatments.
Assuntos
Fotoquimioterapia , Fármacos Fotossensibilizantes , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Fotoquimioterapia/métodos , RetroalimentaçãoRESUMO
Photo-induced dark transient states of fluorophores can pose a problem in fluorescence spectroscopy. However, their typically long lifetimes also make them highly environment sensitive, suggesting fluorophores with prominent dark-state formation yields to be used as microenvironmental sensors in bio-molecular spectroscopy and imaging. In this work, we analyzed the singlet-triplet transitions of fluorescein and three synthesized carboxy-fluorescein derivatives, with one, two or four bromines linked to the anthracence backbone. Using transient state (TRAST) spectroscopy, we found a prominent internal heavy atom (IHA) enhancement of the intersystem crossing (ISC) rates upon bromination, inferred by density functional theory calculations to take place via a higher triplet state, followed by relaxation to the lowest triplet state. A corresponding external heavy atom (EHA) enhancement was found upon adding potassium iodide (KI). Notably, increased KI concentrations still resulted in lowered triplet state buildup in the brominated fluorophores, due to relatively lower enhancements in ISC, than in the triplet decay. Together with an antioxidative effect on the fluorophores, adding KI thus generated a fluorescence enhancement of the brominated fluorophores. By TRAST measurements, analyzing the average fluorescence intensity of fluorescent molecules subject to a systematically varied excitation modulation, dark state transitions within very high triplet yield (>90%) fluorophores can be directly analyzed under biologically relevant conditions. These measurements, not possible by other techniques such as fluorescence correlation spectroscopy, opens for bio-sensing applications based on high triplet yield fluorophores, and for characterization of high triplet yield photodynamic therapy agents, and how they are influenced by IHA and EHA effects.
Assuntos
Corantes Fluorescentes , Corantes Fluorescentes/química , Fluoresceína , Espectrometria de Fluorescência/métodosRESUMO
Lanthanide upconversion nanoparticles (UCNPs) have been extensively explored as biomarkers, energy transducers, and information carriers in wide-ranging applications in areas from healthcare and energy to information technology. In promoting the brightness and enriching the functionalities of UCNPs, core-shell structural engineering has been well-established as an important approach. Despite its importance, a strong limiting issue has been identified, namely, cation intermixing in the interfacial region of the synthesized core-shell nanoparticles. Currently, there still exists confusion regarding this destructive phenomenon and there is a lack of facile means to reach a delicate control of it. By means of a new set of experiments, we identify and provide in this work a comprehensive picture for the major physical mechanism of cation intermixing occurring in synthesis of core-shell UCNPs, i.e., partial or substantial core nanoparticle dissolution followed by epitaxial growth of the outer layer and ripening of the entire particle. Based on this picture, we provide an easy but effective approach to tackle this issue that enables us to produce UCNPs with highly boosted optical properties.
RESUMO
The frequency-domain (FD) method provides an alternative to the commonly used time-domain (TD) approach in characterizing the luminescence kinetics of luminophores, with its own strengths, e.g., the capability to decouple multiple lifetime components with higher reliability and accuracy. While extensively explored for characterizing luminophores with down-shifted emission, this method has not been investigated for studying nonlinear luminescent materials such as lanthanide-doped upconversion nanoparticles (UCNPs), featuring more complicated kinetics. In this work, employing a simplified rate-equation model representing a standard two-photon energy-transfer upconversion process, we thoroughly analyzed the response of the luminescence of UCNPs in the FD method. We found that the FD method can potentially obtain from a single experiment the effective decay rates of three critical energy states of the sensitizer/activator ions involved in the upconversion process. The validity of the FD method is demonstrated by experimental data, agreeing reasonably well with the results obtained by TD methods.
RESUMO
Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7. We show how this red-emissive photoisomerized state contributes to the blinking kinetics in different emission bands of NIR cyanine dyes, and how it can influence single-molecule, super-resolution, as well as Förster resonance energy transfer (FRET) and multicolor readouts. Since this state can also be populated at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also readouts not relying on high excitation conditions. However, this additional red-emissive state and its photodynamics, as identified and characterized in this work, can also be used as a strategy to push the emission of NIR cyanine dyes further into the NIR and to enhance photosensitization of nanoparticles with absorption spectra further into the NIR. Finally, we show that the photoisomerization kinetics of SCy7 and the formation of its redshifted photoisomer depend strongly on local environmental conditions, such as viscosity, polarity, and steric constraints, which suggests the use of SCy7 and other NIR cyanine dyes as environmental sensors. Such environmental information can be monitored by TRAST, in the NIR, with low autofluorescence and scattering conditions and on a broad range of samples and experimental conditions.
RESUMO
Cyanine fluorophores are extensively used in fluorescence spectroscopy and imaging. Upon continuous excitation, especially at excitation conditions used in single-molecule and super-resolution experiments, photo-isomerized states of cyanines easily reach population probabilities of around 50%. Still, effects of photo-isomerization are largely ignored in such experiments. Here, we studied the photo-isomerization of the pentamethine cyanine 5 (Cy5) by two similar, yet complementary means to follow fluorophore blinking dynamics: fluorescence correlation spectroscopy (FCS) and transient-state (TRAST) excitation-modulation spectroscopy. Additionally, we combined TRAST and spectrofluorimetry (spectral-TRAST), whereby the emission spectra of Cy5 were recorded upon different rectangular pulse-train excitations. We also developed a framework for analyzing transitions between multiple emissive states in FCS and TRAST experiments, how the brightness of the different states is weighted, and what initial conditions that apply. Our FCS, TRAST, and spectral-TRAST experiments showed significant differences in dark-state relaxation amplitudes for different spectral detection ranges, which we attribute to an additional red-shifted, emissive photo-isomerized state of Cy5, not previously considered in FCS and single-molecule experiments. The photo-isomerization kinetics of this state indicate that it is formed under moderate excitation conditions, and its population and emission may thus deserve also more general consideration in fluorescence imaging and spectroscopy experiments.
Assuntos
Corantes Fluorescentes , Imagem Óptica , Espectrometria de Fluorescência/métodos , Carbocianinas/química , Corantes Fluorescentes/químicaRESUMO
Guanine (G)-rich nucleic acids are prone to assemble into four-stranded structures, so-called G-quadruplexes. Abnormal GGGGCC repeat elongations, and in particular their folding states, are associated with amyotrophic lateral sclerosis and frontotemporal dementia. Due to methodological constraints however, most studies of G quadruplex structures are restricted to in vitro conditions. Evidence of how GGGGCC repeats form into G-quadruplexes in vivo is sparse. We devised a readout strategy, exploiting the sensitivity of trans-cis isomerization of cyanine dyes to local viscosity and sterical constraints. Thereby, folding states of cyanine-labeled RNA, and in particular G-quadruplexes, can be identified in a sensitive manner. The isomerization kinetics, monitored via fluorescence blinking generated upon transitions between a fluorescent trans isomer and a non-fluorescent cis isomer, was first characterized for RNA with GGGGCC repeats in aqueous solution using fluorescence correlation spectroscopy and transient state (TRAST) monitoring. With TRAST, monitoring the isomerization kinetics from how the average fluorescence intensity varies with laser excitation modulation characteristics, we could then detect folding states of fluorescently tagged RNA introduced into live cells.
Assuntos
Demência Frontotemporal , Quadruplex G , Humanos , Corantes Fluorescentes , Demência Frontotemporal/genética , Isomerismo , RNA/químicaRESUMO
Optical nanoscopy, also known as super-resolution optical microscopy, has provided scientists with the means to surpass the diffraction limit of light microscopy and attain new insights into nanoscopic structures and processes that were previously inaccessible. In recent decades, numerous studies have endeavored to enhance super-resolution microscopy in terms of its spatial (lateral) resolution, axial resolution, and temporal resolution. In this review, we discuss recent efforts to push the resolution limit of stimulated emission depletion (STED) optical nanoscopy across multiple dimensions, including lateral resolution, axial resolution, temporal resolution, and labeling precision. We introduce promising techniques and methodologies building on the STED concept that have emerged in the field, such as MINSTED, isotropic STED, and event-triggered STED, and evaluate their respective strengths and limitations. Moreover, we discuss trade-off relationships that exist in far-field optical microscopy and how they come about in STED optical nanoscopy. By examining the latest developments addressing these aspects, we aim to provide an updated overview of the current state of STED nanoscopy and its potential for future research.
Assuntos
MicroscopiaRESUMO
Lanthanide-doped upconversion nanoparticles (UCNPs) have rich photophysics exhibiting complex luminescence kinetics. In this work, we thoroughly investigated the luminescence response of UCNPs to excitation pulse durations. Analyzing this response opens new opportunities in optical encoding/decoding and the assignment of transitions to emission peaks and provides advantages in applications of UCNPs, e.g., for better optical sectioning and improved luminescence nanothermometry. Our work shows that monitoring the UCNP luminescence response to excitation pulse durations (while keeping the duty cycle constant) by recording the average luminescence intensity using a low-time resolution detector such as a spectrometer offers a powerful approach for significantly extending the utility of UCNPs.
RESUMO
BACKGROUND: Increasing evidence suggests that platelets play a central role in cancer progression, with altered storage and selective release from platelets of specific tumor-promoting proteins as a major mechanism. Fluorescence-based super-resolution microscopy (SRM) can resolve nanoscale spatial distribution patterns of such proteins, and how they are altered in platelets upon different activations. Analysing such alterations by SRM thus represents a promising, minimally invasive strategy for platelet-based diagnosis and monitoring of cancer progression. However, broader applicability beyond specialized research labs will require objective, more automated imaging procedures. Moreover, for statistically significant analyses many SRM platelet images are needed, of several different platelet proteins. Such proteins, showing alterations in their distributions upon cancer progression additionally need to be identified. RESULTS: A fast, streamlined and objective procedure for SRM platelet image acquisition, analysis and classification was developed to overcome these limitations. By stimulated emission depletion SRM we imaged nanoscale patterns of six different platelet proteins; four different SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) mediating protein secretion by membrane fusion of storage granules, and two angiogenesis regulating proteins, representing cargo proteins within these granules coupled to tumor progression. By a streamlined procedure, we recorded about 100 SRM images of platelets, for each of these six proteins, and for five different categories of platelets; incubated with cancer cells (MCF-7, MDA-MB-231, EFO-21), non-cancer cells (MCF-10A), or no cells at all. From these images, structural similarity and protein cluster parameters were determined, and probability functions of these parameters were generated for the different platelet categories. By comparing these probability functions between the categories, we could identify nanoscale alterations in the protein distributions, allowing us to classify the platelets into their correct categories, if they were co-incubated with cancer cells, non-cancer cells, or no cells at all. CONCLUSIONS: The fast, streamlined and objective acquisition and analysis procedure established in this work confirms the role of SNAREs and angiogenesis-regulating proteins in platelet-mediated cancer progression, provides additional fundamental knowledge on the interplay between tumor cells and platelets, and represent an important step towards using tumor-platelet interactions and redistribution of nanoscale protein patterns in platelets as a basis for cancer diagnostics.
Assuntos
Neoplasias , Proteínas SNARE , Plaquetas/metabolismo , Fusão de Membrana , Microscopia de Fluorescência/métodos , Neoplasias/metabolismo , Proteínas SNARE/metabolismoRESUMO
Stimulated emission depletion (STED) microscopy is a powerful diffraction-unlimited technique for fluorescence imaging. Despite its rapid evolution, STED fundamentally suffers from high-intensity light illumination, sophisticated probe-defined laser schemes, and limited photon budget of the probes. Here, we demonstrate a versatile strategy, stimulated-emission induced excitation depletion (STExD), to deplete the emission of multi-chromatic probes using a single pair of low-power, near-infrared (NIR), continuous-wave (CW) lasers with fixed wavelengths. With the effect of cascade amplified depletion in lanthanide upconversion systems, we achieve emission inhibition for a wide range of emitters (e.g., Nd3+, Yb3+, Er3+, Ho3+, Pr3+, Eu3+, Tm3+, Gd3+, and Tb3+) by manipulating their common sensitizer, i.e., Nd3+ ions, using a 1064-nm laser. With NaYF4:Nd nanoparticles, we demonstrate an ultrahigh depletion efficiency of 99.3 ± 0.3% for the 450 nm emission with a low saturation intensity of 23.8 ± 0.4 kW cm-2. We further demonstrate nanoscopic imaging with a series of multi-chromatic nanoprobes with a lateral resolution down to 34 nm, two-color STExD imaging, and subcellular imaging of the immunolabelled actin filaments. The strategy expounded here promotes single wavelength-pair nanoscopy for multi-chromatic probes and for multi-color imaging under low-intensity-level NIR-II CW laser depletion.
Assuntos
Elementos da Série dos Lantanídeos , Nanopartículas , Lasers , Luz , Imagem Óptica/métodosRESUMO
A photon avalanche (PA) effect that occurs in lanthanide-doped solids gives rise to a giant nonlinear response in the luminescence intensity to the excitation light intensity. As a result, much weaker lasers are needed to evoke such PAs than for other nonlinear optical processes. Photon avalanches are mostly restricted to bulk materials and conventionally rely on sophisticated excitation schemes, specific for each individual system. Here we show a universal strategy, based on a migrating photon avalanche (MPA) mechanism, to generate huge optical nonlinearities from various lanthanide emitters located in multilayer core/shell nanostructrues. The core of the MPA nanoparticle, composed of Yb3+ and Pr3+ ions, activates avalanche looping cycles, where PAs are synchronously achieved for both Yb3+ and Pr3+ ions under 852 nm laser excitation. These nanocrystals exhibit a 26th-order nonlinearity and a clear pumping threshold of 60 kW cm-2. In addition, we demonstrate that the avalanching Yb3+ ions can migrate their optical nonlinear response to other emitters (for example, Ho3+ and Tm3+) located in the outer shell layer, resulting in an even higher-order nonlinearity (up to the 46th for Tm3+) due to further cascading multiplicative effects. Our strategy therefore provides a facile route to achieve giant optical nonlinearity in different emitters. Finally, we also demonstrate applicability of MPA emitters to bioimaging, achieving a lateral resolution of ~62 nm using one low-power 852 nm continuous-wave laser beam.
Assuntos
Elementos da Série dos Lantanídeos , Íons , Elementos da Série dos Lantanídeos/química , Lasers , Luz , LuminescênciaRESUMO
Mitochondrial membranes and their microenvironments directly influence and reflect cellular metabolic states but are difficult to probe on site in live cells. Here, we demonstrate a strategy, showing how the widely used mitochondrial membrane localization fluorophore 10-nonyl acridine orange (NAO) can be transformed into a multifunctional probe of membrane microenvironments by monitoring its blinking kinetics. By transient state (TRAST) studies of NAO in small unilamellar vesicles (SUVs), together with computational simulations, we found that NAO exhibits prominent reversible singlet-triplet state transitions and can act as a light-induced Lewis acid forming a red-emissive doublet radical. The resulting blinking kinetics are highly environment-sensitive, specifically reflecting local membrane oxygen concentrations, redox conditions, membrane charge, fluidity, and lipid compositions. Here, not only cardiolipin concentration but also the cardiolipin acyl chain composition was found to strongly influence the NAO blinking kinetics. The blinking kinetics also reflect hydroxyl ion-dependent transitions to and from the fluorophore doublet radical, closely coupled to the proton-transfer events in the membranes, local pH, and two- and three-dimensional buffering properties on and above the membranes. Following the SUV studies, we show by TRAST imaging that the fluorescence blinking properties of NAO can be imaged in live cells in a spatially resolved manner. Generally, the demonstrated blinking imaging strategy can transform existing fluorophore markers into multiparametric sensors reflecting conditions of large biological relevance, which are difficult to retrieve by other means. This opens additional possibilities for fundamental membrane studies in lipid vesicles and live cells.
Assuntos
Piscadela , Cardiolipinas , Fluorescência , Corantes Fluorescentes , CinéticaRESUMO
Metastasising cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumours in the clinic. We aimed to clarify how vimentin regulates the motility of metastasising fibroblasts. STED super-resolution microscopy, live-cell imaging and quantitative proteomics revealed that oncogene-expressing and metastasising fibroblasts show a less-elongated cell shape, reduced cell spreading, increased cell migration speed, reduced directionality, and stronger coupling between these migration parameters compared to normal control cells. In total, we identified and compared 555 proteins in the vimentin interactome. In metastasising cells, the levels of keratin 18 and Rab5C were increased, while those of actin and collagen were decreased. Inhibition of HDAC6 reversed the shape, spreading and migration phenotypes of metastasising cells back to normal. Inhibition of HDAC6 also decreased the levels of talin 1, tropomyosin, Rab GDI ß, collagen and emilin 1 in the vimentin interactome, and partially reversed the nanoscale vimentin organisation in oncogene-expressing cells. These findings describe the changes in the vimentin interactome and nanoscale distribution that accompany the defective cell shape, spreading and migration of metastasising cells. These results support the hypothesis that oncogenes can act through HDAC6 to regulate the vimentin binding of the cytoskeletal and cell-extracellular matrix adhesion components that contribute to the defective motility of metastasising cells.
Assuntos
Movimento Celular/fisiologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Vimentina/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Forma Celular/fisiologia , Junções Célula-Matriz/metabolismo , Células Cultivadas , Colágeno/metabolismo , Citoesqueleto/metabolismo , Desacetilase 6 de Histona/metabolismo , Humanos , Camundongos , Oncogenes/fisiologiaRESUMO
The possible targeting functionality and low immunogenicity of exosomes and exosome-like nanovesicles make them promising as drug-delivery carriers. To tap into this potential, accurate non-destructive methods to load them and characterize their contents are of utmost importance. However, the small size, polydispersity, and aggregation of nanovesicles in solution make quantitative characterizations of their loading particularly challenging. Here, an ad-hoc methodology is developed based on burst analysis of dual-color confocal fluorescence microscopy experiments, suited for quantitative characterizations of exosome-like nanovesicles and of their fluorescently-labeled loading. It is applied to study exosome-mimetic nanovesicles derived from animal extracellular-vesicles and human red blood cell detergent-resistant membranes, loaded with fluorescently-tagged dUTP cargo molecules. For both classes of nanovesicles, successful loading is proved and by dual-color coincident fluorescence burst analysis, size statistics and loading yields are retrieved and quantified. The procedure affords single-vesicle characterizations well-suited for the investigation of a variety of cargo molecules and biological nanovesicle combinations besides the proof-of-principle demonstrations of this study. The results highlight a powerful characterization tool essential for optimizing the loading process and for advanced engineering of biomimetic nanovesicles for therapeutic drug delivery.