Modern drug formulations often require, besides the active drug molecule, auxiliaries to enhance their pharmacological properties. Tailor-made, biocompatible polymers covalently connected to the drug molecule can fulfill this function by increasing its solubility, reducing its toxicity, and guiding it to a specific target. If targeting membrane-bound proteins, localization of the drug close to the cell membrane and its target is beneficial to increase drug efficiency and residence time. In this study, we present the synthesis of highly defined, branched polymeric structures with membrane-binding properties. One to three hydrophilic poly(ethylene oxide) or poly(2-ethyloxazoline) side chains were connected via a peptoid backbone using a two-step iterative protocol for solid-phase peptoid synthesis. Additional groups, e.g., a hydrophobic anchor for membrane attachment, were introduced. Due to the nature of solid-phase synthesis, the number and order of the side chains and additional units can be precisely defined. The method proved to be versatile for the generation of multifunctional, branched polymeric structures of molecular weights up to approximately 7000 g mol-1. The behavior of all compounds towards biological membranes and cells was investigated using liposomes as cell membrane models, HEK293 and U251-MG cell lines, and red blood cells, thereby demonstrating their potential value as drug auxiliaries with cell membrane affinity.

• Publikační typ
• časopisecké články MeSH

The ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Fluorescence-based methods like two-color fluorescence cross-correlation spectroscopy can provide this information, but measurement precision is hampered by various sources of errors caused by instrumental or optical limitations such as imperfect overlap of detection volumes or detector cross talk. Furthermore, the nature and properties of used fluorescent proteins or fluorescent dyes, such as labeling efficiency, fluorescent protein maturation, photostability, bleaching, and fluorescence brightness can have an impact. Here, we take advantage of previously published fluorescence lifetime correlation spectroscopy which relies on lifetime differences as a mean to discriminate fluorescent proteins with similar spectral properties and to use them for single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS). By using only one excitation and one detection wavelength, this setup avoids all sources of errors resulting from chromatic aberrations and detector cross talk. To establish sc-FLCCS, we first engineered and tested multiple green fluorescent protein (GFP)-like fluorescent proteins for their suitability. This identified a novel, to our knowledge, GFP variant termed short-lifetime monomeric GFP with the so-far shortest lifetime. Monte-Carlo simulations were employed to explore the suitability of different combinations of GFP variants. Two GFPs, Envy and short-lifetime monomeric GFP, were predicted to constitute the best performing couple for sc-FLCCS measurements. We demonstrated application of this GFP pair for measuring protein interactions between the proteasome and interacting proteins and for measuring protein interactions between three partners when combined with a red florescent protein. Together, our findings establish sc-FLCCS as a valid alternative for conventional dual-color fluorescence cross-correlation spectroscopy measurements.

• MeSH
• fluorescence MeSH
• fluorescenční barviva * MeSH
• fluorescenční spektrometrie MeSH
• luminescentní proteiny genetika   MeSH
• zelené fluorescenční proteiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fluorescenční barviva * MeSH
• luminescentní proteiny MeSH
• zelené fluorescenční proteiny MeSH

Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.

• MeSH
• buněčná membrána metabolismus   MeSH
• dimerizace MeSH
• lidé MeSH
• membránové lipidy metabolismus   MeSH
• membránové proteiny * metabolismus   MeSH
• multimerizace proteinu MeSH
• rezonanční přenos fluorescenční energie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové lipidy MeSH
• membránové proteiny * MeSH

The coexistence of lipid domains with different degrees of lipid packing in the plasma membrane of mammalian cells has been postulated, but direct evidence has so far been challenging to obtain because of the small size and short lifetime of these domains in live cells. Here, we use fluorescence spectral correlation spectroscopy in conjunction with a probe sensitive to the membrane environment to quantify spectral fluctuations associated with dynamics of membrane domains in live cells. With this method, we show that membrane domains are present in live COS-7 cells and have a lifetime lower bound of 5.90 and 14.69 ms for the ordered and disordered phases, respectively. Comparisons to simulations indicate that the underlying mechanism of these fluctuations is complex but qualitatively described by a combination of dye diffusion between membrane domains as well as the motion of domains within the membrane.

• MeSH
• benzoxaziny chemie   MeSH
• buněčná membrána chemie   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• fluorescenční spektrometrie MeSH
• kvartérní amoniové sloučeniny chemie   MeSH
• membránové lipidy chemie   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzoxaziny MeSH
• kvartérní amoniové sloučeniny MeSH
• membránové lipidy MeSH
• NR12S compound MeSH Prohlížeč

To facilitate efficient drug delivery to tumor tissue, several nanomaterials have been designed, with combined diagnostic and therapeutic properties. In this work, we carried out fundamental in vitro and in vivo experiments to assess the labeling efficacy of our novel theranostic nanoprobe, consisting of glycogen conjugated with a red fluorescent probe and gadolinium. Microscopy and resazurin viability assays were used to study cell labeling and cell viability in human metastatic melanoma cell lines. Fluorescence lifetime correlation spectroscopy (FLCS) was done to investigate nanoprobe stability. Magnetic resonance imaging (MRI) was performed to study T₁ relaxivity in vitro, and contrast enhancement in a subcutaneous in vivo tumor model. Efficient cell labeling was demonstrated, while cell viability, cell migration, and cell growth was not affected. FLCS showed that the nanoprobe did not degrade in blood plasma. MRI demonstrated that down to 750 cells/μL of labeled cells in agar phantoms could be detected. In vivo MRI showed that contrast enhancement in tumors was comparable between Omniscan contrast agent and the nanoprobe. In conclusion, we demonstrate for the first time that a non-toxic glycogen-based nanoprobe may effectively visualize tumor cells and tissue, and, in future experiments, we will investigate its therapeutic potential by conjugating therapeutic compounds to the nanoprobe.

• Klíčová slova
• fluorescence lifetime correlation spectroscopy, fluorescence microscopy, high throughput microscopy, magnetic resonance imaging, melanoma brain metastasis, nanoprobe, theranostics, zeta potential,
• MeSH
• barvení a značení MeSH
• cytoplazma metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• glykogen metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• kontrastní látky chemie   MeSH
• lidé MeSH
• lyzozomy metabolismus   MeSH
• magnetická rezonanční tomografie metody   MeSH
• melanom metabolismus   patologie   MeSH
• molekulární sondy * MeSH
• molekulární zobrazování metody   MeSH
• multimodální zobrazování * MeSH
• nádorové buněčné linie MeSH
• nanotechnologie * MeSH
• pohyb buněk MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glykogen MeSH
• kontrastní látky MeSH
• molekulární sondy * MeSH