Synthesis of Asymmetric Ionic Hybrid Detergents enables Micelles with Scalable Properties including Cell Compatibility.

Ionic detergents enable applications and cause harm in biospheres due to cell toxicity. The utility of covalent combinations between ionic and non-ionic detergent headgroups in modulating cell toxicity remains speculative due to the yet rarely explored synthesis. We close this gap and establish the modular synthesis of ionic/non-ionic hybrid detergents. We restructure a combinatorial methallyl dichloride one-pot coupling into a two-step coupling, which reduces by-products, improves product yields, and enables the gram-scale preparation of asymmetric, cationic/non-ionic and anionic/non-ionic hybrid detergents. Our modular synthesis delivers new modalities for the design of ionic detergents, including an unprecedented scaling of properties that determine applications, such as charge, critical micelle concentration, solubilizing properties, hard water tolerance, and cell compatibility. We uncover that shielding the charge in ionic headgroups can switch the detergent species that is toxic to cells from monomers to mixtures of monomers and micellar assemblies. Establishing the chemistry of ionic/non-ionic hybrid detergents provides a missing evolutionary link in the structural comparison of ionic and non-ionic detergents, enables an easy synthesis access to yet unexplored chemical spaces of asymmetric hybrid materials, and delivers new modalities for designing the toxicity of supramolecular nanomaterials.

Detergent Chemistry Modulates the Transgression of Planetary Boundaries including Antimicrobial Resistance and Drug Discovery.

Detergent chemistry enables applications in the world today while harming safe operating spaces that humanity needs for survival. Aim of this review is to support a holistic thought process in the design of detergent chemistry. We harness the planetary boundary concept as a framework for literature survey to identify progresses and knowledge gaps in context with detergent chemistry and five planetary boundaries that are currently transgressed, i.e., climate, freshwater, land system, novel entities, biosphere integrity. Our survey unveils the status of three critical challenges to be addressed in the years to come, including (i) the implementation of a holistically, climate-friendly detergent industry; (ii) the alignment of materialistic and social aspects in creating technical solutions by means of sustainable chemistry; (iii) the development of detergents that serve the purpose of applications but do not harm the biosphere in their role as novel entities. Specifically, medically relevant case reports revealed that even the most sophisticated detergent design cannot sufficiently accelerate drug discovery to outperform the antibiotic resistance development that detergents simultaneously promote as novel entities. Safe operating spaces that humanity needs for its survival may be secured by directing future efforts beyond sustainable chemistry, resource efficiency, and net zero emission targets.

Trends in the Diversification of the Detergentome.

Detergents are amphiphilic molecules that serve as enabling steps for today's world applications. The increasing diversity of the detergentome is key to applications enabled by detergent science. Regardless of the application, the optimal design of detergents is determined empirically, which leads to failed preparations, and raising costs. To facilitate project planning, here we review synthesis strategies that drive the diversification of the detergentome. Synthesis strategies relevant for industrial and academic applications include linear, modular, combinatorial, bio-based, and metric-assisted detergent synthesis. Scopes and limitations of individual synthesis strategies in context with industrial product development and academic research are discussed. Furthermore, when designing detergents, the selection of molecular building blocks, i. e., head, linker, tail, is as important as the employed synthesis strategy. To facilitate the design of safe-to-use and tailor-made detergents, we provide an overview of established head, linker, and tail groups and highlight selected scopes and limitations for applications. It becomes apparent that most recent contributions to the increasing chemical diversity of detergent building blocks originate from the development of detergents for membrane protein studies. The overview of synthesis strategies and molecular blocks will bring us closer to the ability to predictably design and synthesize optimal detergents for challenging future applications.

A Photoswitchable Solvatochromic Dye for Probing Membrane Ordering by RESOLFT Super-resolution Microscopy.

A switchable solvatochromic fluorescent dyad can be used to map ordering of lipids in vesicle membranes at a resolution better than the diffraction limit. Combining a Nile Red fluorophore with a photochromic spironaphthoxazine quencher allows the fluorescence to be controlled using visible light, via photoswitching and FRET quenching. Synthetic lipid vesicles of varying composition were imaged with an average 2.5-fold resolution enhancement, compared to the confocal images. Ratiometric detection was used to probe the membrane polarity, and domains of different lipid ordering were distinguished within the same membrane.

Protocol to test the utility of detergents for E. coli membrane protein extraction and delipidation.

We present a protocol to evaluate the utility of detergents for purification and delipidation of E. coli membrane proteins. We determine the critical aggregation concentration of detergents. Furthermore, we compare the ability of detergents to extract membrane proteins and to maintain protein-lipid interactions during purification. The protocol describes steps for isolating and delipidating membrane proteins from E. coli membranes by extraction and affinity purification using detergents. The protocol does not enable an absolute quantification of purification outcomes. For complete details on the use and execution of this protocol, please refer to Urner et al.1.

Towards Probing Conformational States of Y2 Receptor Using Hyperpolarized 129Xe NMR.

G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor-cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand.

Aggregation of Amphiphilic Carbocyanines: Fluorination Favors Cylindrical Micelles over Bilayered Tubes.

The synthesis of a new amphiphilic 5,5',6,6'-tetrachlorobenzimidacarbocyanine dye derivative with -(CH2)2-(CF2)5-CF3 chains attached to the nitrogen atoms in the 1,1'-position, CF8O3, is reported. Depending on the dye concentration and the addition of MeOH, CF8O3 forms J- and H-aggregates in aqueous solutions. The aggregation behavior was investigated using steady-state absorption, linear dichroism, and fluorescence spectroscopy, as well as by cryogenic transmission electron microscopy (cryo-TEM). The J-band of the MeOH-free solution is monomer-like, rather broad, and less red-shifted with respect to the monomer absorption, indicating weak excitonic coupling and disorder effects. Cryo-TEM reveals a diversity of supramolecular structures, wherein linear and branched cylindrical micelles dominate. It is concluded that the high stiffness of fluoroalkyl chains does not allow the chains to splay and completely fill up the hydrophobic gap between opposing chromophores. This destabilizes the bilayers and favors the micellar structure motifs instead. The aggregates appearing at 30% MeOH show a split absorption spectrum consisting of a broad blue-shifted H-band and an accompanying sharp red-shifted J-band with perpendicular polarizations. These HJ-type aggregates are also composed of micellar fibers, but these bundle into rope-like strands. For 10% MeOH, a narrow bilayered tube is the dominating morphology. The observed MeOH dependence of aggregation reveals a clear cosolvent effect.

Stereochemistry-Controlled Supramolecular Architectures of New Tetrahydroxy-Functionalised Amphiphilic Carbocyanine Dyes.

The syntheses of novel amphiphilic 5,5',6,6'-tetrachlorobenzimidacarbocyanine (TBC) dye derivatives with aminopropanediol head groups, which only differ in stereochemistry (chiral enantiomers, meso form and conformer), are reported. For the achiral meso form, a new synthetic route towards asymmetric cyanine dyes was established. All compounds form J aggregates in water, the optical properties of which were characterised by means of spectroscopic methods. The supramolecular structure of the aggregates is investigated by means of cryo-transmission electron microscopy, cryo-electron tomography and AFM, revealing extended sheet-like aggregates for chiral enantiomers and nanotubes for the mesomer, respectively, whereas the conformer forms predominately needle-like crystals. The experiments demonstrate that the aggregation behaviour of compounds can be controlled solely by head group stereochemistry, which in the case of enantiomers enables the formation of extended hydrogen-bond chains by the hydroxyl functionalities. In case of the achiral meso form, however, such chains turned out to be sterically excluded.

Cells Undergo Major Changes in the Quantity of Cytoplasmic Organelles after Uptake of Gold Nanoparticles with Biologically Relevant Surface Coatings.

Here, we use cryo soft X-ray tomography (cryo-SXT), which delivers 3D ultrastructural volumes of intact cells without chemical fixation or staining, to gain insight about nanoparticle uptake for nanomedicine. We initially used dendritic polyglycerol sulfate (dPGS) with potential diagnostic and therapeutic applications in inflammation. Although dPGS-coated gold nanoparticle (dPGS-AuNP) uptake followed a conventional endocytic/degradative pathway in human lung epithelial cell lines (A549), with cryo-SXT, we detected ∼5% of dPGS-AuNPs in the cytoplasm, a level undetectable by confocal light microscopy. We also observed ∼5% of dPGS-AuNPs in a rarely identified subcellular site, namely, lipid droplets, which are important for cellular energy metabolism. Finally, we also found substantial changes in the quantity of cytoplasmic organelles upon dPGS-AuNP uptake over the 1-6 h incubation period; the number of small vesicles and mitochondria significantly increased, and the number of multivesicular bodies and the number and volume of lipid droplets significantly decreased. Although nearly all organelle numbers at 6 h were still significantly different from controls, most appeared to be returning to normal levels. To test for generality, we also examined cells after uptake of gold nanoparticles coated with a different agent, polyethylenimine (PEI), used for nucleic acid delivery. PEI nanoparticles did not enter lipid droplets, but they induced similar, albeit less pronounced, changes in the quantity of cytoplasmic organelles. We confirmed these changes in organelle quantities for both nanoparticle coatings by confocal fluorescence microscopy. We suggest this cytoplasmic remodeling could reflect a more common cellular response to coated gold nanoparticle uptake.

Super-resolution RESOLFT microscopy of lipid bilayers using a fluorophore-switch dyad.

Dyads consisting of a photochromic switch covalently linked to a fluorescent dye allow the emission from the dye to be controlled by reversible photoisomerization of the switch; one form of the switch quenches fluorescence by accepting energy from the dye. Here we investigate the use of dyads of this type for super-resolution imaging of lipid bilayers. Giant unilamellar vesicles stained with the dyads were imaged with about a two-fold resolution-enhancement compared with conventional confocal microscopy. This was achieved by exciting the fluorophore at 594 nm, using a switch activated by violet and red light (405/640 nm).

Functionalized graphene sheets for intracellular controlled release of therapeutic agents.

Since therapeutic agents target specific compartments inside the cells, their efficiency depends on their intracellular release from drug delivery systems (DDS). However, control over the intracellular release of therapeutic agents is a challenging issue and can only be achieved by governing their interactions with the DDS. In this work, polyglycerol amine- and polyglycerol sulfate-functionalized graphene sheets as positively and negatively charged 2D nanomaterials with 150 nm lateral size were used to deliver and control the release of doxorubicin (DOX) inside cells. A pH-sensitive dye was conjugated onto the surfaces of graphene sheets and used as an antenna to obtain specific signals from the acidic cell compartments. It was found that both positively and negatively charged graphene sheets undergo similar acidification processes after cellular uptake. Nevertheless, the intracellular drug release of these DOX-loaded nanomaterials was distinctly different. As an overall effect of the π-π stacking and electrostatic interactions, the release of DOX from the positively charged graphene sheets was much faster than that from their analogs with a negative surface charge. Therefore, therapeutic efficiency in the first case was much higher than that in the latter. Based on our findings, the intracellular release of drugs from the surfaces of graphene sheets can be finely tuned by manipulating their functionalities, which is of great importance in the designing of the future graphene-based nanomedicines.

Heterobifunctional Dyes: Highly Fluorescent Linkers Based on Cyanine Dyes.

Herein, we present a new synthetic route to cyanine-based heterobifunctional dyes and their application as fluorescent linkers between polymers and biomolecules. The synthesized compounds, designed in the visible spectral range, are equipped with two different reactive groups for highly selective conjugation under physiological conditions. By applying indolenine precursors with functionalized benzenes, we achieved water-soluble asymmetric cyanine dyes bearing maleimido and N-hydroxysuccinimidyl functionalities in a three-step synthesis. Spectroscopic characterization revealed good molar absorption coefficients and moderate fluorescence quantum yields. Further reaction with polyethylene glycol yielded dye-polymer conjugates that were subsequently coupled to the antibody cetuximab, often applied in cancer therapy. Successful coupling was confirmed by mass shifts detected by gel electrophoresis. Receptor-binding studies and live-cell imaging revealed that labeling did not alter the biological function. In sum, we provided a successful synthetic pathway to rigid heterobifunctional cyanine dyes that are applicable as fluorescent linkers, for example, for connecting antibodies with macromolecules. Our approach contributes to the field of bioconjugation chemistry, such as antibody-drug conjugates by combining diagnostic and therapeutic approaches.

Fullerene Polyglycerol Amphiphiles as Unimolecular Transporters.

Due to their unique structure and properties, water-soluble fullerene derivatives are of great interest for various biomedical purposes. In this work, solution behavior, encapsulation and release properties, biocompatibility, and cellular uptake pathways of fullerene-polyglycerol amphiphiles (FPAs) with defined structures are investigated. The number of polyglycerol branches attached to the surface of fullerene affects the physicochemical properties of FPAs dramatically but not their cellular uptake. Release of encapsulated hydrophobic dyes from FPAs strongly depends on the number of their branches. Conjugation of a pH-sensitive dye to the FPAs as a probe showed that their self-assemblies are taken up through endocytotic pathways. It was observed that FPAs are able to transfer small molecules into cells both above and below their critical aggregation concentration. Taking advantage of the water solubility, biocompatibility, and transfer-ability of FPAs, they might find use as unimolecular carriers for future biomedical applications.

Synthesis and Validation of Functional Nanogels as pH-Sensors in the Hair Follicle.

In the present study, a pH responsive dendritic polyglycerol nanogel (dPG-NG) is developed to measure the pH values inside the hair follicle (HF) using an ex vivo porcine ear model. The macromolecular precursors are labeled with a pH sensitive indodicarbocyanine dye (pH-IDCC) and a control dye (indocarbocyanine dye: ICC) and crosslinked via a mild and surfactant-free Thiol-Michael reaction using an inverse nanoprecipitation method. With this method, it is possible to prepare tailor-made particles in the range of 100 nm to 1 µm with a narrow polydispersity. The dPG-NGs are characterized using dynamic light scattering, nanoparticle tracking analysis, and atomic force microscopy. Systematic analysis of confocal microscope images of histological sections of the skin enables accurate determination of the pH gradient inside the HF. The results show that these novel pH-nanosensors deeply penetrate the skin via the follicular pathway and the pH of the pig hair follicles increase from 6.5 at the surface of the skin to 7.4 in deeper areas of the HF. The pH-nanosensor shows no toxicity potentials.

Synthesis, self-assembly, and photocrosslinking of fullerene-polyglycerol amphiphiles as nanocarriers with controlled transport properties.

In this work, we report a new, simple, gram-scale method for synthesizing water-soluble fullerene-polyglycerol amphiphiles (FPAs) that self-assemble into partially and fully crosslinked nanoclusters with the ability to controllably transport hydrophobic and hydrophilic agents.

Responsive Contrast Agents: Synthesis and Characterization of a Tunable Series of pH-Sensitive Near-Infrared Pentamethines.

The demand for responsive dyes in optical imaging is high to achieve a better signal-to-noise ratio and, more specifically, to visualize acidic compartments of the endocytic pathway. Herein, we present a new synthetic route, with a step-by-step synthesis of water-soluble pH-sensitive cyanine dyes exhibiting pKa values in the region of physiological pH, as confirmed by absorption and fluorescence spectra. Moreover, modification of pKa values was achieved by two different substitution patterns, creating tunable pH-sensitive dyes. We demonstrated the functionality of the pH-sensitive dyes and their suitability as contrast agents for cellular uptake studies by preparing dye-labeled cetuximab and transferrin conjugates. Sulfonated head chains increased water solubility and prevented the formation of dimers, even in the context of dye-labeled bioconjugates. Confocal microscopy images of living cells revealed their pH-responsiveness, as specific fluorescence signal enhancements were observed in acidic compartments of the endocytic pathway (endosomes and lysosomes), although the background signal was low in a pH-neutral environment. Using mixtures of conjugates labeled with either a pH-sensitive or non-pH-sensitive dye for the uptake studies, we could follow the receptor binding and distinguish it from the endocytic uptake process of the conjugates in a simultaneous manner. Moreover, we used flow cytometry to quantify the fluorescence and observed a 3-fold signal enhancement for the pH-sensitive dye conjugates over a period of 3 h.

Glycerol-based contrast agents: a novel series of dendronized pentamethine dyes.

The synthesis of water-soluble dyes, which absorb and emit in the range between 650 and 950 nm and display high extinction coefficients (ε) as well as high fluorescence quantum yields (Φf), is still a demand for optical imaging. We now present a synthetic route for the preparation of a new group of glycerol-substituted cyanine dyes from dendronized indole precursors that have been functionalized as N-hydroxysuccinimide (NHS) esters. High Φf values of up to 0.15 and extinction coefficients of up to 189 000 L mol(-1) cm(-1) were obtained for the pure dyes. Furthermore, conjugates of the new dendronized dyes with the antibody cetuximab (ctx) that were directed against the epidermal growth factor receptor (EGFR) of tumor cells could be prepared with dye to protein ratios between 0.3 and 2.2 to assess their potential as imaging probes. For the first time, ctx conjugates could be achieved without showing a decrease in Φf and with an increasing labeling degree that exceeded the value of the pure dye even at a labeling degree above 2. The incorporation of hydrophilically and sterically demanding dendrimers into cyanines prevented dimer formation after covalent conjugation to the antibody. The binding functionality of the resulting ctx conjugates to the EGFR was successfully demonstrated by cell microscopy studies using EGFR expressing cell lines. In summary, the combination of hydrophilic glycerol dendrons with reactive dye labels has been established for the first time and is a promising approach toward more powerful fluorescent labels with less dimerization.