Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors.

Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids.

Supramolecular nanogels fabricated via host-guest molecular recognition as penetration enhancer for dermal drug delivery.

Nanogels that are assembled by supramolecular interactions as compared to covalent crosslinked nanogels, exhibit new functionalities with potential for easy processability, recycling and self-healing due to the nature of dynamic and reversible non-covalent interactions. Here we design a supramolecular polymer nanogel that utilize host-guest interactions between the groups pillar [5] arene and alkyl chains on hyperbranched polyglycerol backbone as crosslinking agents for a new dermal drug delivery system. The anti-inflammatory drug Dexamethasone (Dexa) can be efficiently loaded into the nanogels and released from the assemblies. Besides, the supramolecular polymer nanogels exhibit better drug loading capacity and skin penetration enhancement than the individual host polymer and guest polymer. In vitro skin permeation studies show that supramolecular polymer nanogels can improve the Nile red penetration through the skin by up to 9 fold, compared to the individual polymers or a conventional cream formulation on a barrier deficient skin model.

Positively Charged Nanoaggregates Based on Zwitterionic Pillar[5]arene that Combat Planktonic Bacteria and Disrupt Biofilms.

Bacterial biofilms are difficult to eradicate because they are less susceptible to antibiotics and more easily develop resistance. Therefore, there is an urgent need for new materials that can combat planktonic bacteria and disrupt established biofilms. To tackle this challenge, we design a multifunctional zwitterionic pillar[5]arene, which can self-assemble into weakly positively charged nanoaggregates that exhibit antibacterial activity against Gram-negative Escherichia coli (DH5α) and Gram-positive Staphylococcus aureus (SH1000) bacterial strains in solution. In addition, the zwitterionic pillar[5]arene can efficiently disrupt pre-existing Escherichia coli (DH5α) biofilms and kill the biofilm-enclosed bacteria without rapid generation of resistance.

Multivalent Flexible Nanogels Exhibit Broad-Spectrum Antiviral Activity by Blocking Virus Entry.

The entry process of viruses into host cells is complex and involves stable but transient multivalent interactions with different cell surface receptors. The initial contact of several viruses begins with attachment to heparan sulfate (HS) proteoglycans on the cell surface, which results in a cascade of events that end up with virus entry. The development of antiviral agents based on multivalent interactions to shield virus particles and block initial interactions with cellular receptors has attracted attention in antiviral research. Here, we designed nanogels with different degrees of flexibility based on dendritic polyglycerol sulfate to mimic cellular HS. The designed nanogels are nontoxic and broad-spectrum, can multivalently interact with viral glycoproteins, shield virus surfaces, and efficiently block infection. We also visualized virus-nanogel interactions as well as the uptake of nanogels by the cells through clathrin-mediated endocytosis using confocal microscopy. As many human viruses attach to the cells through HS moieties, we introduce our flexible nanogels as robust inhibitors for these viruses.

Supramolecular Copolymerization as a Strategy to Control the Stability of Self-Assembled Nanofibers.

A major challenge in supramolecular polymerization is controlling the stability of the polymers formed, that is, controlling the rate of monomer exchange in the equilibrium between monomer and polymer. The exchange dynamics of supramolecular polymers based on benzene-1,3,5-tricarboxamide (BTA) can be regulated by copolymerizing molecules with dendronized (dBTA) and linear (nBTA) ethylene glycol-based water-soluble side chains. Whereas nBTAs form long nanofibers in water, dBTAs do not polymerize, forming instead small spherical aggregates. The copolymerization of the two BTAs results in long nanofibers. The exchange dynamics of both the BTA monomers in the copolymer are significantly slowed down in the mixed systems, leading to a more stable copolymer, while the morphology and spectroscopic signature of the copolymers are identical to that of nBTA homopolymer. This copolymerization is the supramolecular counterpart of styrene/ maleic anhydride copolymerization.

A toolbox approach for multivalent presentation of ligand-receptor recognition on a supramolecular scaffold.

A supramolecular toolbox approach for multivalent ligand-receptor recognition was established based on ß-cyclodextrin vesicles (CDVs). A series of bifunctional ligands for CDVs was synthesised. These ligands comprise on one side adamantane, enabling the functionalisation of CDVs with these ligands, and either mannose or sulphate group moieties on the other side for biological receptor recognition. The physicochemical properties of the host-guest complexes formed by ß-cyclodextrin (ß-CD) and adamantane were determined by isothermal titration calorimetry (ITC). Ligand-lectin interactions were investigated by surface plasmon resonance experiments (SPR) for the mannose ligands and the lectin Concanavalin A (ConA). Microscale thermophoresis (MST) measurements were applied for sulphate-dependent binding to L-selectin. In both cases, a multivalent affinity enhancement became apparent when the ligands were presented on the CDV scaffold. Furthermore, not only the clustering between our supramolecular mannosylated complex and Escherichia coli (E. coli), expressing the lectin FimH, was visualised by cryo-TEM, but also the competitive character to detach bound E. coli from a cell line, representing the uroepithelial cell surface, was demonstrated. In summary, a facile and effective supramolecular toolbox was established for various ligand-receptor recognition applications.

Defined pH-sensitive nanogels as gene delivery platform for siRNA mediated in vitro gene silencing.

In the present study, a pH sensitive nanogel platform for gene delivery was developed. The cationic nanogels based on dendritic polyglycerol (dPG) and low molecular weight polyethylenimine units were able to encapsulate siRNA during the manufacturing process. The thiol-Michael nanoprecipitation method, which operates under mild conditions and did not require any catalyst or surfactant, was used to develop tailor-made nanogels in the sub-100 nm range. The incorporation of pH sensitive benzacetal-bonds inside the nanogel network enables the controlled intracellular release of the cargo. The functionality to transport therapeutic biomolecules was tested by an in vitro GFP-siRNA transfection assay. Encapsulated siRNA could silence GFP expressing HeLa cells (up to 71% silencing in GFP). Furthermore, significantly reduced toxicity of the nanogel platform compared to the non-degradable PEI was observed. These properties realize a new carrier platform in the field of gene therapy.

Ethyl Lithiodiazoacetate: Extremely Unstable Intermediate Handled Efficiently in Flow.

Ethyl diazoacetate (EDA) is one of the most prominent diazo reagents. It is frequently used in metal-carbene-type reactions. However, EDA can also be used as a nucleophile under base catalysis. Whilst the addition of EDA to aldehydes can be performed using organic bases, the addition of EDA to other carbonyl electrophiles requires the use of organometallics such as lithium diisopropylamide (LDA). The generated ethyl lithiodiazoacetate is highly reactive and decomposes rapidly, even at low temperatures. Herein, we report a continuous flow protocol that overcomes the problems associated with the instantaneous decomposition of ethyl lithiodiazoacetate. The addition of ethyl lithiodiazoacetate to ketones provides direct access to tertiary diazoalcohols in good yields.