Bioorthogonal Cleavage and Exchange of Major Histocompatibility Complex Ligands by Employing Azobenzene‐Containing Peptides

Bioorthogonal cleavable linkers are attractive building blocks for compounds that can be manipulated to study biological and cellular processes. Sodium dithionite sensitive azobenzene‐containing (Abc) peptides were applied for the temporary stabilization of recombinant MHC complexes, which can then be employed to generate libraries of MHC tetramers after exchange with a novel epitope. This technology represents an important tool for high‐throughput studies of disease‐specific T cell responses.     

A General Strategy for Nano‐Encapsulation via Interfacially Confined Living/Controlled Radical Miniemulsion Polymerization

Summary: We propose and demonstrate the utility of an interfacial living/controlled (reversible addition fragmentation chain transfer, RAFT) radical miniemulsion polymerization in nano‐encapsulation. The principles and methodology behind this technique are readily scalable and highly efficient. The living/controlled nature of the system offers great opportunities to tune the properties of the polymer shell‐like thickness, surface functionality, molecular weight, and inner‐wall functionality by simply using a semi‐continuous polymerization technique.

Illustration of encapsulation principles by RAFT interfacial miniemulsion polymerization.     

pH‐Triggered Charge‐Reversal Polyurethane Micelles for Controlled Release of Doxorubicin

A series of pH‐triggered charge‐reversal polyurethane copolymers (PS‐PUs) containing methoxyl‐poly(ethylene glycol) (mPEG), carboxylic acid groups, and piperazine groups is presented in this work. The obtained PS‐PUs copolymers can form into stable micelles at pH 7.4, which response to a narrow pH change (5.5–7.5) and show a tunable pH‐triggered charge‐reversal property. Doxorubicin (DOX) is encapsulated into the PS‐PU micelles as a model drug. The drug release of DOX‐loaded PS‐PU micelles shows an obviously stepped‐up with reducing the pH. Meanwhile, it is found that the charge‐reversal property can improve the cellular uptake behavior and intracellular drug release in both HeLa cells and MCF‐7 cells. Additionally, the time‐dependent cytotoxicity of the DOX‐loaded PS‐PU micelles is confirmed by MTT assay. Attributed to the tunable charge‐reversal property through changing the molar ratio of piperazine/carboxyl, the PS‐PU micelles will be a potential candidate as an intelligent drug delivery system in future studies.

     

A Cyclodextrin Microgel for Controlled Release Driven by Inclusion Effects

Summary: A novel intelligent delivery system based on the environmental dependence of the inclusion effect of β‐cyclodextrin (β‐CD) with guest molecules, using a β‐CD polymer (CDP) microgel as carrier, is proposed. Compared with smart hydrogels, which are driven by the phase‐volume transition, controlled release from the CDP microgel is driven by “host‐guest” inclusion effects. With the pH‐dependent inclusion complexation of methyl orange (MO) with β‐CD as a model system, the behavior of the controlled release of a CDP microgel was tested by changing the pH, showing that the mechanism is reasonable.

Schematic illustration of the pH‐dependent inclusion complexation of MO with β‐CD in the CDP microgel.     

Nitrile Oxide‐Norbornene Cycloaddition as a Bioorthogonal Crosslinking Reaction for the Preparation of Hydrogels

This communication describes the first application of cycloaddition between an in situ generated nitrile oxide with norbornene leading to a polymer crosslinking reaction for the preparation of poly(ethylene glycol) hydrogels under physiological conditions. Hydrogels with high water content and robust physical strength are readily formed within 2–5 min by a simple two‐solution mixing method which allows 3D encapsulation of neuronal cells. This bioorthogonal crosslinking reaction provides a simple yet highly effective method for preparation of hydrogels to be used in bioengineering.

     

A biuret‐derived,MS‐cleavable cross‐linking reagent for protein structural analysis: A proof‐of‐principle study

Chemical cross‐linking combined with mass spectrometry (XL‐MS) and computational modeling has evolved as an alternative method to derive protein 3D structures and to map protein interaction networks. Special focus has been laid recently on the development and application of cross‐linkers that are cleavable by collisional activation as they yield distinct signatures in tandem mass spectra. Building on our experiences with cross‐linkers containing an MS‐labile urea group, we now present the biuret‐based, CID‐MS/MS‐cleavable cross‐linker imidodicarbonyl diimidazole (IDDI) and demonstrate its applicability for protein cross‐linking studies based on the four model peptides angiotensin II, MRFA, substance P, and thymopentin.     

Dual thermo‐ and pH‐sensitive network‐grafted hydrogels formed by macrocrosslinker as drug delivery system

Dual thermo‐ and pH‐sensitive network‐grafted hydrogels made of poly(N,N‐dimethylaminoethyl methacrylate) (PDMAEMA) network and poly(N‐isopropylacrylamide) (PNIPAM) grafting chains were successfully synthesized by the combination of atom transfer radical polymerization (ATRP), reversible addition‐fragmentation chain transfer (RAFT) polymerization, and click chemistry. PNIPAM having two azide groups at one chain end [PNIPAM‐(N₃)₂] was prepared with an azide‐capped ATRP initiator of N,N‐di(β‐azidoethyl) 2‐chloropropionylamide. Alkyne‐pending poly(N,N‐dimethylaminoethyl methacrylate‐co‐propargyl acrylate) [P(DMAEMA‐co‐ProA)] was obtained through RAFT copolymerization using dibenzyltrithiocarbonate as chain transfer agent. The subsequent click reaction led to the formation of the network‐grafted hydrogels. The influences of the chemical composition of P(DMAEMA‐co‐ProA) on the properties of the hydrogels were investigated in terms of morphology and swelling/deswelling kinetics. The dual stimulus‐sensitive hydrogels exhibited fast response, high swelling ratio, and reproducible swelling/deswelling cycles under different temperatures and pH values. The uptake and release of ceftriaxone sodium by these hydrogels showed both thermal and pH dependence, suggesting the feasibility of these hydrogels as thermo‐ and pH‐dependent drug release devices. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Super‐Resolution Imaging of the Golgi in Live Cells with a Bioorthogonal Ceramide Probe

We report a lipid‐based strategy to visualize Golgi structure and dynamics at super‐resolution in live cells. The method is based on two novel reagents: a trans‐cyclooctene‐containing ceramide lipid (Cer‐TCO) and a highly reactive, tetrazine‐tagged near‐IR dye (SiR‐Tz). These reagents assemble via an extremely rapid “tetrazine‐click” reaction into Cer‐SiR, a highly photostable “vital dye” that enables prolonged live‐cell imaging of the Golgi apparatus by 3D confocal and STED microscopy. Cer‐SiR is nontoxic at concentrations as high as 2 μM and does not perturb the mobility of Golgi‐resident enzymes or the traffic of cargo from the endoplasmic reticulum through the Golgi and to the plasma membrane.     

Metallo‐Supramolecular Nanogels for Intracellular pH‐Responsive Drug Release

A simple process is developed to fabricate metallo‐supramolecular nanogels (MSNs) by the metallo‐supramolecular‐coordinated interaction between histidine and iron‐meso‐tetraphenylporphin. MSNs are composed of histidine‐modified dextran (DH) and iron‐meso‐tetraphenylporphin (Fe–Por) and exhibit excellent biocompatibility and stability. MSNs show pH responsiveness in the intracellular mildly acidic environment, which has great potential for acid‐triggered drug release delivery. In vitro drug release profiles demonstrate that the pH‐dependent disassembly of MSNs to histidine and Por results in a quicker release rate of loaded‐DOX at pH 5.3, while at pH 7.4 MSNs could hinder the release of loaded‐DOX due to the enhanced stability of MSNs.

     

Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels–Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)-a method that allows to incorporate noncanonical amino acids (ncAAs) site-specifically into proteins in vivo. These reactions enable residue-specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to β-elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo β-elimination and b) a fluorescence flow cytometry based FRET-assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE-specific experimental conditions, such as long-term exposure of the ring-strained ncAA to living cells, that are not taken into account in other assays.     

Redox‐Triggered Self‐Rolling Robust Hydrogel Tubes for Cell Encapsulation

In this study, mechanically strong hydrogels are synthesized by photopolymerization of 2‐vinyl‐4,6‐diamino‐1,3,5‐triazine, poly(ethylene glycol) methacrylate, and disulfide‐containing cross‐linker, N′N‐bis(acryloyl)cystamine. The bilayer hydrogel with distinct cross‐linking density is shown to self‐roll into a 3D tube, which could still be well reinforced by hydrogen bondings, upon exposing reductants such as 1,4‐dithio‐DL‐threitol (DTT) or L‐glutathione (GSH), because the redox‐induced cleavage of disulfide bonds results in the imbalanced internal shrinking stress between two layers. At an intracellular level of GSH, model L929 cells‐seeded bilayer gel sheet could curl up into a 3D tubular scaffold where the cells maintained good viability.

     

A Synthetic Toolbox for the In Situ Formation of Functionalized Homo‐ and Heteropolysaccharide‐Based Hydrogel Libraries

A synthetic toolbox for the introduction of aldehydo and hydrazido groups into the polysaccharides hyaluronic acid, alginate, dextran, pullulan, glycogen, and carboxymethyl cellulose and their use for hydrogel formation is reported. Upon mixing differently functionalized polysaccharides derived from the same natural precursor, hydrazone cross‐linking takes place, which results in formation of a hydrogel composed of one type of polysaccharide backbone. Likewise, hydrogels based on two different polysaccharide strands can be formed after mixing the corresponding aldehydo‐ and hydrazido‐modified polysaccharides. A second line of these studies paves the way to introduce a biomedically relevant ligand, namely, the adhesion factor cyclic RGD pentapeptide, by using an orthogonal click reaction. This set of modified polysaccharides served to create a library of hydrogels that differ in the combination of polysaccharide strands and the degree of cross‐linking. The different hydrogels were evaluated with respect to their rheological properties, their ability to absorb water, and their cytotoxicity towards human fibroblast cell cultures. None of the hydrogels studied were cytotoxic, and, hence, they are in principal biocompatible for applications in tissue engineering.     

pH‐Tunable Hydrogelators for Water Purification: Structural Optimisation and Evaluation

A focused library of potential hydrogelators each containing two substituted aromatic residues separated by a urea or thiourea linkage have been synthesised and characterized. Six of these novel compounds are highly efficient hydrogelators, forming gels in aqueous solution at low concentrations (0.03–0.60 wt %). Gels were formed through a pH switching methodology, by acidification of a basic solution (pH 14 to ≈4) either by addition of HCl or via the slow hydrolysis of glucono‐δ‐lactone. Frequently, gelation was accompanied by a dramatic switch in the absorption spectra of the gelators, resulting in a significant change in colour, typically from a vibrant orange to pale yellow. Each of the gels was capable of sequestering significant quantities of the aromatic cationic dye, methylene blue, from aqueous solution (up to 1.02 g of dye per gram of dry gelator). Cryo‐transmission electron microscopy of two of the gels revealed an extensive network of high aspect ratio fibers. The structure of the fibers altered dramatically upon addition of 20 wt % of the dye, resulting in aggregation and significant shortening of the fibrils. This study demonstrates the feasibility for these novel gels finding application as inexpensive and effective water purification platforms.     

A Double‐Clicking Bis‐Azide Fluorogenic Dye for Bioorthogonal Self‐Labeling Peptide Tags

Herein, we give the very first example for the development of a fluorogenic molecular probe that combines the two‐point binding specificity of biarsenical‐based dyes with the robustness of bioorthogonal click‐chemistry. This proof‐of‐principle study reports on the synthesis and fluorogenic characterization of a new, double‐quenched, bis‐azide fluorogenic probe suitable for bioorthogonal two‐point tagging of small peptide tags by double strain‐promoted azide–alkyne cycloaddition. The presented probe exhibits remarkable increase in fluorescence intensity when reacted with bis‐cyclooctynylated peptide sequences, which could also serve as possible self‐labeling small peptide tag motifs.     

Photo‐ and pH‐Responsive Polycarbonate Block Copolymer Prodrug Nanomicelles for Controlled Release of Doxorubicin

Photo/pH dual‐responsive amphiphilic diblock copolymers with alkyne functionalized pendant o‐nitrobenzyl ester group are synthesized using poly(ethylene glycol) as a macroinitiator. The pendant alkynes are functionalized as aldehyde groups by the azide‐alkyne Huisgen cycloaddition. The anticancer drug doxorubicin (DOX) molecules are then covalently conjugated through acid‐sensitive Schiff‐base linkage. The resultant prodrug copolymers self‐assemble into nanomicelles in aqueous solution. The prodrug nanomicelles have a well‐defined morphology with an average size of 20–40 nm. The dual‐stimuli are applied individually or simultaneously to study the release behavior of DOX. Under UV light irradiation, nanomicelles are disassembled due to the ONB ester photocleavage. The light‐controlled DOX release behavior is demonstrated using fluorescence spectroscopy. Due to the pH‐sensitive imine linkage the DOX molecules are released rapidly from the nanomicelles at the acidic pH of 5.0, whereas only minimal amount of DOX molecules is released at the pH of 7.4. The DOX release rate is tunable by applying the dual‐stimuli simultaneously. In vitro studies against colon cancer cells demonstrate that the nanomicelles show the efficient cellular uptake and the intracellular DOX release, indicating that the newly designed copolymers with dual‐stimuli‐response have significant potential applications as a smart nanomedicine against cancer.