Chemoselective Dual Labeling of Native and Recombinant Proteins.

The attachment of two different functionalities in a site-selective fashion represents a great challenge in protein chemistry. We report site specific dual functionalizations of peptides and proteins capitalizing on reactivity differences of cysteines in their free (thiol) and protected, oxidized (disulfide) forms. The dual functionalization of interleukin 2 and EYFP proceeded with no loss of bioactivity in a stepwise fashion applying maleimide and disulfide rebridging allyl-sulfone groups. In order to ensure broader applicability of the functionalization strategy, a novel, short peptide sequence that introduces a disulfide bridge was designed and site-selective dual labeling in the presence of biogenic groups was successfully demonstrated.

Fluorescent Nanodiamond-Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging.

There is a continuous demand for imaging probes offering excellent performance in various microscopy techniques for comprehensive investigations of cellular processes by more than one technique. Fluorescent nanodiamond-gold nanoparticles (FND-Au) constitute a new class of "all-in-one" hybrid particles providing unique features for multimodal cellular imaging including optical imaging, electron microscopy, and, and potentially even quantum sensing. Confocal and optical coherence microscopy of the FND-Au allow fast investigations inside living cells via emission, scattering, and photothermal imaging techniques because the FND emission is not quenched by AuNPs. In electron microscopy, transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) analysis of FND-Au reveals greatly enhanced contrast due to the gold particles as well as an extraordinary flickering behavior in three-dimensional cellular environments originating from the nanodiamonds. The unique multimodal imaging characteristics of FND-Au enable detailed studies inside cells ranging from statistical distributions at the entire cellular level (micrometers) down to the tracking of individual particles in subcellular organelles (nanometers). Herein, the processes of endosomal membrane uptake and release of FNDs were elucidated for the first time by the imaging of individual FND-Au hybrid nanoparticles with single-particle resolution. Their convenient preparation, the availability of various surface groups, their flexible detection modalities, and their single-particle contrast in combination with the capability for endosomal penetration and low cytotoxicity make FND-Au unique candidates for multimodal optical-electronic imaging applications with great potential for emerging techniques, such as quantum sensing inside living cells.

PEGylated Cationic Serum Albumin for Boosting Retroviral Gene Transfer.

Retroviral vectors are common tools for introducing genes into the genome of a cell. However, low transduction rates are a major limitation in retroviral gene transfer, especially in clinical applications. We generated cationic human serum albumin (cHSA) protected by a shell of poly(ethylene glycol) (PEG); this significantly enhanced retroviral gene transduction with potentially attractive pharmacokinetics and low immunogenicity. By screening a panel of chemically optimized HSA compounds, we identified a very potent enhancer that boosted the transduction rates of viral vectors. Confocal microscopy revealed a drastically increased number of viral particles attached to the surfaces of target cells. In accordance with the positive net charge of cationic and PEGylated HSA, this suggests a mechanism of action in which the repulsion of the negatively charged cellular and viral vector membranes is neutralized, thereby promoting attachment and ultimately transduction. Importantly, the transduction-enhancing PEGylated HSA derivative evaded recognition by HSA-specific antibodies and macrophage activation. Our findings hold great promise for facilitating improved retroviral gene transfer.

Encoding function into polypeptide-oligonucleotide precision biopolymers.

We report a novel synthesis strategy to prepare precision polymers providing exact chain lengths, molecular weights and monomer sequences that allow post modifications by convenient DNA hybridization. Two grafted single strand DNA (ssDNA) side chains serve as a versatile platform for sequence-specific attachment of chromophores, proteins, cell-targeting peptide, and a Y-shape DNA linker. This approach resembles a LEGO®-type incorporation of functionalities to create functional biopolymers of high structure definition under mild conditions.

Spatiotemporally Controlled Release of Rho-Inhibiting C3 Toxin from a Protein-DNA Hybrid Hydrogel for Targeted Inhibition of Osteoclast Formation and Activity.

In osteoporosis, bone structure can be improved by the introduction of therapeutic molecules inhibiting bone resorption by osteoclasts. Here, biocompatible hydrogels represent an excellent option for the delivery of pharmacologically active molecules to the bone tissue because of their biodegradability, injectability, and manifold functionalization capacity. The present study reports the preparation of a multifunctional hybrid hydrogel from chemically modified human serum albumin and rationally designed DNA building blocks. The hybrid hydrogel combines advantageous characteristics, including rapid gelation through DNA hybridization under physiological conditions and a self-healing and injectable nature with the possibility of specific loading and spatiotemporally controlled release of active proteins, making it an advanced biomaterial for the local treatment of bone diseases, for example, osteoporosis. The hydrogels are loaded with a recombinant Rho-inhibiting C3 toxin, C2IN-C3lim-G205C. This toxin selectively targets osteoclasts and inhibits Rho-signaling and, thereby, actin-dependent processes in these cells. Application of C2IN-C3lim-G205C toxin-loaded hydrogels effectively reduces osteoclast formation and resorption activity in vitro, as demonstrated by tartrate-resistant acid phosphatase staining and the pit resorption assay. Simultaneously, osteoblast activity, viability, and proliferation are unaffected, thus making C2IN-C3lim-G205C toxin-loaded hybrid hydrogels an attractive pharmacological system for spatial and selective modulation of osteoclast functions to reduce bone resorption.

Converting Human Proteins into Precision Polymer Therapeutics.

Cells as the smallest unit of life rely on precise macromolecules and programmable supramolecular interactions to accomplish the various vital functions. To translate such strategies to precisely control architectures and interactions into the synthetic world represents an exciting endeavor. Polymers with distinct structures, sequences and architectures are still challenging to achieve. However, in particular for biomedical applications, reproducible synthesis, narrow dispersities, tunable functionalities and additionally biocompatibility of the polymeric materials are crucial. Polymers derived from protein precursors provide many advantages of proteins such as precise monomer sequences and contour lengths, biodegradability and multiple functionalities, which can be synergistically combined with the valuable features of synthetic polymers e.g. stability, tunable solubility and molecular weights. The resulting polymeric biohybrid materials offer many applications ranging from drug delivery to biosensing and therapeutic hydrogels. This minireview summarizes the most recent advances in this field.

Programmable protein-DNA hybrid hydrogels for the immobilization and release of functional proteins.

A modular approach for the precise assembly of multi-component hydrogels consisting of protein and DNA building blocks is described for the first time. Multi-arm DNA is designed for crosslinking and stepwise, non-covalent assembly of active proteins inside the hydrogel.