Kasper Renggli

Horseradish Peroxidase as a Catalyst for Atom Transfer Radical Polymerization

The hemoprotein horseradish peroxidase (HRP) catalyzes the polymerization of N-isopropylacrylamide with an alkyl bromide initiator under conditions of activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) in the absence of any peroxide. This is a novel activity of HRP, which we propose to name ATRPase activity. Bromine-terminated polymers with polydispersity indices (PDIs) as low as 1.44 are obtained. The polymerization follows first order kinetics, but the evolution of molecular weight and the PDI upon increasing conversion deviate from the results expected for an ATRP mechanism. Conversion, M(n) and PDI depend on the pH and on the concentration of the reducing agent, sodium ascorbate. HRP is stable during the polymerization and does not unfold or form conjugates.

Hemoglobin and red blood cells catalyze atom transfer radical polymerization.

Hemoglobin (Hb) is a promiscuous protein that not only transports oxygen, but also catalyzes several biotransformations. A novel in vitro catalytic activity of Hb is described. Bovine Hb and human erythrocytes were found to display ATRPase activity, i.e., they catalyzed the polymerization of vinyl monomers under conditions typical for atom transfer radical polymerization (ATRP). N-isopropylacrylamide (NIPAAm), poly(ethylene glycol) methyl ether acrylate (PEGA), and poly(ethylene glycol) methyl ether methacrylate (PEGMA) were polymerized using organobromine initiators and the reducing agent ascorbic acid in acidic aqueous solution. In order to avoid chain transfer from polymer radicals to Hbs cysteine residues, the accessible cysteines were blocked by a reaction with a maleimide. The formation of polymers with bromine chain ends, relatively low polydispersity indices (PDI), first order kinetics and an increase in the molecular weight of poly(PEGA) and poly(PEGMA) upon conversion indicate that control of the polymerization by Hb occurred via reversible atom transfer between the protein and the growing polymer chain. For poly(PEGA) and poly(PEGMA), the reactions proceeded with a good to moderate degree of control. Sodium dodecyl sulfate (SDS) gel electrophoresis, circular dichroism spectroscopy, and time-resolved ultraviolet-visible (UV-vis) spectroscopy revealed that the protein was stable during polymerization, and only underwent minor conformational changes. As Hb and erythrocytes are readily available, environmentally friendly, and nontoxic, their ATRPase activity is a useful tool for synthetic polymer chemistry. Moreover, this novel activity enhances the understanding of Hbs redox chemistry in the presence of organobromine compounds.

Real-time viscosity and mass density sensors requiring microliter sample volume based on nanomechanical resonators.

A microcantilever based method for fluid viscosity and mass density measurements with high temporal resolution and microliter sample consumption is presented. Nanomechanical cantilever vibration is driven by photothermal excitation and detected by an optical beam deflection system using two laser beams of different wavelengths. The theoretical framework relating cantilever response to the viscosity and mass density of the surrounding fluid was extended to consider higher flexural modes vibrating at high Reynolds numbers. The performance of the developed sensor and extended theory was validated over a viscosity range of 1-20 mPa·s and a corresponding mass density range of 998-1176 kg/m(3) using reference fluids. Separating sample plugs from the carrier fluid by a two-phase configuration in combination with a microfluidic flow cell, allowed samples of 5 μL to be sequentially measured under continuous flow, opening the method to fast and reliable screening applications. To demonstrate the study of dynamic processes, the viscosity and mass density changes occurring during the free radical polymerization of acrylamide were monitored and compared to published data. Shear-thinning was observed in the viscosity data at higher flexural modes, which vibrate at elevated frequencies. Rheokinetic models allowed the monomer-to-polymer conversion to be tracked in spite of the shear-thinning behavior, and could be applied to study the kinetics of unknown processes.

Filling polymersomes with polymers by peroxidase-catalyzed atom transfer radical polymerization.

Polymersomes that encapsulate a hydrophilic polymer are prepared by conducting biocatalytic atom transfer radical polymerization (ATRP) in these hollow nanostructures. To this end, ATRPase horseradish peroxidase (HRP) is encapsulated into vesicles self-assembled from poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) diblock copolymers. The vesicles are turned into nanoreactors by UV-induced permeabilization with a hydroxyalkyl phenone and used to polymerize poly(ethylene glycol) methyl ether acrylate (PEGA) by enzyme-catalyzed ATRP. As the membrane of the polymersomes is only permeable for the reagents of ATRP but not for macromolecules, the polymerization occurs inside of the vesicles and fills the polymersomes with poly(PEGA), as evidenced by (1) H NMR. Dynamic and static light scattering show that the vesicles transform from hollow spheres to filled spheres during polymerization. Transmission electron microscopy (TEM) and cryo-TEM imaging reveal that the polymersomes are stable under the reaction conditions. The polymer-filled nanoreactors mimic the membrane and cytosol of cells and can be useful tools to study enzymatic behavior in crowded macromolecular environments.

Covalent Modification of Synthetic Hydrogels with Bioactive Proteins via Sortase-Mediated Ligation

Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic postgrafting approach is presented. Sortase-mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG-EGF) to poly(ethylene glycol) (PEG) hydrogels containing the sortase LPRTG substrate. The reversibility of the sortase reaction was then exploited to cleave tethered EGF from the hydrogels for analysis. Analyses of the reaction supernatant and the postligation hydrogels showed that the amount of tethered EGF increases with increasing LPRTG in the hydrogel or GGG-EGF in the supernatant. Sortase-tethered EGF was biologically active, as demonstrated by stimulation of DNA synthesis in primary human hepatocytes and endometrial epithelial cells. The simplicity, specificity, and reversibility of sortase-mediated ligation and cleavage reactions make it an attractive approach for modification of hydrogels.

A Chaperonin as Protein Nanoreactor for Atom‐Transfer Radical Polymerization

The group II chaperonin thermosome (THS) from the archaea Thermoplasma acidophilum is reported as nanoreactor for atom-transfer radical polymerization (ATRP). A copper catalyst was entrapped into the THS to confine the polymerization into this protein cage. THS possesses pores that are wide enough to release polymers into solution. The nanoreactor favorably influenced the polymerization of N-isopropyl acrylamide and poly(ethylene glycol)methylether acrylate. Narrowly dispersed polymers with polydispersity indices (PDIs) down to 1.06 were obtained in the protein nanoreactor, while control reactions with a globular protein-catalyst conjugate only yielded polymers with PDIs above 1.84.

Tumor-Targeted Synergistic Blockade of MAPK and PI3K from a Layer-by-Layer Nanoparticle

Purpose: Cross-talk and feedback between the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR cell signaling pathways is critical for tumor initiation, maintenance, and adaptive resistance to targeted therapy in a variety of solid tumors. Combined blockade of these pathways—horizontal blockade—is a promising therapeutic strategy; however, compounded dose-limiting toxicity of free small molecule inhibitor combinations is a significant barrier to its clinical application. Experimental Design: AZD6244 (selumetinib), an allosteric inhibitor of Mek1/2, and PX-866, a covalent inhibitor of PI3K, were co-encapsulated in a tumor-targeting nanoscale drug formulation—layer-by-layer (LbL) nanoparticles. Structure, size, and surface charge of the nanoscale formulations were characterized, in addition to in vitro cell entry, synergistic cell killing, and combined signal blockade. In vivo tumor targeting and therapy was investigated in breast tumor xenograft–bearing NCR nude mice by live animal fluorescence/bioluminescence imaging, Western blotting, serum cytokine analysis, and immunohistochemistry. Results: Combined MAPK and PI3K axis blockade from the nanoscale formulations (160 ± 20 nm, −40 ± 1 mV) was synergistically toxic toward triple-negative breast (MDA-MB-231) and RAS-mutant lung tumor cells (KP7B) in vitro, effects that were further enhanced upon encapsulation. In vivo, systemically administered LbL nanoparticles preferentially targeted subcutaneous MDA-MB-231 tumor xenografts, simultaneously blocked tumor-specific phosphorylation of the terminal kinases Erk and Akt, and elicited significant disease stabilization in the absence of dose-limiting hepatotoxic effects observed from the free drug combination. Mice receiving untargeted, but dual drug–loaded nanoparticles exhibited progressive disease. Conclusions: Tumor-targeting nanoscale drug formulations could provide a more safe and effective means to synergistically block MAPK and PI3K in the clinic. Clin Cancer Res; 21(19); 4410–9. ©2015 AACR.

On-demand dissolution of modular, synthetic extracellular matrix reveals local epithelial-stromal communication networks

Methods to parse paracrine epithelial-stromal communication networks are a vital need in drug development, as disruption of these networks underlies diseases ranging from cancer to endometriosis. Here, we describe a modular, synthetic, and dissolvable extracellular matrix (MSD-ECM) hydrogel that fosters functional 3D epithelial-stromal co-culture, and that can be dissolved on-demand to recover cells and paracrine signaling proteins intact for subsequent analysis. Specifically, synthetic polymer hydrogels, modified with cell-interacting adhesion motifs and crosslinked with peptides that include a substrate for cell-mediated proteolytic remodeling, can be rapidly dissolved by an engineered version of the microbial transpeptidase Sortase A (SrtA) if the crosslinking peptide includes a SrtA substrate motif and a soluble second substrate. SrtA-mediated dissolution affected only 1 of 31 cytokines and growth factors assayed, whereas standard protease degradation methods destroyed about half of these same molecules. Using co-encapsulated endometrial epithelial and stromal cells as one model system, we show that the dynamic cytokine and growth factor response of co-cultures to an inflammatory cue is richer and more nuanced when measured from SrtA-dissolved gel microenvironments than from the culture supernate. This system employs accessible, reproducible reagents and facile protocols; hence, has potential as a tool in identifying and validating therapeutic targets in complex diseases.

Chaperonin–Dendrimer Conjugates for siRNA Delivery

The group II chaperonin thermosome (THS) is a hollow protein nanoparticle that can encapsulate macromolecular guests. Two large pores grant access to the interior of the protein cage. Poly(amidoamine) (PAMAM) is conjugated into THS to act as an anchor for small interfering RNA (siRNA), allowing to load the THS with therapeutic payload. THS–PAMAM protects siRNA from degradation by RNase A and traffics KIF11 and GAPDH siRNA into U87 cancer cells. By modification of the protein cage with the cell‐penetrating peptide TAT, RNA interference is also induced in PC‐3 cells. THS–PAMAM protein–polymer conjugates are therefore promising siRNA transfection reagents and greatly expand the scope of protein cages in drug delivery applications.

Combining polymers with the functionality of proteins : new concepts for atom transfer radical polymerization, nanoreactors and damage self-reporting materials

Proteins are macromolecules with a great diversity of functions. By combining these biomolecules with polymers, exciting opportunities for new concepts in polymer sciences arise. This highlight exemplifies the aforementioned with current research results of our group. We review our discovery that the proteins horseradish peroxidase and hemoglobin possess ATRPase activity, i.e. they catalyze atom transfer radical polymerizations. Moreover, a permeabilization method for polymersomes is presented, where the photo-reaction of an α-hydroxyalkylphenone with block copolymer vesicles yields enzyme-containing nanoreactors. A further intriguing possibility to obtain functional nanoreactors is to enclose a polymerization catalyst into the thermosome, a protein cage from the family of chaperonins. Last but not least, fluorescent proteins are discussed as mechanoresponsive molecular sensors that report microdamages within fiber-reinforced composite materials.