Protein nanoreactors and native enzymes for controlled/living radical polymerization

The present PhD thesis entitled ‚Protein Nanoreactors and Native Enzymes for Controlled/Living Radical Polymerization‘ began with the hypothesis that protein-catalyst conjugates are able to beneficially influence controlled/living radical polymerization, i.e. atom transfer radical polymerization (ATRP). The general motivation for this work is driven by problems that occur when transition metal catalyst are used for ATRP. These most commonly used catalyst are only biocompatible to a limited extend and the resulting polymers show unwanted coloration due to the remaining catalyst. Moreover, by conjugating the catalyst into the cavities of a protein cage, we could gain insights into the catalytic mechanism of ATRP as well as into effects of the confined space. Polymer chemistry in particular enables us to perform successful research with proteins since synthetic strategies in a biocompatible environment, i.e. aqueous solution are well established. Inspired by problems addressed earlier in our research group, we developed a robust conjugation and purification method to attach transition metal catalysts to proteins or enclose them into protein cages using bis-aryl hydrazone linker chemistry. Successively, we determined good performing ATRP conditions that allowed for significant lower catalyst concentrations. Thus, activators regenerated by electron transfer (ARGET) ATRP was used to constantly regenerate the catalyst into its active form by the means of a reducing agent. We described one of these protein-catalyst conjugates in full detail. The ATRP catalyst was conjugated to the globular protein bovine serum albumin (BSA) and the complex was extensively characterized using biological and physical methods. The resulting conjugate was used to polymerize N-isopropyl acrylamide (NiPAAm) and poly(ethylene glycol) methyl ether acrylate (PEGA) in aqueous solution and was subsequently analyzed upon its structural integrity after polymerization. The ARGET ATRP of NiPAAm and PEGA yielded polymers with a moderate control over the molecular weight and the polydispersity of the polymers. However, our focus was to reduce the residual copper in polymers. Thus, BSA that served as a functional handle was used to remove the copper containing catalyst effectively from solution. We showed a reduction of residual copper to ppb levels, either by precipitation or by Dynabead removal. Further, our findings showed that some metalloproteins can mediate ATRP. Enzymes have been introduced into synthetic chemistry as green and very selective alternatives to conventional catalysts. In polymer chemistry enzymes have been used as catalysts for polycondensation, ring-opening polymerizations, free radical polymerizations of vinyl-type monomers and the polymerization of aromatic compounds by radical-induced oxidative coupling. However, controlled/living radical polymerizations catalyzed by enzymes have not been exploited. Our results and the ones from di Lena represent the first reports of biocatalytic, controlled/living radical polymerization. Bringing those enzymes into organic solution, e.g. by conjugation of end-group-reactive polymers such as PEG, poly(oxazolines) and amphiphilic blockcopolymers to the surface-exposed lysines or cysteins of the enzymes, could lead to interesting new routes towards environmentally friendly catalysis, functional materials or functional nanosystems. The group II chaperonin thermosome (THS) from the archaea Thermoplasma acidophilum is reported as protein nanoreactor for ATRP. For that purpose, a copper catalyst was entrapped into the THS. The confined space within the protein nanoreactor favorably influenced the polymerization of NiPAAm and PEGA under ARGET ATRP conditions in comparison to polymerizations carried out with the globular protein BSA. This concept was adapted and instead of the copper-complex, we covalently entrapped an enzyme as ATRP catalyst. We demonstrated that the space constriction in the THS has similar effects on the final polymer product, as shown for THS-LxCu. Protein nanoreactors with encapsulated enzymes, i.e. ATRPases, could help to understand the reaction mechanisms behind ATRPases. Further, we described a way to render protein cages, i.e. THS, gated nanoreactors. The gated behavior of THS driven by the hydrolysis of ATP and ATP analogues was shown by enzymatic assays. The incorporation of HRP into THS allowed to study the opening and closing of the cage by converting the non-fluorescent dihydro rhodamine 6G into its fluorescent form rhodamine 6G. In addition, nanomechanical sensing with cantilever arrays measured the surface stress induced by the opening and closing of the protein cage in response to ATP analogues. The field of gated nanoreactors is still in its infancy. Thus, gated nanoreactors could be used in the areas of medicine, sensing, synthesis of drugs and other chemical products, and as analytical tools to study reaction mechanisms in confined volumes

Tubing-Free Microfluidic Microtissue Culture System Featuring Gradual, in vivo-Like Substance Exposure Profiles

In vitro screening methods for compound efficacy and toxicity to date mostly include cell or tissue exposure to preset constant compound concentrations over a defined testing period. Such concentration profiles, however, do not represent realistic in vivo situations after substance uptake. Absorption, distribution, metabolism and excretion of administered substances in an organism or human body entail gradually changing pharmacokinetic concentration profiles. As concentration profile dynamics can influence drug effects on the target tissues, it is important to be able to reproduce realistic concentration profiles in in vitro systems. We present a novel design that can be integrated in tubing-free, microfluidic culture chips. These chips are actuated by tilting so that gravity-driven flow and perfusion of culture chambers can be established between reservoirs at both ends of a microfluidic channel. The design enables the realization of in vivo-like substance exposure scenarios. Compound gradients are generated through an asymmetric Y-junction of channels with different hydrodynamic resistances. Six microtissues (MTs) can be cultured and exposed in compartments along the channel. Changes of the chip design or operation parameters enable to alter the dosing profile over a large range. Modulation of, e.g., the tilting angle, changes the slope of the dosing curves, so that concentration curves can be attained that resemble the pharmacokinetic characteristics of common substances in a human body. Human colorectal cancer (HCT 116) MTs were exposed to both, gradually decreasing and constant concentrations of Staurosporine. Measurements of apoptosis induction and viability after 5 h and 24 h showed different short- and long-term responses of the MTs to dynamic and linear dosing regime

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.National Institutes of Health (U.S.) (5R01EB010246)National Institutes of Health (U.S.) (5UH2TR000496)Institute for Collaborative Biotechnologies (W911NF-09-0001)National Institutes of Health (U.S.) (1T32GM008334)United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039)Begg New Horizon Fund for Undergraduate Research at MITNational Institutes of Health (U.S.) (Biotechnology Training Program NIH/NIGMS 5T32GM008334))Biophysical Instrumentation FacilityVirginia and Daniel K. Ludwig Graduate FellowshipSwiss National Science Foundation (Postdoctoral Fellowship

Microphysiological Drug-Testing Platform for Identifying Responses to Prodrug Treatment in Primary Leukemia

Despite increasing survival rates of pediatric leukemia patients over the past decades, the outcome of some leukemia subtypes has remained dismal. Drug sensitivity and resistance testing on patient-derived leukemia samples provide important information to tailor treatments for high-risk patients. However, currently used well-based drug screening platforms have limitations in predicting the effects of prodrugs, a class of therapeutics that require metabolic activation to become effective. To address this issue, a microphysiological drug-testing platform is developed that enables co-culturing of patient-derived leukemia cells, human bone marrow mesenchymal stromal cells, and human liver microtissues within the same microfluidic platform. This platform also enables to control the physical interaction between the diverse cell types. Herein, it is made possible to recapitulate hepatic prodrug activation of ifosfamide in their platform, which is very difficult in traditional well-based assays. By testing the susceptibility of primary patient-derived leukemia samples to the prodrug ifosfamide, sample-specific sensitivities to ifosfamide in primary leukemia samples are identified. The microfluidic platform is found to enable the recapitulation of physiologically relevant conditions and the testing of prodrugs including short-lived and unstable metabolites. The platform holds great potential for clinical translation and precision chemotherapy selection

Biocatalytic atom transfer radical polymerization in a protein cage nanoreactor

Incorporation of the ATRP-catalyzing enzyme horseradish peroxidase (HRP) into the cavities of the group II chaperonin thermosome is demonstrated. The resulting nanoreactor was used to polymerize an acrylate under ARGET ATRP conditions. The confined space within the protein cage results in poly(ethylene glycol) methyl ether acrylate (PEGA) with lower molecular weights (poly(styrene)-apparent M-n = 4400 g mol(-1)) as well as narrower molecular weight distributions (D = 1.08) compared to polymerizations with the free ATRPase (M-n = 43 700 g mol(-1) and a D of 1.23)

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.United States. Department of Defense (OCRP Teal Innovator Award)National Institutes of Health (U.S.) (Grant NIBIB 1F32EB017614-02)Misrock FoundationNational Science Foundation (U.S.)Swiss National Science FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT (Support Grant P30-CA14051)National Cancer Institute (U.S.)National Science Foundation (U.S.) (Massachusetts Institute of Technology. Materials Research Science and Engineering Center. Shared Experimental Facilities Grant DMR-0819762)Breast Cancer Alliance (Exceptional Project Grant

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 1H 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

Solid or swollen polymer-protein hybrid materials

Hybrid materials comprising synthetic polymers and proteins or active enzymes combine the best of two worlds: The structural properties, the processibility and the moldability of man-made plastics or gels and the highly evolved functionality and responsiveness of nature?s polypeptides. In this chapter we review the body of literature on these smart hybrid materials and classify them according to their function. Biocatalytic plastics and polymers, stimuli-responsive hybrid hydrogels, self-assembled hydrogels with protein crosslinks, hybrid materials for selective binding of heavy metal ions, materials for tissue engineering, materials for controlled drug release, biodegradable materials, smart hydrogels with improved mechanical properties, and self-reporting materials are covered

Controlled radical polymerizations in protein nanoreactors : protein-conjugates as green alternatives to conventional catalysts

Conjugation chem. allows the attachment of a fully functional ATRP catalyst to the inside wall of the thermosome (THS) nanoreactor, thus confining ATRP into a nanoscale cavity defined by the nanoreactor. Expts. with a simple model system as well as ligand analogs underline these findings. The THS systems allow a significant redn. in the copper concn. in polymers synthesized by ATRP as compared to conventional ATRP. Moreover, the nanoreactor allows the synthesis of well-defined polymers in aq. soln., because ATRP inside a protein cage enhances the degree of control of ATRP. [on SciFinder(R)

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

Proteins are macromols. with a great diversity of functions. By combining these biomols. 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 Hb possess ATRPase activity, i.e. they catalyze atom transfer radical polymns. Moreover, a permeabilization method for polymersomes is presented, where the photo-reaction of an α-hydroxyalkylphenone with block copolymer vesicles yields enzyme-contg. nanoreactors. A further intriguing possibility to obtain functional nanoreactors is to enclose a polymn. catalyst into the thermosome, a protein cage from the family of chaperonins. Last but not least, fluorescent proteins are discussed as mechanoresponsive mol. sensors that report microdamages within fiber-reinforced composite materials. [on SciFinder(R)