Polymer–protein conjugates: an enzymatic activity perspective

Proteins have been modified with polymers in diverse manners over the past 30 years. However, while proteins have been used to prepare many functional constructs, they are sensitive biomolecules and their bioactivity can be either positively or negatively influenced by many different aspects of polymer modification. The primary focus of this review article is to highlight the opportunities offered by new trends in protein modification, and specifically how they influence the overall biological activity of the conjugate, including its dependence on temperature and pH. We survey the effect of polymer molecular weight, number of conjugated polymer chains, polymer coupling strategy (including random versus site-specific coupling, "grafting from", and multi-point covalent attachment), polymer architecture (including branched and comb-type), polymer interactions with the protein (including electrostatic and host-guest interactions), polymer interactions with enzyme substrate, and polymer biodegradability. We have selected six enzymes, which have been extensively modified with polymers in diverse fashions in the literature, as basis for this discussion. These proteins are L-asparaginase, alpha-chymotrypsin, trypsin, lysozyme, bovine serum albumin, and papain. This review includes polymers such as poly(ethylene glycol) (PEG), polysaccharides, polypeptides, and other synthetic (vinyl) polymers. From the discussed literature we attempt to extract tentative general trends observed between state-of-the-art methods of preparing protein-polymer conjugates and the activity of the conjugate.

Photopatterning of Synthetic Hydrogels

Katarzyna Mosiewicz

In recent years sophisticated biomaterials have made a significant impact on our understanding of cellular behavior, particularly in the fields of stem cell research and tissue engineering. Nevertheless, the promising potential of stem cells for regenerative medicine has remained largely unmet. One reason for such limitations could be that there is still incomplete understanding of stem cell behavior due to a lack of advanced in vitro tools suitable to achieve adequate control over cellular processes. By mimicking the dynamic and complex biophysical and biochemical interactions between cells and their natural extracellular matrixes (ECMs), it is possible that we may bridge this gap between current limited in vitro models and complex in vivo organisms. In this thesis, an innovative class of biomaterials is presented that enables the flexible in vitro recapitulation of the dynamic biochemical and biophysical signaling involved in controlling cell fate. Such systems of increasing fidelity to real physiological processes could bring new insights into fundamental cell biology as well as bring us to closer towards cell-based therapeutics applications. In order to achieve a dynamic artificial ECM (aECM), we designed chemical schemes which would allow us to control the biochemical and biophysical properties of the hydrogel in space, time and intensity. The chosen concept is built on a synthetic hydrogel providing static physicochemical support, while spatiotemporal control over a single or multiple signals is achieved by spatiotemporal photoactivation reactions within this hydrogel. Indeed, by modulating the duration, location and intensity of illumination, desired heterogeneities in biochemical and biophysical signal can be introduced in the otherwise homogeneous and static hydrogel network. To implement the designed hydrogel photo-patterning tool, new chemical components were synthesized. Synthetic hydrogels based on poly (ethylene glycol) (PEG) were utilized due to their inert properties, cell biocompatibility and lack of unspecific protein binding. Biochemical signal tethering was rendered spatiotemporally controllable by adding photosensitivity to the existing enzyme-based crosslinking scheme. This was achieved by functionalizing one of enzymatic substrate with a photo-labile “caging” moiety, thereby preventing crosslinking to occur until light illumination. This caged substrate was incorporated into the hydrogel network and served as a light-controllable and specific ligand-binding site. Importantly, it was possible to photo-pattern proteins, which are much complex than peptide-based biomolecules, and their bioactivity was fully preserved with this site-specific immobilization scheme. It was also possible to pattern biophysical cues by making the Michael-type (MT) addition reaction for crosslinking PEG-based hydrogel photosensitive. In this case, the thiol moieties of one of the reactive PEG macromers undergoing crosslinking, were equipped with a caging group which prevented its susceptibility to the counter-reactive functionality, the vinyl sulfone group. Thus, the crosslinking density of the hydrogel backbone could be controlled by light, which was directly translated into differential patterns of hydrogel stiffness. Using this approach, user-defined biochemical and biophysical patterns and even gradients could be obtained in the spatiotemporal manner within the same hydrogel. The crosslinking reaction is the critical parameter in the success of hydrogel photo-patterning, and must rely on a well-controlled and highly selective chemical scheme. Therefore, we also developed a novel enzyme-based cross-linking scheme for synthetic PEG hydrogels. The reaction originates from the naturally occurring post-translational modification of proteins by phosphopantetheine (Ppant) group performed by a family of enzymes known as Ppant transferases (PPTases). By synthesizing biochemical components of this reaction, we implemented a PPTase-catalyzed reaction to form and bio-functionalize PEG hydrogel. The utility of the developed hydrogel platforms was determined in the context of 2D and 3D cell culture. Photo-directed patterns of a cell-adhesive fibronectin fragment triggered adhesion and spreading of muscle progenitor cells, a phenomenon which occurred only within the areas of patterned signaling protein. A photo-patterned elasticity gradient was used to direct mesenchymal stem cell spreading and migration. Finally, the PPTase-based hydrogel was proven to be suitable for 3D culture, as shown by encapsulation of primary human fibroblasts. In this thesis, we demonstrated that a combination of chemical schemes could be assembled to achieve a functional materials toolbox which would begin to reconstruct the complexity of the dynamic in vivo ECM. Such a tool is expected to contribute to the field of cell biology by allowing us to study questions which previously could not be addressed in vitro, and could ultimately contribute to cell-based regenerative medicine.

EPFL2012

Functional polypeptide and hybrid materials: Precision synthesis via alpha-amino acid N-carboxyanhydride polymerization and emerging biomedical applications

Harm-Anton Klok

Polypeptides derived from naturally occurring a-amino acids have emerged as a unique and versatile family of bio-inspired biomaterials that can be tailor-made for varying biomedical applications such as controlled drug release, gene delivery, tissue engineering and regenerative medicine. In contrast to traditional biodegradable polymers such as aliphatic polyesters and polycarbonates, polypeptides are inherently functional, allow precise control over polarity and charges, show excellent stability against hydrolysis, and are prone to rapid biodegradation in vivo by specific enzymes. Ring-opening polymerization (ROP) of alpha-amino acid N-carboxyanhydrides (NCAs) is the most straightforward and practical approach for large-scale production of high molecular weight polypeptides. In the past decade, a remarkable progress has been made in controlled NCA polymerization, which offers an unprecedented access to precision polypeptide and hybrid materials by combining with living radical polymerization, click chemistry, and/or post-polymerization modification. Notably, several micellar anti-cancer drugs based on poly(ethylene glycol)-polypeptides have been already advanced to the clinical evaluation. In this review paper, we give an overview on de novo design, controlled synthesis and emerging biomedical applications of functional polypeptide and hybrid materials. (C) 2013 Elsevier Ltd. All rights reserved.

Elsevier2014

Mechanochemically Responsive Surfaces and Soft Tissue Adhesive Interfaces Generated via Controlled Radical Polymerization

Nariye Cavusoglu Ataman

Thin coatings applied to surfaces dramatically improve the properties of materials. Thus, they have found application in a broad variety of areas including biomedicine, nanotechnology, bioelectronics and optics, amongst others. Polymer brushes prepared via surface-initiated controlled radical polymerization (SI-CRP) techniques represent a versatile class of thin polymer coatings that provide manifold opportunities to engineer surface and interface properties. Densely grafted polymer brushes prepared via SI-CRP possess several attractive material properties such as low friction coefficients and the ability to prevent biofouling, which are related to the stretched conformation of the surface tethered polymer chains. Those distinctive characteristics of polymer brushes make them highly appealing to engineer the properties of functional interfaces. A first aim of this Thesis is to investigate the mechanochemistry of hydrophilic polymer brushes prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) from silica substrates. The second aim of the Thesis is to explore poly(2-hydroxyethyl methacrylate) (PHEMA) based polymer brushes as soft-tissue adhesives in wet tissue environment. This Thesis is divided into four chapters, which are briefly summarized below. Chapter 1 provides a summary of recent literature that has reported detachment or degrafting of densely grafted hydrophilic polymer brushes from substrates upon exposure to aqueous media. The collective results from these reports suggest that swelling-induced stretching of hydrophilic polymer brushes lead to mechanochemical activation of the bonds near the brush-substrate interface and subsequently facilitates hydrolytic chain cleavage. Chapter 2 aims to shed light on the influence of two fundamental structural polymer brush parameters on the degrafting of surface-tethered, densely grafted polymer chains, viz. (i) the chemical composition of the ATRP initiator that is used to grow the polymer brushes and (ii) the effect of surface curvature. To this end, a series of poly(poly(ethylene glycol) methacrylate) (PPEGMA) and poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMEMA) brushes were grafted from both flat silica substrates as well as silica nanoparticles. Subsequently, the degrafting of the surface-tethered polymer chains was investigated in cell culture medium and phosphate buffered saline (PBS) using various techniques. The aim of the final two Chapters of this Thesis is less on exploring the chain conformation of polymer chains grafted by SI-CRP, but more to use the ability of SI-CRP to prepare thin polymer films that present a high surface concentration of functional groups to mediate soft tissue adhesion. Chapter 3 describes the preparation of photo-active poly(2-hydroxyethyl methacrylate) (PHEMA) brushes as soft tissue bioadhesives. Tissue adhesion in this Chapter is induced as UV-irradiation of PHEMA brushes converts the initially bio-inert side chain hydroxyl groups of these brushes into (putatively) aldehyde groups, which can react with amine groups present in the tissue. The prepared PHEMA brush coated substrates are biocompatible, which avoids fibrous capsule formation and presents functionalities that can be transformed, on-demand, from a non-reactive, non-adhesive state to a reactive, adhesive state in wet tissue environment. While the photoactivation of the PHEMA side chain functional groups is very efficient, it is not very well defined from a chemistry point of view. Chapter 4 therefore, explores an alternative photoactivation chemistry, which is based on the use of the diazirine motif. These diazirine motifs were incorporated in poly(2-hydroxyethyl methacrylate-co-poly(ethylene glycol) methyl ether methacrylate) (P(HEMA-co-PEGMEMA)) copolymer brushes via post-polymerization modification. These diazirine modified brushes are attractive as they may not only be activated by UV-light but also by electrochemical means.