Intrinsically Disordered Osteopontin Fragment Orders During Interfacial Calcium Oxalate Mineralization.

Calcium oxalate (CaC2 O4 ) is the major component of kidney stone. The acidic osteopontin (OPN) protein in human urine can effectively inhibit the growth of CaC2 O4 crystals, thereby acting as a potent stone preventer. Previous studies in bulk solution all attest to the importance of binding and recognition of OPN at the CaC2 O4 mineral surface, yet molecular level insights into the active interface during CaC2 O4 mineralization are still lacking. Here, we probe the structure of the central OPN fragment and its interaction with Ca2+ and CaC2 O4 at the water-air interface using surface-specific non-linear vibrational spectroscopy. While OPN peptides remain largely disordered in solution, our results reveal that the bidentate binding of Ca2+ ions refold the interfacial peptides into well-ordered and assembled ß-turn motifs. One critical intermediate directs mineralization by releasing structural freedom of backbone and binding side chains. These insights into the mineral interface are crucial for understanding the pathological development of kidney stones and possibly relevant for calcium oxalate biomineralization in general.

Precision Anisotropic Brush Polymers by Sequence Controlled Chemistry.

The programming of nanomaterials at molecular length-scales to control architecture and function represents a pinnacle in soft materials synthesis. Although elusive in synthetic materials, Nature has evolutionarily refined macromolecular synthesis with perfect atomic resolution across three-dimensional space that serves specific functions. We show that biomolecules, specifically proteins, provide an intrinsic macromolecular backbone for the construction of anisotropic brush polymers with monodisperse lengths via grafting-from strategy. Using human serum albumin as a model, its sequence was exploited to chemically transform a single cysteine, such that the expression of said functionality is asymmetrically placed along the backbone of the eventual brush polymer. This positional monofunctionalization strategy was connected with biotin-streptavidin interactions to demonstrate the capabilities for site-specific self-assembly to create higher ordered architectures. Supported by systematic experimental and computational studies, we envisioned that this macromolecular platform provides unique avenues and perspectives in macromolecular design for both nanoscience and biomedicine.

Programming supramolecular biohybrids as precision therapeutics.

CONSPECTUS: Chemical programming of macromolecular structures to instill a set of defined chemical properties designed to behave in a sequential and precise manner is a characteristic vision for creating next generation nanomaterials. In this context, biopolymers such as proteins and nucleic acids provide an attractive platform for the integration of complex chemical design due to their sequence specificity and geometric definition, which allows accurate translation of chemical functionalities to biological activity. Coupled with the advent of amino acid specific modification techniques, "programmable" areas of a protein chain become exclusively available for any synthetic customization. We envision that chemically reprogrammed hybrid proteins will bridge the vital link to overcome the limitations of synthetic and biological materials, providing a unique strategy for tailoring precision therapeutics. In this Account, we present our work toward the chemical design of protein- derived hybrid polymers and their supramolecular responsiveness, while summarizing their impact and the advancement in biomedicine. Proteins, in their native form, represent the central framework of all biological processes and are an unrivaled class of macromolecular drugs with immense specificity. Nonetheless, the route of administration of protein therapeutics is often vastly different from Nature's biosynthesis. Therefore, it is imperative to chemically reprogram these biopolymers to direct their entry and activity toward the designated target. As a consequence of the innate structural regularity of proteins, we show that supramolecular interactions facilitated by stimulus responsive chemistry can be intricately designed as a powerful tool to customize their functions, stability, activity profiles, and transportation capabilities. From another perspective, a protein in its denatured, unfolded form serves as a monodispersed, biodegradable polymer scaffold decorated with functional side chains available for grafting with molecules of interest. Additionally, we are equipped with analytical tools to map the fingerprint of the protein chain, directly elucidating the structure at the molecular level. Contrary to conventional polymers, these biopolymers facilitate a more systematic avenue to investigate engineered macromolecules, with greater detail and accuracy. In this regard, we focus on denaturing serum albumin, an abundant blood protein, and exploit its peptidic array of functionalities to program supramolecular architectures for bioimaging, drug and gene delivery. Ultimately, we seek to assimilate the evolutionary advantage of these protein based biopolymers with the limitless versatility of synthetic chemistry to merge the best of both worlds.

Polydopamine at biological interfaces.

In the last years coating of surfaces in the presence of dopamine or other catecholamines in oxidative conditions to yield "polydopamine" films has become a popular, easy and versatile coating methodology. Polydopamine(s) offer(s) also a rich chemistry allowing to post-functionalize the obtained coatings with metal nanoparticles with polymers and proteins. However, the interactions either of covalent or non-covalent nature between polydopamine and biomolecules has only been explored more recently. They allow polydopamine to become a material, in the form of nanoparticles, membranes and other assemblies, in its own right not just as a coating. It is the aim of this review to describe the most recent advances in the design of composites between polydopamine and related eumelanin like materials with biomolecules like proteins, nucleotides, oligosaccharides and lipid assemblies. Furthermore, the interactions between polydopamine and living cells will be also reported.

Facile synthesis of ultrasmall polydopamine-polyethylene glycol nanoparticles for cellular delivery.

Very small polydopamine (PDA) polyethylene glycol (PEG) crosslinked copolymer (PDA-PEG) nanoparticles have been prepared following a convenient one-step procedure in aqueous solution. Particle sizes and colloidal stabilities have been optimized by varying PEG in view of chain length and end group functionalities. In particular, amine-terminated PEG3000 [PEG3000(NH2)2] reacted with polydopamine intermediates so that very small, crosslinked PDA-PEG nanoparticles with sizes of less than 50 nm were formed. These nanoparticles remained stable in buffer solution and no sedimentation occurred. Chemical functionalization was straight-forward as demonstrated by the attachment of fluorescent dyes. The PDA-PEG nanoparticles revealed efficient cellular uptake via endocytosis and high cytocompatibility, thus rendering them attractive candidates for cell imaging or for drug delivery applications.

Boosting Antitumor Drug Efficacy with Chemically Engineered Multidomain Proteins.

A facile chemical approach integrating supramolecular chemistry, site-selective protein chemistry, and molecular biology is described to engineer synthetic multidomain protein therapeutics that sensitize cancer cells selectively to significantly enhance antitumor efficacy of existing chemotherapeutics. The desired bioactive entities are assembled via supramolecular interactions at the nanoscale into structurally ordered multiprotein complexes comprising a) multiple copies of the chemically modified cyclic peptide hormone somatostatin for selective targeting and internalization into human A549 lung cancer cells expressing SST-2 receptors and b) a new cysteine mutant of the C3bot1 (C3) enzyme from Clostridium botulinum, a Rho protein inhibitor that affects and influences intracellular Rho-mediated processes like endothelial cell migration and blood vessel formation. The multidomain protein complex, SST3-Avi-C3, retargets C3 enzyme into non-small cell lung A549 cancer cells and exhibits exceptional tumor inhibition at a concentration ≈100-fold lower than the clinically approved antibody bevacizumab (Avastin) in vivo. Notably, SST3-Avi-C3 increases tumor sensitivity to a conventional chemotherapeutic (doxorubicin) in vivo. These findings show that the integrated approach holds vast promise to expand the current repertoire of multidomain protein complexes and can pave the way to important new developments in the area of targeted and combination cancer therapy.

A Supramolecular Approach toward Bioinspired PAMAM-Dendronized Fusion Toxins.

Nature has provided a highly optimized toolbox in bacterial endotoxins with precise functions dictated by their clear structural division. Inspired by this streamlined design, a supramolecular approach capitalizing on the strong biomolecular (streptavidin (SA))-biotin interactions is reported herein to prepare two multipartite fusion constructs, which involves the generation 2.0 (D2) or generation 3.0 (D3) polyamidoamine-dendronized transporter proteins (dendronized streptavidin (D3SA) and dendronized human serum albumin (D2HSA)) non-covalently fused to the C3bot1 enzyme from Clostridium botulinum, a potent and specific Rho-inhibitor. The fusion constructs, D3SA-C3 and D2HSA-C3, represent the first examples of dendronized protein transporters that are fused to the C3 enzyme, and it is successfully demonstrated that the C3 Rho-inhibitor is delivered into the cytosol of mammalian cells as determined from the characteristic C3-mediated changes in cell morphology and confocal microscopy. The design circumvents the low uptake of the C3 enzyme by eukaryotic cells and holds great promise for reprogramming the properties of toxin enzymes using a supramolecular approach to broaden their therapeutic applications.

Efficient delivery of p53 and cytochrome c by supramolecular assembly of a dendritic multi-domain delivery system.

Versatile nanocarrier systems facilitating uptake of exogenous proteins are highly alluring in evaluating these proteins for therapeutic applications. The self-assembly of an efficient nano-sized protein transporter consisting of three different entities is presented: A streptavidin protein core functioning as an adapter, second generation polyamidoamine dendrons for facilitating cell uptake as well as two different therapeutic proteins (tumor suppressor p53 or pro-apoptotic cytochrome c as cargo). Well-defined dendrons containing a biotin core are prepared and display no cytotoxic behavior upon conjugation to streptavidin. The integration of biotinylated human recombinant p53 (B-p53) into the three component system allows excellent internalization into HeLa, A549 and SaOS osteosarcoma cells monitored via confocal microscopy, immunoblot analysis and co-localization studies. In addition, the conjugation of B-p53 to dendronized streptavidin preserves its specific DNA-binding in vitro, and its delivery into SaOS cells impairs cell viability with concomitant activation of caspases 3 and 7. The versatility of this system is further exhibited by the significant enhancement of the pro-apoptotic effects of internalized cytochrome c which is analyzed by flow cytometry and cell viability assays. These results demonstrate that the "bio-click" self-assembly of biotinylated dendrons and proteins on a streptavidin adapter yields a stable supramolecular complex. This efficient bionanotransporter provides an attractive platform for mediating the delivery of functional proteins of interest into living mammalian cells in a facile and rapid way.