Self‐Assembling Proteins as High‐Performance Substrates for Embryonic Stem Cell Self‐Renewal

The development of extracellular matrix mimetics that imitate niche stem cell microenvironments and support cell growth for technological applications is intensely pursued. Specifically, mimetics are sought that can enact control over the self‐renewal and directed differentiation of human pluripotent stem cells (hPSCs) for clinical use. Despite considerable progress in the field, a major impediment to the clinical translation of hPSCs is the difficulty and high cost of large‐scale cell production under xeno‐free culture conditions using current matrices. Here, a bioactive, recombinant, protein‐based polymer, termed ZT^Fn, is presented that closely mimics human plasma fibronectin and serves as an economical, xeno‐free, biodegradable, and functionally adaptable cell substrate. The ZT^Fn substrate supports with high performance the propagation and long‐term self‐renewal of human embryonic stem cells while preserving their pluripotency. The ZT^Fn polymer can, therefore, be proposed as an efficient and affordable replacement for fibronectin in clinical grade cell culturing. Further, it can be postulated that the ZT polymer has significant engineering potential for further orthogonal functionalization in complex cell applications.     

Nanoparticle‐Induced Folding and Fibril Formation of Coiled‐Coil‐Based Model Peptides

Nanomedicine is a rapidly growing field that has the potential to deliver treatments for many illnesses. However, relatively little is known about the biological risks of nanoparticles. Some studies have shown that nanoparticles can have an impact on the aggregation properties of proteins, including fibril formation. Moreover, these studies also show that the capacity of nanoscale objects to induce or prevent misfolding of the proteins strongly depends on the primary structure of the protein. Herein, light is shed on the role of the peptide primary structure in directing nanoparticle‐induced misfolding by means of two model peptides. The design of these peptides is based on the α‐helical coiled‐coil folding motif, but also includes features that enable them to respond to pH changes, thus allowing pH‐dependent β‐sheet formation. Previous studies showed that the two peptides differ in the pH range required for β‐sheet folding. Time‐dependent circular dichroism spectroscopy and transmission electron microscopy are used to characterize peptide folding and aggregate morphology in the presence of negatively charged gold nanoparticles (AuNPs). Both peptides are found to undergo nanoparticle‐induced fibril formation. The determination of binding parameters by isothermal titration calorimetry further reveals that the different propensities of both peptides to form amyloid‐like structures in the presence of AuNPs is primarily due to the binding stoichiometry to the AuNPs. Modification of one of the peptide sequences shows that AuNP‐induced β‐sheet formation is related to the structural propensity of the primary structure and is not a generic feature of peptide sequences with a sufficiently high binding stoichiometry to the nanoparticles.     

Protein‐Corona‐by‐Design in 2D: A Reliable Platform to Decode Bio–Nano Interactions for the Next‐Generation Quality‐by‐Design Nanomedicines

Hard corona (HC) protein, i.e., the environmental proteins of the biological medium that are bound to a nanosurface, is known to affect the biological fate of a nanomedicine. Due to the size, curvature, and specific surface area (SSA) 3‐factor interactions inherited in the traditional 3D nanoparticle, HC‐dependent bio–nano interactions are often poorly probed and interpreted. Here, the first HC‐by‐design case study in 2D is demonstrated that sequentially and linearly changes the HC quantity using functionalized graphene oxide (GO) nanosheets. The HC quantity and HC quality are analyzed using NanoDrop and label‐free liquid chromatography–mass spectrometry (LC‐MS) followed by principal component analysis (PCA). Cellular responses (uptake and cytotoxicity in J774 cell model) are compared using imaging cytometry and the modified lactate dehydrogenase assays, respectively. Cellular uptake linearly and solely correlates with HC quantity (R² = 0.99634). The nanotoxicity, analyzed by retrospective design of experiment (DoE), is found to be dependent on the nanomaterial uptake (primary), HC composition (secondary), and nanomaterial exposure dose (tertiary). This unique 2D design eliminates the size–curvature–SSA multifactor interactions and can serve as a reliable screening platform to uncover HC‐dependent bio–nano interactions to enable the next‐generation quality‐by‐design (QbD) nanomedicines for better clinical translation.     

Designing Non‐Native Iron‐Binding Site on a Protein Cage for Biological Synthesis of Nanoparticles

In biomineralization processes, a supramolecular organic structure is often used as a template for inorganic nanomaterial synthesis. The E2 protein cage derived from Geobacillus stearothermophilus pyruvate dehydrogenase and formed by the self‐assembly of 60 subunits, has been functionalized with non‐native iron‐mineralization capability by incorporating two types of iron‐binding peptides. The non‐native peptides introduced at the interior surface do not affect the self‐assembly of E2 protein subunits. In contrast to the wild‐type, the engineered E2 protein cages can serve as size‐ and shape‐constrained reactors for the synthesis of iron nanoparticles. Electrostatic interactions between anionic amino acids and cationic iron molecules drive the formation of iron oxide nanoparticles within the engineered E2 protein cages. The work expands the investigations on nanomaterial biosynthesis using engineered host‐guest encapsulation properties of protein cages.     

Metal Ion‐Induced Self‐Assembly of a Multi‐Responsive Block Copolypeptide into Well‐Defined Nanocapsules

Protein cages are an interesting class of biomaterials with potential applications in bionanotechnology. Therefore, substantial effort is spent on the development of capsule‐forming designer polypeptides with a tailor‐made assembly profile. The expanded assembly profile of a triblock copolypeptide consisting of a metal ion chelating hexahistidine‐tag, a stimulus‐responsive elastin‐like polypeptide block, and a pH‐responsive morphology‐controlling viral capsid protein is presented. The self‐assembly of this multi‐responsive protein‐based block copolymer is triggered by the addition of divalent metal ions. This assembly process yields monodisperse nanocapsules with a 20 nm diameter composed of 60 polypeptides. The well‐defined nanoparticles are the result of the emergent properties of all the blocks of the polypeptide. These results demonstrate the feasibility of hexahistidine‐tags to function as supramolecular cross‐linkers. Furthermore, their potential for the metal ion‐mediated encapsulation of hexahistidine‐tagged proteins is shown.     

One‐Pot Synthesis of Multiple Protein‐Encapsulated DNA Flowers and Their Application in Intracellular Protein Delivery

Inspired by biological systems, many biomimetic methods suggest fabrication of functional materials with unique physicochemical properties. Such methods frequently generate organic–inorganic composites that feature highly ordered hierarchical structures with intriguing properties, distinct from their individual components. A striking example is that of DNA–inorganic hybrid micro/nanostructures, fabricated by the rolling circle technique. Here, a novel concept for the encapsulation of bioactive proteins in DNA flowers (DNF) while maintaining the activity of protein payloads is reported. A wide range of proteins, including enzymes, can be simultaneously associated with the growing DNA strands and Mg₂PPi crystals during the rolling circle process, ultimately leading to the direct immobilization of proteins into DNF. The unique porous structure of this construct, along with the abundance of Mg ions and DNA molecules present, provides many interaction sites for proteins, enabling high loading efficiency and enhanced stability. Further, as a proof of concept, it is demonstrated that the DNF can deliver payloads of cytotoxic protein (i.e., RNase A) to the cells without a loss in its biological function and structural integrity, resulting in highly increased cell death compared to the free protein.     

Effect of high‐protein supplemental therapy on subjective global assessment of CKD‐5D patients

Adequate nutrition in patients on hemodialysis is an important step for improving the quality of life. This prospective study was undertaken to monitor the nutritional status of patients who were given high‐protein supplements on malnutrition inflammation score (MIS) and to correlate with biochemical parameters in maintenance hemodialysis (MHD) patients. This prospective study was conducted on 55 chronic kidney disease patients on MHD (37 women, 18 men), aged between 21 and 67 years. Of the 55 patients, 26 patients received high‐protein commercial nutritional supplements, whereas 29 patients received high‐protein kitchen feeding. Every patient had their MIS, 24‐hour dietary recall, hand grip, mid arm circumference, triceps skin‐fold thickness at 0, 3, and 6 months. Each of the above parameters was compared between the high‐protein commercial nutritional supplement cohort and high‐protein kitchen feeding cohort, and the data were analyzed. Of the 55 patients, 82.61% of patients on high‐protein kitchen feeding group and 66.67% in high‐protein commercial nutritional supplement group were nonvegetarian (P = 0.021). According to the MIS, improvement was observed in malnutrition status from 3‐ to 6‐month period in 38.1% of patients in high‐protein commercial supplement group, whereas only in 8.7% in high‐protein kitchen feeding group (P = 0.04). Assessment showed improvement in malnutrition status with high‐protein commercial nutritional supplement, which was marked in patients with age group >65 years (P = 0.03) and in those in whom serum albumin is <35 g/L (P = 0.02). Both high‐protein kitchen feeding and high‐protein commercial nutritional supplement cohorts were observed to have improvement in overall nutritional status. Older patients >65 years with lower serum albumin levels (<3.5 g/dL) were observed to have significant improvement in nutritional status with high‐protein commercial nutritional supplements.     

Configurable Low‐Cost Plotter Device for Fabrication of Multi‐Color Sub‐Cellular Scale Microarrays

The construction and operation of a low‐cost plotter for fabrication of microarrays for multiplexed single‐cell analyses is reported. The printing head consists of polymeric pyramidal pens mounted on a rotation stage installed on an aluminium frame. This construction enables printing of microarrays onto glass substrates mounted on a tilt stage, controlled by a Lab‐View operated user interface. The plotter can be assembled by typical academic workshops from components of less than 15 000 Euro. The functionality of the instrument is demonstrated by printing DNA microarrays on the area of 0.5 cm² using up to three different oligonucleotides. Typical feature sizes are 5 μm diameter with a pitch of 15 μm, leading to densities of up to 10⁴–10⁵ spots/mm². The fabricated DNA microarrays are used to produce sub‐cellular scale arrays of bioactive epidermal growth factor peptides by means of DNA‐directed immobilization. The suitability of these biochips for cell biological studies is demonstrated by specific recruitment, concentration, and activation of EGF receptors within the plasma membrane of adherent living cells. This work illustrates that the presented plotter gives access to bio‐functionalized arrays usable for fundamental research in cell biology, such as the manipulation of signal pathways in living cells at subcellular resolution.     

Multifunctional Transmembrane Protein Ligands for Cell‐Specific Targeting of Plasma Membrane‐Derived Vesicles

Liposomes and nanoparticles that bind selectively to cell‐surface receptors can target specific populations of cells. However, chemical conjugation of ligands to these particles is difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. Toward harnessing cellular capabilities, here it is demonstrated that plasma membrane vesicles (PMVs) derived from donor cells can display engineered transmembrane protein ligands that precisely target cells on the basis of receptor expression. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs that display targeting proteins with affinity for the epidermal growth factor receptor (EGFR) bind at increasing concentrations to breast cancer cells that express increasing levels of EGFR. Further, as an example of the generality of this approach, PMVs expressing a single‐domain antibody against green fluorescence protein (eGFP) bind to cells expressing eGFP‐tagged receptors with a selectivity of ≈50:1. The results demonstrate the versatility of PMVs as cell targeting systems, suggesting diverse applications from drug delivery to tissue engineering.     

The Orientations of Large Aspect‐Ratio Coiled‐Coil Proteins Attached to Gold Nanostructures

Methods for patterning biomolecules on a substrate at the single molecule level have been studied as a route to sensors with single‐molecular sensitivity or as a way to probe biological phenomena at the single‐molecule level. However, the arrangement and orientation of single biomolecules on substrates has been less investigated. Here, the arrangement and orientation of two rod‐like coiled‐coil proteins, cortexillin and tropomyosin, around patterned gold nanostructures is examined. The high aspect ratio of the coiled coils makes it possible to study their orientations and to pursue a strategy of protein orientation via two‐point attachment. The proteins are anchored to the surfaces using thiol groups, and the number of cysteine residues in tropomyosin is varied to test how this variation affects the structure and arrangement of the surface‐attached proteins. Molecular dynamics studies are used to interpret the observed positional distributions. Based on initial studies of protein attachment to gold post structures, two 31‐nm‐long tropomyosin molecules are aligned between the two sidewalls of a trench with a width of 68 nm. Because the approach presented in this study uses one of twenty natural amino acids, this method provides a convenient way to pattern biomolecules on substrates using standard chemistry.     

Thermal transitions and heat‐sealing of glycerol‐plasticized whey protein films

Transparent, flexible whey protein‐based edible films plasticized with glycerol were produced by wet (solution‐casting) and dry (twin‐screw extrusion followed by compression‐molding) processes. The relationship between the thermal transitions and the heat‐sealability of the whey protein‐based edible films was investigated. Differential Scanning Calorimetry showed the existence of endothermic peaks with an onset transition temperature of 156.3 ± 1.4°C for both the solution‐cast and extruded films. Films were heat‐sealed using an impulse heat‐sealer at an effective jaw pressure of 293.31 kPa, a voltage of 15 V and a cooling time of 4 s. Various impulse times, ranging from 1.5 to 2.5 s, were used to heat‐seal the films. A thermocouple was used to measure the heat‐sealing temperatures at each impulse time. Heat‐sealing temperatures ranged between 126.1 ± 9.0 and 204.0 ± 5.4°C for the shortest and longest impulse times, respectively. Seal strengths were determined using an Instron Universal Testing Machine. Film thickness appeared to have an effect on seal strength; higher strengths were achieved for thinner films, which, in this case, were the solution‐cast films. Thicker (extruded) films required a longer minimum impulse time to achieve a heat‐seal. The highest seal strength (433.07 ± 39.37 N/m) was obtained with solution cast films (thickness of 0.13 ± 0.01 mm) sealed with an impulse time of 2 s (164.6 ± 5.1°C). Impulse times above 2.5 s (204.0 ± 5.4°C) resulted in degradation of both solution‐cast and extruded films. Copyright © 2009 John Wiley & Sons, Ltd.     

Logic‐Based Delivery of Site‐Specifically Modified Proteins from Environmentally Responsive Hydrogel Biomaterials

The controlled presentation of proteins from and within materials remains of significant interest for many bioengineering applications. Though “smart” platforms offer control over protein release in response to a single external cue, no strategy has been developed to trigger delivery in response to user‐specified combinations of environmental inputs, nor to independently control the release of multiple species from a homogenous material. Here, a modular semisynthetic scheme is introduced to govern the release of site‐specifically modified proteins from hydrogels following Boolean logic. A sortase‐mediated transpeptidation reaction is used to generate recombinant proteins C‐terminally tethered to gels through environmentally sensitive degradable linkers. By varying the connectivity of multiple stimuli‐labile moieties within these customizable linkers, YES/OR/AND control of protein release is exhaustively demonstrated in response to one and two‐input combinations involving enzyme, reductant, and light. Tethering of multiple proteins each through a different stimuli‐sensitive linker permits their independent and sequential release from a common material. It is expected that these methodologies will enable new opportunities in tissue engineering and therapeutic delivery.