Self-assembled Biopolymer Research Articles

Inulin-Based Polymeric Micelles Functionalized with Ocular Permeation Enhancers: Improvement of Dexamethasone Permeation/Penetration through Bovine Corneas.

Ophthalmic drug delivery is still a challenge due to the protective barriers of the eye. A common strategy to promote drug absorption is the use of ocular permeation enhancers, while an innovative approach is the use of polymeric micelles. In the present work, the two mentioned approaches were coupled by conjugating ocular permeation enhancers (PEG2000, carnitine, creatine, taurine) to an inulin-based co-polymer (INU-EDA-RA) in order to obtain self-assembling biopolymers with permeation enhancer properties for the hydrophobic drug dexamethasone (DEX). Inulin derivatives were properly synthetized, were found to expose about 2% mol/mol of enhancer molecules in the side chain, and resulted able to self-assemble at various concentrations by varying the pH and the ionic strength of the medium. Moreover, the ability of polymeric micelles to load dexamethasone was demonstrated, and size, mucoadhesiveness, and cytocompatibility against HCE cells were evaluated. Furthermore, the efficacy of the permeation enhancer was evaluated by ex vivo permeation studies to determine the performance of the used enhancers, which resulted in PEG2000 > CAR > TAU > CRE, while entrapment ability studies resulted in CAR > TAU > PEG2000 > CRE, both for fluorescent-labelled and DEX-loaded micelles. Finally, an increase in terms of calculated Kp and Ac parameters was demonstrated, compared with the values calculated for DEX suspension.

Bilingual Peptide Nucleic Acids: Encoding the Languages of Nucleic Acids and Proteins in a Single Self-Assembling Biopolymer.

Nucleic acids and proteins are the fundamental biopolymers that support all life on Earth. Nucleic acids store large amounts of information in nucleobase sequences while peptides and proteins utilize diverse amino acid functional groups to adopt complex structures and perform wide-ranging activities. Although nature has evolved machinery to read the nucleic acid code and translate it into amino acid code, the extant biopolymers are restricted to encoding amino acid or nucleotide sequences separately, limiting their potential applications in medicine and biotechnology. Here we describe the design, synthesis, and stimuli-responsive assembly behavior of a bilingual biopolymer that integrates both amino acid and nucleobase sequences into a single peptide nucleic acid (PNA) scaffold to enable tunable storage and retrieval of tertiary structural behavior and programmable molecular recognition capabilities. Incorporation of a defined sequence of amino acid side-chains along the PNA backbone yields amphiphiles having a "protein code" that directs self-assembly into micellar architectures in aqueous conditions. However, these amphiphiles also carry a "nucleotide code" such that subsequent introduction of a complementary RNA strand induces a sequence-specific disruption of assemblies through hybridization. Together, these properties establish bilingual PNA as a powerful biopolymer that combines two information systems to harness structural responsiveness and sequence recognition. The PNA scaffold and our synthetic system are highly generalizable, enabling fabrication of a wide array of user-defined peptide and nucleotide sequence combinations for diverse future biomedical and nanotechnology applications.

Synthetic Biology for Multiscale Designed Biomimetic Assemblies: From Designed Self-Assembling Biopolymers to Bacterial Bioprinting.

Nature is based on complex self-assembling systems that span from the nanoscale to the macroscale. We have already begun to design biomimetic systems with properties that have not evolved in nature, based on designed molecular interactions and regulation of biological systems. Synthetic biology is based on the principle of modularity, repurposing diverse building modules to design new types of molecular and cellular assemblies. While we are currently able to use techniques from synthetic biology to design self-assembling molecules and re-engineer functional cells, we still need to use guided assembly to construct biological assemblies at the macroscale. We review the recent strategies for designing biological systems ranging from molecular assemblies based on self-assembly of (poly)peptides to the guided assembly of patterned bacteria, spanning 7 orders of magnitude.

A Genetically Encoded Protein Polymer for Uranyl Binding and Extraction Based on the SpyTag-SpyCatcher Chemistry.

A defining goal of synthetic biology is to develop biomaterials with superior performance and versatility. Here we introduce a purely genetically encoded and self-assembling biopolymer based on the SpyTag-SpyCatcher chemistry. We show the application of this polymer for highly efficient uranyl binding and extraction from aqueous solutions, by embedding two functional modules-the superuranyl binding protein and the monomeric streptavidin-to the polymer via genetic fusion. We further provide a modeling strategy for predicting the polymer's physical properties, and experimentally demonstrate the autosecretion of component monomers from bacterial cells. The potential of multifunctionalization, in conjunction with the genetic design and production pipeline, underscores the advantage of the SpyTag-SpyCatcher biopolymers for applications beyond trace metal enrichment and environmental remediation.

Modification and Assembly of a Versatile Lactonase for Bacterial Quorum Quenching.

This work sets out to provide a self-assembled biopolymer capsule activated with a multi-functional enzyme for localized delivery. This enzyme, SsoPox, which is a lactonase and phosphotriesterase, provides a means of interrupting bacterial communication pathways that have been shown to mediate pathogenicity. Here we demonstrate the capability to express, purify and attach SsoPox to the natural biopolymer chitosan, preserving its activity to “neutralize” long-chain autoinducer-1 (AI-1) communication molecules. Attachment is shown via non-specific binding and by engineering tyrosine and glutamine affinity ‘tags’ at the C-terminus for covalent linkage. Subsequent degradation of AI-1, in this case N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL), serves to “quench” bacterial quorum sensing (QS), silencing intraspecies communication. By attaching enzymes to pH-responsive chitosan that, in turn, can be assembled into various forms, we demonstrate device-based flexibility for enzyme delivery. Specifically, we have assembled quorum-quenching capsules consisting of an alginate inner core and an enzyme “decorated” chitosan shell that are shown to preclude bacterial QS crosstalk, minimizing QS mediated behaviors.

Interfacial rheological properties of self-assembling biopolymer microcapsules.

Tuning the mechanical properties of microcapsules through a cost-efficient route of fabrication is still a challenge. The traditional method of layer-by-layer assembly of microcapsules allows building a tailored composite multi-layer membrane but is technically complex as it requires numerous steps. The objective of this article is to characterize the interfacial rheological properties of self-assembling biopolymer microcapsules that were obtained in one single facile step. This thorough study provides new insights into the mechanics of these weakly cohesive membranes. Firstly, suspensions of water-in-oil microcapsules were formed in microfluidic junctions by self-assembly of two oppositely charged polyelectrolytes, namely chitosan (water soluble) and phosphatidic fatty acid (oil soluble). In this way, composite membranes of tunable thickness (between 40 and 900 nm measured by AFM) were formed at water/oil interfaces in a single step by changing the composition. Secondly, microcapsules were mechanically characterized by stretching them up to break-up in an extensional flow chamber which extends the relevance and convenience of the hydrodynamic method to weakly cohesive membranes. Finally, we show that the design of microcapsules can be 'engineered' in an extensive way since they present a wealth of interfacial rheological properties in terms of elasticity, plasticity and yield stress whose magnitudes can be controlled by the composition. These behaviors are explained by the variation of the membrane thickness with the physico-chemical parameters of the process.

Fusing Sensor Paradigms to Acquire Chemical Information: An Integrative Role for Smart Biopolymeric Hydrogels.

The Information Age transformed our lives but it has had surprisingly little impact on the way chemical information (e.g., from our biological world) is acquired, analyzed and communicated. Sensor systems are poised to change this situation by providing rapid access to chemical information. This access will be enabled by technological advances from various fields: biology enables the synthesis, design and discovery of molecular recognition elements as well as the generation of cell-based signal processors; physics and chemistry are providing nano-components that facilitate the transmission and transduction of signals rich with chemical information; microfabrication is yielding sensors capable of receiving these signals through various modalities; and signal processing analysis enhances the extraction of chemical information. The authors contend that integral to the development of functional sensor systems will be materials that (i) enable the integrative and hierarchical assembly of various sensing components (for chemical recognition and signal transduction) and (ii) facilitate meaningful communication across modalities. It is suggested that stimuli-responsive self-assembling biopolymers can perform such integrative functions, and redox provides modality-spanning communication capabilities. Recent progress toward the development of electrochemical sensors to manage schizophrenia is used to illustrate the opportunities and challenges for enlisting sensors for chemical information processing.

Structural advances on titin: towards an atomic understanding of multi-domain functions in myofilament mechanics and scaffolding.

Titin is a gigantic filamentous protein of the muscle sarcomere that plays roles in myofibril mechanics and homoeostasis. 3D-structures of multi-domain fragments of titin are now available that start revealing the molecular mechanisms governing its mechanical and scaffolding functions. This knowledge is now being translated into the fabrication of self-assembling biopolymers. Here we review the structural advances on titin, the novel concepts derived from these and the emerging translational avenues.

Improved viscoelasticity of xanthan gum through self-association with surfactant: β-cyclodextrin inclusion complexes for applications in enhanced oil recovery

This article presents the chemical formulation and rheological properties of a novel self-assembling biopolymer (SAP) system, which could be a useful alternative in polymer flooding for oil recovery, especially under harsh reservoir conditions of temperature, salinity, and hardness. The nonbonding associations of the side branches of xanthan gum with the surfactant: β-cyclodextrin inclusion complexes in aqueous solution generate an improved viscoelastic SAP network system. This system exhibits superior mechanical and thermal stability, and also tolerance to elevated brine salinity and hardness due to the “interlocking effect.” Sandpack flood tests conducted at reservoir conditions (Pelican Lake reservoir, Alberta, Canada) demonstrate that the SAP system can produce considerably higher resistance factors than the baseline xanthan gum during flow in porous media, which indicates its effectiveness in controlling the mobility of the displacing fluid. Moreover, this system shows potential as in situ permeability modifier. POLYM. ENG. SCI., 55:523–532, 2015. © 2014 Society of Plastics Engineers

Design and characterization of soluble biopolymer complexes produced by electrostatic self-assembly of a whey protein isolate and sodium alginate

The aim of this study was to design and characterize soluble biopolymer complexes formed by self-assembly of an anionic polysaccharide (sodium alginate, SA) and a whey protein isolate (WPI). First, conditions for producing stable protein aggregates were studied by analysing the effect of concentration (2, 4, 6% w/w) and heating temperature (55, 70, 85 °C) on WPI structural characteristics, using spectroscopic techniques UV–Vis and fluorescence. As heat treatment of WPI at the highest temperature induced the formation of soluble protein aggregates with a greater exposure of hydrophobic patches on their surface, this condition was selected to form protein particles. Secondly, from aqueous solutions of 6% w/w heat-treated WPI at 85 °C, WPI/SA systems were obtained at different ratios (2:1, 4:1 and 6:1) and transmittance measurements as a function of pH (6.0–3.0) were made. Dynamic behaviour of biopolymers in WPI/SA systems was discussed in terms of transition pHs, pHc and pHϕ, corresponding to the formation of WPI-SA soluble complexes and the beginning of associative phase separation (coacervation), respectively. Finally, with these pH values, phase diagrams were constructed for each system. From these diagrams, pH 4.0 was selected to perform deposition of SA onto WPI aggregates surface, as at this pH value formation of WPI-SA soluble complexes was observed for all WPI:SA ratios. Resultant self-assembled biopolymer complexes were then characterized by the same set of spectroscopic techniques. Higher WPI:SA ratios produced soluble self-assembled biopolymer particles where hydrophobic patches of protein aggregates would be more occluded ensuring the protection of potential lipophilic bioactive agents attached inside.

Spatially-Interactive Biomolecular Networks Organized by Nucleic Acid Nanostructures

Living systems have evolved a variety of nanostructures to control the molecular interactions that mediate many functions including the recognition of targets by receptors, the binding of enzymes to substrates, and the regulation of enzymatic activity. Mimicking these structures outside of the cell requires methods that offer nanoscale control over the organization of individual network components. Advances in DNA nanotechnology have enabled the design and fabrication of sophisticated one-, two- and three-dimensional (1D, 2D, and 3D) nanostructures that utilize spontaneous and sequence-specific DNA hybridization. Compared with other self-assembling biopolymers, DNA nanostructures offer predictable and programmable interactions and surface features to which other nanoparticles and biomolecules can be precisely positioned. The ability to control the spatial arrangement of the components while constructing highly organized networks will lead to various applications of these systems. For example, DNA nanoarrays with surface displays of molecular probes can sense noncovalent hybridization interactions with DNA, RNA, and proteins and covalent chemical reactions. DNA nanostructures can also align external molecules into well-defined arrays, which may improve the resolution of many structural determination methods, such as X-ray diffraction, cryo-EM, NMR, and super-resolution fluorescence. Moreover, by constraint of target entities to specific conformations, self-assembled DNA nanostructures can serve as molecular rulers to evaluate conformation-dependent activities. This Account describes the most recent advances in the DNA nanostructure directed assembly of biomolecular networks and explores the possibility of applying this technology to other fields of study. Recently, several reports have demonstrated the DNA nanostructure directed assembly of spatially interactive biomolecular networks. For example, researchers have constructed synthetic multienzyme cascades by organizing the position of the components using DNA nanoscaffolds in vitro or by utilizing RNA matrices in vivo. These structures display enhanced efficiency compared with the corresponding unstructured enzyme mixtures. Such systems are designed to mimic cellular function, where substrate diffusion between enzymes is facilitated and reactions are catalyzed with high efficiency and specificity. In addition, researchers have assembled multiple choromophores into arrays using a DNA nanoscaffold that optimizes the relative distance between the dyes and their spatial organization. The resulting artificial light-harvesting system exhibits efficient cascading energy transfers. Finally, DNA nanostructures have been used as assembly templates to construct nanodevices that execute rationally designed behaviors, including cargo loading, transportation, and route control.

Sequence-structure correlations in silk: Poly-Ala repeat of N. clavipes MaSp1 is naturally optimized at a critical length scale

Spider silk is a self-assembling biopolymer that outperforms many known materials in terms of its mechanical performance despite being constructed from simple and inferior building blocks. While experimental studies have shown that the molecular structure of silk has a direct influence on the stiffness, toughness, and failure strength of silk, few molecular-level analyses of the nanostructure of silk assemblies in particular under variations of genetic sequences have been reported. Here we report atomistic-level structures of the MaSp1 protein from the Nephila Clavipes spider dragline silk sequence, obtained using an in silico approach based on replica exchange molecular dynamics (REMD) and explicit water molecular dynamics. We apply this method to study the effects of a systematic variation of the poly-alanine repeat lengths, a parameter controlled by the genetic makeup of silk, on the resulting molecular structure of silk at the nanoscale. Confirming earlier experimental and computational work, a structural analysis reveals that poly-alanine regions in silk predominantly form distinct and orderly β-sheet crystal domains while disorderly regions are formed by glycine-rich repeats that consist of 310-helix type structures and β-turns. Our predictions are directly validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots combined with an analysis of the secondary structure content. The key result of our study is our finding of a strong dependence of the resulting silk nanostructure depending on the poly-alanine length. We observe that the wildtype poly-alanine repeat length of six residues defines a critical minimum length that consistently results in clearly defined β-sheet nanocrystals. For poly-alanine lengths below six, the β-sheet nanocrystals are not well-defined or not visible at all, while for poly-alanine lengths at and above six, the characteristic nanocomposite structure of silk emerges with no significant improvement of the quality of the β-sheet nanocrystal geometry. We present a simple biophysical model that explains these computational observations based on the mechanistic insight gained from the molecular simulations. Our findings set the stage for understanding how variations in the spidroin sequence can be used to engineer the structure and thereby functional properties of this biological superfiber, and present a design strategy for the genetic optimization of spidroins for enhanced mechanical properties. The approach used here may also find application in the design of other self-assembled molecular structures and fibers and in particular biologically inspired or completely synthetic systems.

Nanostructure and molecular mechanics of spider dragline silk protein assemblies.

Spider silk is a self-assembling biopolymer that outperforms most known materials in terms of its mechanical performance, despite its underlying weak chemical bonding based on H-bonds. While experimental studies have shown that the molecular structure of silk proteins has a direct influence on the stiffness, toughness and failure strength of silk, no molecular-level analysis of the nanostructure and associated mechanical properties of silk assemblies have been reported. Here, we report atomic-level structures of MaSp1 and MaSp2 proteins from the Nephila clavipes spider dragline silk sequence, obtained using replica exchange molecular dynamics, and subject these structures to mechanical loading for a detailed nanomechanical analysis. The structural analysis reveals that poly-alanine regions in silk predominantly form distinct and orderly beta-sheet crystal domains, while disorderly regions are formed by glycine-rich repeats that consist of 3₁-helix type structures and beta-turns. Our structural predictions are validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots, alpha-carbon atomic distances, as well as secondary structure content. Mechanical shearing simulations on selected structures illustrate that the nanoscale behaviour of silk protein assemblies is controlled by the distinctly different secondary structure content and hydrogen bonding in the crystalline and semi-amorphous regions. Both structural and mechanical characterization results show excellent agreement with available experimental evidence. Our findings set the stage for extensive atomistic investigations of silk, which may contribute towards an improved understanding of the source of the strength and toughness of this biological superfibre.

Atomistic model of the spider silk nanostructure

Spider silk is an ultrastrong and extensible self-assembling biopolymer that outperforms the mechanical characteristics of many synthetic materials including steel. Here we report atomic-level structures that represent aggregates of MaSp1 proteins from the N. Clavipes silk sequence based on a bottom-up computational approach using replica exchange molecular dynamics. We discover that poly-alanine regions predominantly form distinct and orderly beta-sheet crystal domains while disorderly structures are formed by poly-glycine repeats, resembling 31-helices. These could be the molecular source of the large semicrystalline fraction observed in silks, and also form the basis of the so-called “prestretched” molecular configuration. Our structures are validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots, alpha-carbon atomic distances, as well as secondary structure content.

Global Energy Matching Method for Atomistic-to-Continuum Modeling of Self-Assembling Biopolymer Aggregates

This paper studies mathematical models of biopolymer supramolecular aggregates that are formed by the self-assembly of single monomers. We develop a new multiscale numerical approach to model the structural properties of such aggregates. This theoretical approach establishes micro-macro relations between the geometrical and mechanical properties of the monomers and supramolecular aggregates. Most atomistic-to-continuum methods are constrained by a crystalline order or a periodic setting and therefore cannot be directly applied to modeling of soft matter. By contrast, the energy matching method developed in this paper does not require crystalline order and, therefore, can be applied to general microstructures with strongly variable spatial correlations. In this paper we use this method to compute the shape and the bending stiffness of their supramolecular aggregates from known chiral and amphiphilic properties of the short chain peptide monomers. Numerical implementation of our approach demonstrates consistency with results obtained by molecular dynamics simulations.

From natural to designer self-assembling biopolymers, the structural characterisation of fibrous proteins & peptides using fibre diffraction

Protein and peptide self-assembly is integral to a growing number of misfolding diseases that involve the ordered assembly and deposition of misfolded proteins as amyloid fibrils. However, it is becoming clear that these highly stable assemblies can be exploited as potential bionanomaterials. Recent work reveals that amyloid-like assembly is carefully controlled by a number of organisms to form functional amyloid. It is essential to gain an understanding of how sequence influences the assembly mechanism, as well as the final structure, to enable the control that is required to fully exploit these materials. In this tutorial review, we will discuss the different types of fibrous protein assembly from structural and cytoskeletal proteins through to misfolded proteins and finally designed self-assembling peptides. In the second part, we will discuss the advances in structure determination, with particular reference to the use of X-ray fibre diffraction.

Potentials of bionanotechnology in the study and manufacturing of self-assembled biopolymer complexes and gels

The self-assembly of biological macromolecules is a crucial prerequisite for life. It is also exploited by man in numerous processes and as a basis for a variety of hydrocolloid-based products. In this paper, recent progress in the development of some nanotechnological tools for the characterization of macromolecular interactions and their application is highlighted. Biopolymer assemblies stabilized by various types of non-covalent forces are used as examples to illustrate the potentials of such tools within the food hydrocolloids field. Control of nanoscale pattern and shape of objects, as well as their further hierarchical assembly has recently shown considerable progress. Further challenges include molecular control of these processes also for molecular species lacking detailed sequence specific interaction sites.

Microrheology of Biopolymer-Membrane Complexes

We create tailored microstructures, consisting of complexes of lipid membranes with self-assembled biopolymer shells, to study the fundamental properties and interactions of these basic components of living cells. We measure the mechanical response of these artificial structures at the micrometer scale, using optical tweezers and single-particle tracking. These systems exhibit rich dynamics that illustrate the viscoelastic character of the quasi-two-dimensional biopolymer network. We present a theoretical model relating the rheological properties of these membranes to the observed dynamics.