Fabrication of novel biomaterials through molecular self-assembly

Two complementary strategies can be used in the fabrication of molecular biomaterials. In the 'top-down' approach, biomaterials are generated by stripping down a complex entity into its component parts (for example, paring a virus particle down to its capsid to form a viral cage). This contrasts with the 'bottom-up' approach, in which materials are assembled molecule by molecule (and in some cases even atom by atom) to produce novel supramolecular architectures. The latter approach is likely to become an integral part of nanomaterials manufacture and requires a deep understanding of individual molecular building blocks and their structures, assembly properties and dynamic behaviors. Two key elements in molecular fabrication are chemical complementarity and structural compatibility, both of which confer the weak and noncovalent interactions that bind building blocks together during self-assembly. Using natural processes as a guide, substantial advances have been achieved at the interface of nanomaterials and biology, including the fabrication of nanofiber materials for three-dimensional cell culture and tissue engineering, the assembly of peptide or protein nanotubes and helical ribbons, the creation of living microlenses, the synthesis of metal nanowires on DNA templates, the fabrication of peptide, protein and lipid scaffolds, the assembly of electronic materials by bacterial phage selection, and the use of radiofrequency to regulate molecular behaviors.     

Emerging biological materials through molecular self-assembly

Understanding of new materials at the molecular level has become increasingly critical for a new generation of nanomaterials for nanotechnology, namely, the design, synthesis and fabrication of nanodevices at the molecular scale. New technology through molecular self-assembly as a fabrication tool will become tremendously important in the coming decades. Basic engineering principles for microfabrication can be learned by understanding the molecular self-assembly phenomena. Self-assembly phenomenon is ubiquitous in nature. The key elements in molecular self-assembly are chemical complementarity and structural compatibility through noncovalent interactions. We have defined the path to understand these principles. Numerous self-assembling systems have been developed ranging from models to the study of protein folding and protein conformational diseases, to molecular electronics, surface engineering, and nanotechnology. Several distinctive types of self-assembling peptide systems have been developed. Type I, "molecular Lego" forms a hydrogel scaffold for tissue engineering; Type II, "molecular switch" as a molecular actuator; Type III, "molecular hook" and "molecular velcro" for surface engineering; Type IV, peptide nanotubes and nanovesicles, or "molecular capsule" for protein and gene deliveries and Type V, "molecular cavity" for biomineralization. These self-assembling peptide systems are simple, versatile and easy to produce. These self-assembly systems represent a significant advance in the molecular engineering for diverse technological innovations.     

Coarse-grained molecular simulation of self-assembly nanostructures of CTAB on nanoscale graphene

Coarse-grained molecular dynamics simulation has been performed to study the aggregated morphology of the cationic surfactant, cetyltrimethylammonium bromide (CTAB), adsorbed on nanoscale graphene surfaces. The CTAB surfactants can self-assemble on graphene to form various supramolecular morphologies and structures. The effect of packing density, thickness of graphene sheet and width of graphene nanoribbon on the CTAB–graphene self-assembly has been investigated. The buoyant densities of various graphene–CTAB assemblies were calculated, which increase with surfactant coverage and number of graphene layers. This result demonstrates that density gradient can be used to isolate graphenes with various layers. This simulation provides larger-scale microscopic insight into the supramolecular self-assembly nanostructures for the CTAB surfactants aggregated on graphene, which could be valuable to guide fabrication of graphene-based hybrid nanocomposites.     

Organocatalytic approach to amphiphilic comb-block copolymers capable of stereocomplexation and self-assembly

Biocompatible amphiphilic block copolymers comprised of poly(ethylene glycol) (PEG) as the hydrophilic component and a poly(methylcarboxytrimethylene carbonate) (PMTC) as a hydrophobic backbone having either poly(L-lactide) (L-PLA) or poly(D-lactide) (D-PLA) branches were prepared by organocatalytic ring-opening polymerization (ROP). The polycarbonate backbone was prepared by copolymerization of two different MTC-type monomers (MTCs) including a tetrahydropyranyloxy protected hydroxyl group, a masked initiator for a subsequent ROP step. Interestingly, the organic catalyst used in the ROP of MTCs was also effective for acetylation of the hydroxyl end-groups by the addition of acetic anhydride added after polymerization. Acidic deprotection of the tetrahydropyranyloxy (THP) protecting group on the carbonate chain generated hydroxyl functional groups that served as initiators for the ROP of either D- or L-lactide. Comb-shaped block copolymers of predictable molecular weights and narrow polydispersities (approximately 1.3) were prepared with up to 8-PLA branches. Mixtures of the D- and L-lactide based copolymers were studied to understand the effect of noncovalent interactions or stereocomplexation on the properties.     

Fabrication of hierarchical hybrid structures using bio-enabled layer-by-layer self-assembly

Development of versatile and flexible assembly systems for fabrication of functional hybrid nanomaterials with well-defined hierarchical and spatial organization is of a significant importance in practical nanobiotechnology applications. Here we demonstrate a bio-enabled self-assembly technique for fabrication of multi-layered protein and nanometallic assemblies utilizing a modular gold-binding (AuBP1) fusion tag. To accomplish the bottom-up assembly we first genetically fused the AuBP1 peptide sequence to the C'-terminus of maltose-binding protein (MBP) using two different linkers to produce MBP-AuBP1 hetero-functional constructs. Using various spectroscopic techniques, surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR), we verified the exceptional binding and self-assembly characteristics of AuBP1 peptide. The AuBP1 peptide tag can direct the organization of recombinant MBP protein on various gold surfaces through an efficient control of the organic-inorganic interface at the molecular level. Furthermore using a combination of soft-lithography, self-assembly techniques and advanced AuBP1 peptide tag technology, we produced spatially and hierarchically controlled protein multi-layered assemblies on gold nanoparticle arrays with high molecular packing density and pattering efficiency in simple, reproducible steps. This model system offers layer-by-layer assembly capability based on specific AuBP1 peptide tag and constitutes novel biological routes for biofabrication of various protein arrays, plasmon-active nanometallic assemblies and devices with controlled organization, packing density and architecture.     

Effects of the sequence and size of non-polar residues on self-assembly of amphiphilic peptides

Peptides with alternating hydrophobic and polar amino acids have been shown to form stable beta-sheet secondary structures and self-assemble into hydrogel-like matrices in the presence of physiological salt concentrations. We hypothesized that the sequence and steric size differences of non-polar residues can affect the balance of peptide intermolecular forces in solution that drive self-assembly. To test this hypothesis, we designed a library of artificial amphiphilic peptides based on the sequence (FEFEFKFK)2 by substituting combinations of the non-polar residues glycine, alanine, valine, leucine and isoleucine for phenylalanine. Peptide structure and self-assembly were characterized using scanning electron microscopy, the Thioflavin T assay, transmission electron microscopy, X-ray fiber diffraction and circular dichroism spectroscopy. The sequence and steric size of non-polar residues are shown to cause variations in peptide secondary structures and create significant differences in the matrix morphology of self-assembled peptides.     

Induction of protein-like molecular architecture by self-assembly processes

One of the most intriguing self-assembly processes is the folding of peptide chains into native protein structures. We have developed a method for building protein-like structural motifs that incorporate sequences of biological interest. A lipophilic moiety is attached onto an N(alpha)-amino group of a peptide chain, resulting in a 'peptide-amphiphile'. The alignment of amphiphilic compounds at the lipid solvent interface is used to facilitate peptide alignment and structure initiation and propagation. Peptide-amphiphiles containing potentially triple-helical structural motifs have been synthesized. The resultant head group structures have been characterized by circular dichroism and NMR spectroscopies. Evidence for a self-assembly process of peptide-amphiphiles has been obtained from: (a) circular dichroism spectra and melting curves characteristic of triple-helices, (b) one- and two-dimensional NMR spectra indicative of stable triple-helical structure at low temperatures and melted triple-helices at high temperatures, and (c) pulsed-field gradient NMR experiments demonstrating different self-diffusion coefficients between proposed triple-helical and non-triple-helical species. The peptide-amphiphiles described here provide a simple approach for building stable protein structural motifs using peptide head groups.     

A coarse-grained model for PCL: conformation,self-assembly of MePEG-b-PCL amphiphilic diblock copolymers

Poly-ε-caprolactone (PCL) is a biodegradable hydrophobic polyester that has been widely used in medical devices, tissue engineering and nanoparticle-based drug delivery. Coarse-grained molecular dynamics (CGMD) has been employed to study and gain insights into the conformational, structural and self-assembly behaviour of polymers, lipids and amphiphilic macromolecules. In this work, we developed a model for PCL within the framework of the MARTINI coarse-grained force field. The non-bonded interactions were based on the existing MARTINI bead types, while the bonded interactions were mapped onto a PCL rendition obtained from atomistic simulations. The model accurately reproduces the structural and dynamic properties of the PCL homopolymer and shows very reasonable temperature and solvent transferability. We also studied self-assembly of MePEG-b-PCL linear diblock copolymers using an existing MARTINI model for MePEG (Methoxy Polyethylene glycol), by analysing the critical micelle concentration (CMC), as well as the shape, size and morphology of the nano-polymeric micelles. We obtained excellent agreement of the CMC, while the size was under-predicted compared to experimental data. This robust model paves the way for CGMD modelling of PCL and serves as a starting point for future designs of PCL-related polymeric systems .     

Spectroscopic studies of the molecular imprinting self-assembly process

A method for the rapid estimation of the extent of complex formation in molecular imprinting prepolymerization mixtures is described. By the use of a UV spectroscopy titration procedure, apparent binding constants for such self-assembly processes have been obtained. This method was used for comparison of the interactions between a dipeptide template (N-acetyl-L-phenylalaninyl-L-tryptophanyl methyl ester) and the functional monomer methacrylic acid, and the monomer analogues acetic acid and trifluoroacetic acid. The importance of template-monomer association during the molecular imprinting prepolymerization phase is discussed with respect to the systems studied.     

Purification and characterization of amphiphilic trehalase from rabbit small intestine

Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase.     

Assembly of multimeric phage nanostructures through leucine zipper interactions

One barrier to the construction of nanoscale devices is the ability to place materials into 2D- and 3D-ordered arrays by controlling the assembly and ordering of connections between nanomaterials. Ordered assembly of nanoscale materials may potentially be achieved using biological tools that direct specific connections between individual components. Recently, viruses were successfully employed as scaffolds for the nucleation of nanoparticles and nanowires (Mao et al., 2004); however, there is a paucity of methods for the higher order assembly of phage-templated materials. Here we describe a general strategy for the assembly of filamentous bacteriophages into long, wire-like or into tripod-like structures. To prepare the linear phage assemblies, dimeric leucine zipper protein domains, fused to the p3 and p9 proteins of M13 bacteriophage, were employed to direct the specific end-to-end self-association of the bacteriophage particles. Electron microscopy revealed that up to 90% of the phage displaying complementary leucine zipper domains formed linear multi-phage assemblies, composed of up to 30 phage in length. To prepare tripod-like assemblies, phage were engineered to express trimeric leucine zippers as p3 fusion proteins. This resulted in 3D assembly with three individual phages attached at a single point. These ordered phage structures should provide a foundation for self-assembly of virally templated nanomaterials into useful devices.     

Solution self-assembly of hybrid block copolymers containing poly(ethylene glycol) and amphiphilic beta-strand peptide sequences

The self-assembly in aqueous solution of hybrid block copolymers consisting of amphiphilic beta-strand peptide sequences flanked by one or two PEG chains was investigated by means of circular dichroism spectroscopy, small-angle X-ray scattering, and transmission electron microscopy. In comparison with the native peptide sequence, it was found that the peptide secondary structure was stabilized against pH variation in the di- and tri-block copolymers with PEG. Small-angle X-ray scattering indicated the presence of fibrillar structures, the dimensions of which are comparable to the estimated width of a beta-strand (with terminal PEG chains in the case of the copolymers). Transmission electron microscopy on selectively stained and dried specimens shows directly the presence of fibrils. It is proposed that these fibrils result from the hierarchical self-assembly of peptide beta-strands into helical tapes, which then stack into fibrils.     

Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins

Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.     

Triplex inducer-directed self-assembly of single-walled carbon nanotubes: a triplex DNA-based approach for controlled manipulation of nanostructures

As a promising strategy for artificially control of gene expression, reversible assembly of nanomaterials and DNA nanomachine, DNA triplex formation has received much attention. Carbon nanotubes as gene and drug delivery vector or as 'building blocks' in nano/microelectronic devices have been successfully explored. Therefore, studies on triplex DNA-based carbon nanotube hybrid materials are important for development of smart nanomaterials and for gene therapy. In this report, a small molecule directed single-walled carbon nanotubes (SWNTs) self-assembly assay has been developed by disproportionation of SWNTs-dT(22)·dA(22) duplex into triplex dT(22)·dA(22)·dT(22) and dA(22) by a triplex formation inducer, coralyne. This has been studied by circular dichroism, light scattering (LS) spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), electrophoretic mobility shift assay and supported by using DNA random sequence. In contrast, SWNTs do not aggregate under the same experimental conditions when the small molecules used can not induce dT(22)·dA(22)·dT(22) triplex formation. Therefore, this novel small molecule-directed SWNTs self-assembly assay has also been used for screening of triplex inducers in our studies.     

Cationic amphiphilic star and linear block copolymers: synthesis, self-assembly, and in vitro gene transfection

A series of amphiphilic star and linear block copolymers were synthesized using ATRP. The core consisted of either polystyrene (PS) or poly(n-butyl acrylate) (PBuA), having different glass-transition (T(g)) values. These polymers were used as macroinitiators in the polymerization of the cationic 2-(dimethylamino)ethyl methacrylate (DMAEMA). The polymers were used to study the effects of polymer architecture and flexibility on the self-assembling properties, DNA complexation, and transfection. All polymers formed core-shell micelles in aqueous solutions and condensed plasmid DNA. Linear PDMAEMA-PBuA-PDMAEMA has transfection efficiency comparable to PEI25K in ARPE19 cell line. Glassy state of the micellar core and star-shaped architecture decreased the DNA transfection compared with the rubbery and linear polymer structures. The polymers showed low cellular toxicity at low nitrogen/phosphate (n/p) ratios.     

Fabrication of molecular materials using peptide construction motifs

Biotechnology has generally been associated with gene cloning and expression, genomics, high throughput drug discovery, biomedical advancement and agricultural development. That is about to change. Biotechnology will expand to encompass discovery and fabrication of biological and molecular materials with diverse structures, functionalities and utilities. The advent of nanobiotechnology and nanotechnology have accelerated this trend. Analogous to the construction of an intricate architectural structure, diverse and numerous structural motifs are used to assemble a sophisticated complex. Nature has selected, produced and evolved numerous molecular architectural motifs over billions of years for particular functions. These molecular motifs can now be used to build materials from the bottom up. Biotechnology will continue to harness nature's enormous power to benefit other disciplines and society as a whole.     

生物大分子自组装合成多维纳米生物结构与器件

生物体通过指导的自组装合成种类繁多、功能特异的天然纳米结构,它们在生命过程中扮演重要角色。按照自组装体的维度,可以分为线状(一维)、层状(二维)、笼状(三维)生物纳米结构。通过设计,这些生物大分子纳米结构可在细胞"工厂"中重组制备,且可通过合成生物学技术对其组装和功能化进行理性设计和调控,成为功能性纳米器件。这类纳米生物结构和器件已经在生物传感、催化、肿瘤热疗、药物递送、组织工程、生物电池等领域获得展示或应用。相关研究正在成为合成生物学和纳米生物学的一个交叉领域,受到关注。     

Design of nanostructured biological materials through self-assembly of peptides and proteins

Several self-assembling peptide and protein systems that form nanotubes, helical ribbons and fibrous scaffolds have recently emerged as biological materials. Peptides and proteins have also been selected to bind metals, semiconductors and ions, inspiring the design of new materials for a wide range of applications in nano-biotechnology.