Probe microscopy methods and applications in imaging of biological materials.

Atomic force microscopy is an emerging tool for investigating the biomolecular aspects of cellular interactions; however, cell and tissue analyses must frequently be performed in aqueous environment, over rough surfaces, and on complex adhesive samples that complicate the imaging process and readily facilitate the blunting or fouling of the AFM probe. In addition, the shape and surface chemistry of the probe determine the quality and types of data that can be acquired from biological materials, with certain information becoming available only within a specific range of tip lengths or diameters, or through the assistance of specific chemical or biological functionalization procedures. Consequently, a broad range of probe modification techniques has been developed to extend the capabilities and overcome the limitations of biological AFM measurements, including the fabrication of AFM tips with specialized morphologies, surface coating with biologically affine molecules, and the attachment of proteins, nucleic acids and cells to AFM probes. In this review, we underline the importance of probe choice and modification for the AFM analysis of biomaterials, discuss the recent literature on the use of non-standard AFM tips in life sciences research, and consider the future utility of tip functionalization methods for the investigation of fundamental cell and tissue interactions.

Force and time-dependent self-assembly, disruption and recovery of supramolecular peptide amphiphile nanofibers.

Biological feedback mechanisms exert precise control over the initiation and termination of molecular self-assembly in response to environmental stimuli, while minimizing the formation and propagation of defects through self-repair processes. Peptide amphiphile (PA) molecules can self-assemble at physiological conditions to form supramolecular nanostructures that structurally and functionally resemble the nanofibrous proteins of the extracellular matrix, and their ability to reconfigure themselves in response to external stimuli is crucial for the design of intelligent biomaterials systems. Here, we investigated real-time self-assembly, deformation, and recovery of PA nanofibers in aqueous solution by using a force-stabilizing double-pass scanning atomic force microscopy imaging method to disrupt the self-assembled peptide nanofibers in a force-dependent manner. We demonstrate that nanofiber damage occurs at tip-sample interaction forces exceeding 1 nN, and the damaged fibers subsequently recover when the tip pressure is reduced. Nanofiber ends occasionally fail to reconnect following breakage and continue to grow as two individual nanofibers. Energy minimization calculations of nanofibers with increasing cross-sectional ellipticity (corresponding to varying levels of tip-induced fiber deformation) support our observations, with high-ellipticity nanofibers exhibiting lower stability compared to their non-deformed counterparts. Consequently, tip-mediated mechanical forces can provide an effective means of altering nanofiber integrity and visualizing the self-recovery of PA assemblies.

Multivalent Presentation of Cationic Peptides on Supramolecular Nanofibers for Antimicrobial Activity.

Noncovalent and electrostatic interactions facilitate the formation of complex networks through molecular self-assembly in biomolecules such as proteins and glycosaminoglycans. Self-assembling peptide amphiphiles (PA) are a group of molecules that can form nanofibrous structures and may contain bioactive epitopes to interact specifically with target molecules. Here, we report the presentation of cationic peptide sequences on supramolecular nanofibers formed by self-assembling peptide amphiphiles for cooperative enhanced antibacterial activity. Antibacterial properties of self-assembled peptide nanofibers were significantly higher than soluble peptide molecules with identical amino acid sequences, suggesting that the tandem presentation of bioactive epitopes is important for designing new materials for bactericidal activity. In addition, bacteria were observed to accumulate more rapidly on peptide nanofibers compared to soluble peptides, which may further enhance antibacterial activity by increasing the number of peptide molecules interacting with the bacterial membrane. The cationic peptide amphiphile nanofibers were observed to attach to bacterial membranes and disrupt their integrity. These results demonstrate that short cationic peptides show a significant improvement in antibacterial activity when presented in the nanofiber form.

Supramolecular Peptide Nanofiber Morphology Affects Mechanotransduction of Stem Cells.

Chirality and morphology are essential factors for protein function and interactions with other biomacromolecules. Extracellular matrix (ECM) proteins are also similar to other proteins in this sense; however, the complexity of the natural ECM makes it difficult to study these factors at the cellular level. The synthetic peptide nanomaterials harbor great promise in mimicking specific ECM molecules as model systems. In this work, we demonstrate that mechanosensory responses of stem cells are directly regulated by the chirality and morphology of ECM-mimetic peptide nanofibers with strictly controlled characteristics. Structural signals presented on l-amino acid containing cylindrical nanofibers (l-VV) favored the formation of integrin ß1-based focal adhesion complexes, which increased the osteogenic potential of stem cells through the activation of nuclear YAP. On the other hand, twisted ribbon-like nanofibers (l-FF and d-FF) guided the cells into round shapes and decreased the formation of focal adhesion complexes, which resulted in the confinement of YAP proteins in the cytosol and a corresponding decrease in osteogenic potential. Interestingly, the d-form of twisted-ribbon like nanofibers (d-FF) increased the chondrogenic potential of stem cells more than their l-form (l-FF). Our results provide new insights into the importance and relevance of morphology and chirality of nanomaterials in their interactions with cells and reveal that precise control over the chemical and physical properties of nanostructures can affect stem cell fate even without the incorporation of specific epitopes.

Biocompatible Electroactive Tetra(aniline)-Conjugated Peptide Nanofibers for Neural Differentiation.

Peripheral nerve injuries cause devastating problems for the quality of patients' lives, and regeneration following damage to the peripheral nervous system is limited depending on the degree of the damage. Use of nanobiomaterials can provide therapeutic approaches for the treatment of peripheral nerve injuries. Electroactive biomaterials, in particular, can provide a promising cure for the regeneration of nerve defects. Here, a supramolecular electroactive nanosystem with tetra(aniline) (TA)-containing peptide nanofibers was developed and utilized for nerve regeneration. Self-assembled TA-conjugated peptide nanofibers demonstrated electroactive behavior. The electroactive self-assembled peptide nanofibers formed a well-defined three-dimensional nanofiber network mimicking the extracellular matrix of the neuronal cells. Neurite outgrowth was improved on the electroactive TA nanofiber gels. The neural differentiation of PC-12 cells was more advanced on electroactive peptide nanofiber gels, and these biomaterials are promising for further use in therapeutic neural regeneration applications.

A Modular Antigen Presenting Peptide/Oligonucleotide Nanostructure Platform for Inducing Potent Immune Response.

The design and development of vaccines, which can induce cellular immunity, particularly CD8+ T cells hold great importance since these cells play crucial roles against cancers and viral infections. Covalent conjugation of antigen and adjuvant molecules has been used for successful promotion of immunogenicity in subunit vaccines; however, the stimulation of the CD8+ T-cell responses by this approach has so far been limited. This study demonstrates a modular system based on noncovalent attachment of biotinylated antigen to a hybrid nanofiber system consisting of biotinylated self-assembling peptide and CpG oligodeoxynucleotides (ODN) molecules, via biotin-streptavidin interaction. These peptide/oligonucleotide hybrid nanosystems are capable of bypassing prior limitations related with inactivated or live-attenuated virus vaccines and achieve exceptionally high CD8+ T-cell responses. The nanostructures are found to trigger strong IgG response and effectively modulate cross-presentation of their antigen "cargo" through close proximity between the antigen and peptide/ODN adjuvant system. In addition, the biotinylated peptide nanofiber system is able to enhance antigen uptake and induce the maturation of antigen-presenting cells. Due to its versatility, biocompatibility, and biodegradability with a broad variety of streptavidin-linked antigens, the nanosystem shown here can be utilized as an efficient strategy for new vaccine development.

Atomic force microscopy for the investigation of molecular and cellular behavior.

The present review details the methods used for the measurement of cells and their exudates using atomic force microscopy (AFM) and outlines the general conclusions drawn by the mechanical characterization of biological materials through this method. AFM is a material characterization technique that can be operated in liquid conditions, allowing its use for the investigation of the mechanical properties of biological materials in their native environments. AFM has been used for the mechanical investigation of proteins, nucleic acids, biofilms, secretions, membrane bilayers, tissues and bacterial or eukaryotic cells; however, comparison between studies is difficult due to variances between tip sizes and morphologies, sample fixation and immobilization strategies, conditions of measurement and the mechanical parameters used for the quantification of biomaterial response. Although standard protocols for the AFM investigation of biological materials are limited and minor differences in measurement conditions may create large discrepancies, the method is nonetheless highly effective for comparatively evaluating the mechanical integrity of biomaterials and can be used for the real-time acquisition of elasticity data following the introduction of a chemical or mechanical stimulus. While it is currently of limited diagnostic value, the technique is also useful for basic research in cancer biology and the characterization of disease progression and wound healing processes.

Virus-like nanostructures for tuning immune response.

Synthetic vaccines utilize viral signatures to trigger immune responses. Although the immune responses raised against the biochemical signatures of viruses are well characterized, the mechanism of how they affect immune response in the context of physical signatures is not well studied. In this work, we investigated the ability of zero- and one-dimensional self-assembled peptide nanostructures carrying unmethylated CpG motifs (signature of viral DNA) for tuning immune response. These nanostructures represent the two most common viral shapes, spheres and rods. The nanofibrous structures were found to direct immune response towards Th1 phenotype, which is responsible for acting against intracellular pathogens such as viruses, to a greater extent than nanospheres and CpG ODN alone. In addition, nanofibers exhibited enhanced uptake into dendritic cells compared to nanospheres or the ODN itself. The chemical stability of the ODN against nuclease-mediated degradation was also observed to be enhanced when complexed with the peptide nanostructures. In vivo studies showed that nanofibers promoted antigen-specific IgG production over 10-fold better than CpG ODN alone. To the best of our knowledge, this is the first report showing the modulation of the nature of an immune response through the shape of the carrier system.