Engineering of pulmonary surfactant corona on inhaled nanoparticles to operate in the lung system

Exposure of inhaled nanoparticles (NPs) to the deep lung tissue results in the adsorption of pulmonary surfactant (PSf) on the surface of NPs and the formation of a biomolecular corona. The adsorption of the peculiar phospholipids (PLs) and surfactant proteins (SPs) provides NPs with a new bio-identity, which likely changes their corresponding interactions with cells and other bio-systems. Exploring the interaction of NPs with the PSf film at the alveolar air-fluid interface can provide valuable insights into the role of biofluids in the cellular uptake of NPs and their nanotoxic effects. Wrapping biomembranes around NPs and the formation of lipoprotein corona regulate viscoelastic changes, NP insertion into the membrane, and cellular uptake of NPs. In this review, a concise overview has been presented on the engineering of PSf on inhaled NPs to operate in lung environment. First, the physiological barriers in the pulmonary delivery of NPs and approaches to regulating their pulmonary fate are introduced and rationalized. Next, a short description is given on the different sources used for exploring the interfacial performance of inhaled NPs in vitro. A discussion is then presented on SP corona formation on the surface of inhaled NPs, coronal proteome/lipidome in respiratory tract lining fluid (RTLF), regulation of NP aggregation and surfactant flow characteristics, PSf corona and its functional role in the cellular uptake of NPs, followed by explanations on the clinical correlations of PSf corona formation/inhibition on the surface of NPs. Finally, the challenges and future perspectives of the field have been discussed. This review can be harnessed to exploit PSf for the development of safe and bio-inspired pulmonary drug delivery strategies.</p

2014 Conference Abstracts: Annual Undergraduate Research Conference at the Interface of Biology and Mathematics

Conference schedule and abstract book for the Sixth Annual Undergraduate Research Conference at the Interface of Biology and Mathematics Date: November 1-2, 2014Plenary Speakers: Joseph Tien, Associate Professor of Mathematics at The Ohio State University; and Jeremy Smith, Governor\u27s Chair at the University of Tennessee and Director of the University of Tennessee/Oak Ridge National Lab Center for Molecular Biophysic

Quantitative analysis of the mechanics of fibrillar fribronectin

Thesis (Ph.D.)--Boston UniversityThe information exchange between cells and their environment is a key mediator of cell behavior that will result in disease or dysfunction if disrupted. A thorough understanding of the in vivo cell environment is critical to relating cell behaviors observed in vitro to cell behaviors in pathogenesis and homeostasis. In addition to neighboring cells, the extracellular matrix (ECM) defines the local cell environment in the body. The protein fibronectin (Fn) is a prominent component of the ECM and a key cell adhesive ligand. Fn is assembled by cells into an extremely extensible, fibrous network through which cells migrate. Fn is also an integral part of the signaling machinery that instructs cell behavior. Cells may bind to Fn through a large number of receptors, in addition Fn binds and presents growth factors to cells, regulating their proliferative and migratory behavior. Stretch, applied to Fn fibers has been demonstrated to alter properties like binding site availability and fiber stiffness. In order to understand how molecular conformations and mechanical stretch regulate these cell instructive properties of Fn fibers, one must build a quantitative understanding of the intermolecular architecture of Fn fibers. In this study we have characterized the physical characteristics of fibronectin, its density, stiffness, extensibility, and viscoelasticity with respect to the extension of the Fn fiber. We have quantified conformational changes within the molecule that regulate both its mechanical properties and the availability of binding sites. In addition, we determined that the configuration of the molecular crosslinks strongly influences the fiber's physical properties. By taking measurements of the Fn fibers under constant tension we have shown that fibronectin is a highly viscoelastic material with extremely slow response times, indicating that in the slow pulling regime of cell tractions Fn material properties may deviate significantly from measurements made at higher pulling rates. A strong quantitative understanding of fibronectin's properties opens the door to new insights into disease and new approaches to creating engineered tissue constructs

Evolutionary Dynamics on Protein Bi-stability Landscapes Can Potentially Resolve Adaptive Conflicts

Experimental studies have shown that some proteins exist in two alternative native-state conformations. It has been proposed that such bi-stable proteins can potentially function as evolutionary bridges at the interface between two neutral networks of protein sequences that fold uniquely into the two different native conformations. Under adaptive conflict scenarios, bi-stable proteins may be of particular advantage if they simultaneously provide two beneficial biological functions. However, computational models that simulate protein structure evolution do not yet recognize the importance of bi-stability. Here we use a biophysical model to analyze sequence space to identify bi-stable or multi-stable proteins with two or more equally stable native-state structures. The inclusion of such proteins enhances phenotype connectivity between neutral networks in sequence space. Consideration of the sequence space neighborhood of bridge proteins revealed that bi-stability decreases gradually with each mutation that takes the sequence further away from an exactly bistable protein. With relaxed selection pressures, we found that bi-stable proteins in our model are highly successful under simulated adaptive conflict. Inspired by these model predictions, we developed a method to identify real proteins in the PDB with bridge-like properties, and have verified a clear bi-stability gradient for a series of mutants studied by Alexander et al. (Proc Nat Acad Sci USA 2009, 106:21149–21154) that connect two sequences that fold uniquely into two different native structures via a bridge-like intermediate mutant sequence. Based on these findings, new testable predictions for future studies on protein bi-stability and evolution are discussed

Folding and Assembly of Cytoskeletal Proteins Under Force - From Single Molecule Studies of Dystrophin to Studies of Intact Cells

Changes in tertiary and quaternary structure of proteins within the actin cytoskeletal network are a likely way cells read mechanical signals from their environment. However, showing that these conformational changes occur as a result of mechanical stress and that such changes are important to the function of the cell is a major challenge. This thesis seeks to address these questions using a cohort of molecular biophysical and cell biological methods applied in increasingly complex contexts. First, the importance of force-driven unfolding to function and how changes in unfolding pathway correlate with diseased states was determined with single molecule Atomic Force Microscopy on nano-constructs of wild-type and mutant forms of dystrophin. Biophysical studies showed that the ability to fold into mechanically stable, spectrin-type helical bundle domains and the preservation of cooperative unfolding were common characteristics of functional truncated dystrophins. Second, a newly developed in-cell cysteine labeling technique demonstrated stress-enhanced repeat unfolding within spectrin in wild-type red blood cells under shear stress versus static conditions, thus demonstrating that forced unfolding is not just an in vitro phenomena. The importance of the cytoskeletal network to spectrin function was also demonstrated in mutant, 4.1R-null red blood cells, where the intrinsic properties of spectrin remain intact but the network integrity is compromised by absence of 4.1R. Loss of network integrity was evident in a decrease in spectrin unfolding under stress. Repeat unfolding was accompanied by changes in associations of spectrin with its binding partners in a time- and stress- dependent manner, indicating that the erythrocyte cytoskeleton exhibits a graded response to stress. Lastly, with cardiomyocytes derived from embryonic stem cells, the importance of stress to quaternary structure of actinin within the sarcomeric cytoskeleton and its effects on cell-wide function was tested in cells adhered to elastic substrates. Substrate stiffness sets the load on these spontaneously contracting cells, and differences in load lead to cytoskeletal reorganization with significant effects on cardiogenic development. Taken together, these findings present evidence of various cytoskeletal proteins – especially in the spectrin superfamily – as mediators of mechanical signaling within cell

Surface Modification of Ti Implant for Enhancing Biotribology and Cells Attachment

Implant success strongly depends on the proper integration of bone to biomaterial surface. By the selected retrieval cases, inadequate integration of bone screws was a dominated factor caused failure. The surface modification technology that improve osteointegration by inducing TiO2 nanotubes (NT) on Ti-based implants has a potential applications in orthopaedic implants. NT generated by anodization method provide a vertically aligned nanotube structure that enhances the integration between bone tissue and implant surface by improving osteoblasts attachment. Although cells to NT is positive, the mechanical weakness of NT has also been well-documented and is an obstacle to its applications. The thesis comprise a detailed method to improve NT mechanical stability, by introducing an interfacial bonding layer at NT bottom and Ti substrate, and retaining vertically aligned nanotubes. The physicochemical properties of this structure optimized TiO2 nanotubes (SO-NT) was systematically characterized, the SO-NT has been demonstrated with improved biotribological and biocorrosive performance. The uniform hyperfine interfacial bonding layer with nano-sized grains exhibited a strong bonding to NT layer and Ti substrate. It was observed, the layer not only effectively dissipates external impacts and shear stress but also acts as a good corrosion resistance barrier to prevent the Ti substrate from corrosion. The SO-NT modified bone screw has also demonstrated with enhanced fretting corrosion resistance than NT and pristine Ti6Al4V on screws. Since the elongated osteoblasts were observed on NT and SO-NT compared with Ti surface, the nanotubes structure has been shown with promoting of osteoblasts attachment. However, the mechanism of cell nanotubes interactions are largely in controversial. In order to reveal the cell-nanomaterial interactions, nanotopographies including Nanoconvex, Nanoconcave and Nanoflat were generated and characterized to evaluate the cell initial attachment behaviour. Human osteoblasts were observed with spindle shape on Nanoconcave, cells on Nanoflat were well-spreading but in sphere shape, while the osteoblasts on Nanoconvex were with the minimum spreading areas. Cell-materials interface is mediated and influenced by the adsorption of ECM on nanomaterials. Thus, a novel fibronectin adsorption model was proposed by calculating Coulomb's force to illustrate the interact mechanism between protein and material that influence cell behaviours. The achievements of thesis are; 1. Retrieval analyzed two cases of implants failure and pointed out one of dominated failure factor, the lack of osteointegration. 2. Introduce the interfacial bonding layer that significantly improve the biotribological and biocorrosive performance of NT, and generated SO-NT. 3. Systematically evaluated the biotribological performance of Ti, NT and SO-NT, and propose a novel methodology to quantify the fretting degradation on bone screws. 4. Propose a novel model to estimate the fibronectin adsorption on Nanoflat, Nanoconvex and Nanoconcave by the Coulomb's force calculation

Collagen-Based Biomimetic Systems to Study the Biophysical Tumour Microenvironment

The extracellular matrix (ECM) is a pericellular network of proteins and other molecules that provides mechanical support to organs and tissues. ECM biophysical properties such as topography, elasticity and porosity strongly influence cell proliferation, differentiation and migration. The cell’s perception of the biophysical microenvironment (mechanosensing) leads to altered gene expression or contractility status (mechanotransduction). Mechanosensing and mechanotransduction have profound implications in both tissue homeostasis and cancer. Many solid tumours are surrounded by a dense and aberrant ECM that disturbs normal cell functions and makes certain areas of the tumour inaccessible to therapeutic drugs. Understanding the cell-ECM interplay may therefore lead to novel and more effective therapies. Controllable and reproducible cell culturing systems mimicking the ECM enable detailed investigation of mechanosensing and mechanotransduction pathways. Here, we discuss ECM biomimetic systems. Mainly focusing on collagen, we compare and contrast structural and molecular complexity as well as biophysical properties of simple 2D substrates, 3D fibrillar collagen gels, cell-derived matrices and complex decellularized organs. Finally, we emphasize how the integration of advanced methodologies and computational methods with collagen-based biomimetics will improve the design of novel therapies aimed at targeting the biophysical and mechanical features of the tumour ECM to increase therapy efficacy