Nanolattice-Forming Hybrid Collagens in Protective Shark Egg Cases.

Nanoscopic structural control with long-range ordering remains a profound challenge in nanomaterial fabrication. The nanoarchitectured egg cases of elasmobranchs rely on a hierarchically ordered latticework for their protective function─serving as an exemplary system for nanoscale self-assembly. Although the proteinaceous precursors are known to undergo intermediate liquid crystalline phase transitions before being structurally arrested in the final nanolattice architecture, their sequences have so far remained unknown. By leveraging RNA-seq and proteomic techniques, we identified a cohort of nanolattice-forming proteins comprising a collagenous midblock flanked by domains typically associated with innate immunity and network-forming collagens. Structurally homologous proteins were found in the genomes of other egg-case-producing cartilaginous fishes, suggesting a conserved molecular self-assembly strategy. The identity and stabilizing role of cross-links were subsequently elucidated using mass spectrometry and in situ small-angle X-ray scattering. Our findings provide a new design approach for protein-based liquid crystalline elastomers and the self-assembly of nanolattices.

The human Descemet's membrane and lens capsule: Protein composition and biomechanical properties.

The Descemet's membrane (DM) and the lens capsule (LC) are two ocular basement membranes (BMs) that are essential in maintaining stability and structure of the cornea and lens. In this study, we investigated the proteomes and biomechanical properties of these two materials to uncover common and unique properties. We also screened for possible protein changes during diabetes. LC-MS/MS was used to determine the proteomes of both BMs. Biomechanical measurements were conducted by atomic force microscopy (AFM) in force spectroscopy mode, and complemented with immunofluorescence microscopy. Proteome analysis showed that all six existing collagen IV chains represent 70% of all LC-protein, and are thus the dominant components of the LC. The DM on the other hand is predominantly composed of a single protein, TGF-induced protein, which accounted for around 50% of all DM-protein. Four collagen IV-family members in DM accounted for only 10% of the DM protein. Unlike the retinal vascular BMs, the LC and DM do not undergo significant changes in their protein compositions during diabetes. Nanomechanical measurements showed that the endothelial/epithelial sides of both BMs are stiffer than their respective stromal/anterior-chamber sides, and both endothelial and stromal sides of the DM were stiffer than the epithelial and anterior-chamber sides of the LC. Long-term diabetes did not change the stiffness of the DM and LC. In summary, our analyses show that the protein composition and biomechanical properties of the DM and LC are different, i.e., the LC is softer than DM despite a significantly higher concentration of collagen IV family members. This finding is unexpected, as collagen IV members are presumed to be responsible for BM stiffness. Diabetes had no significant effect on the protein composition and the biomechanical properties of both the DM and LC.

Artificial Lysosomal Platform to Study Nanoparticle Long-term Stability.

Nanoparticles (NPs) possess unique properties useful for designing specific functionalities for biomedi- cal applications. A prerequisite of a safe-by-design and effective use in any biomedical application is to study NP-cell interactions to gain a better understanding of cellular consequences upon exposure. Cellular uptake of NPs results mainly in the localization of NPs in the complex environment of lysosomes, a compartment which can be mimicked by artificial lysosomal fluid. In this work we showed the applicability of lysosomal fluid as a platform for a fast assessment of gold, iron oxide and silica NP stability over 24 h in a relevant biological fluid, by using multiple analytical methods.

Mimicking the Chemistry of Natural Eumelanin Synthesis: The KE Sequence in Polypeptides and in Proteins Allows for a Specific Control of Nanosized Functional Polydopamine Formation.

The oxidation of dopamine and of other catecholamines leads to the formation of conformal films on the surface of all known materials and to the formation of a precipitate in solution. In some cases, it has been shown that the addition of additives in the dopamine solution, like certain surfactants or polymers, polyelectrolytes, and certain proteins, allows to get polydopamine nanoparticles of controlled size and the concomitant decrease, in an additive/dopamine dependent manner, in film formation on the surface of the reaction beaker. However, the mechanism behind this controlled oxidation and self-assembly of catecholamines is not known. In this article, it is shown that a specific diad of amino acids in proteins, namely KE, allows for specific control in the oxidation-self-assembly of dopamine to obtain polydopamine@protein core-shell nanoparticles which are biocompatible. The interactions between dopamine and the adjacent KE amino acids potentially responsible for the size control of polydopamine aggregates was investigated by molecular dynamics simulations. The obtained core-shell nanoparticles display the biological activity of the protein used to control the self-assembly of PDA. The photon to heat conversion ability of PDA is conserved in the PDA@protein particles.

Assessing the Stability of Fluorescently Encoded Nanoparticles in Lysosomes by Using Complementary Methods.

Nanoparticles (NPs) are promising tools in biomedical research. In vitro testing is still the first method for initial evaluation; however, NP colloidal behavior and integrity, in particular inside cells (that is, in lysosomes), are largely unknown and difficult to evaluate because of the complexity of the environment. Furthermore, while the majority of NPs are usually labeled with fluorescent dyes for tracking purposes, the effect of the lysosomal environment on the fluorophore properties, as well as the ensuing effects on data interpretation, is often only sparsely addressed. In this work, we have employed several complementary analytical methods to better understand the fate of fluorescently encoded NPs and identify potential pitfalls that may arise from focusing primary analysis on a single attribute, for example, fluorophore detection. Our study shows that in a lysosomal environment NPs can undergo significant changes resulting in dye quenching and distorted fluorescence signals.

Filling polymersomes with polymers by peroxidase-catalyzed atom transfer radical polymerization.

Polymersomes that encapsulate a hydrophilic polymer are prepared by conducting biocatalytic atom transfer radical polymerization (ATRP) in these hollow nanostructures. To this end, ATRPase horseradish peroxidase (HRP) is encapsulated into vesicles self-assembled from poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) diblock copolymers. The vesicles are turned into nanoreactors by UV-induced permeabilization with a hydroxyalkyl phenone and used to polymerize poly(ethylene glycol) methyl ether acrylate (PEGA) by enzyme-catalyzed ATRP. As the membrane of the polymersomes is only permeable for the reagents of ATRP but not for macromolecules, the polymerization occurs inside of the vesicles and fills the polymersomes with poly(PEGA), as evidenced by (1) H NMR. Dynamic and static light scattering show that the vesicles transform from hollow spheres to filled spheres during polymerization. Transmission electron microscopy (TEM) and cryo-TEM imaging reveal that the polymersomes are stable under the reaction conditions. The polymer-filled nanoreactors mimic the membrane and cytosol of cells and can be useful tools to study enzymatic behavior in crowded macromolecular environments.

Luminescent nanoparticles with lanthanide-containing poly(ethylene glycol)-Poly(ε-caprolactone) block copolymers.

Lanthanide-containing nanoparticles have attracted much attention due to their unique optical properties and potential in nanotechnological applications. An amphiphilic block copolymer of poly(ethylene glycol)-b-poly(ε-caprolactone) methyl ether (mPEG-PCL) was functionalized with a dipicolinic acid (dpa) moiety and coordinated to lanthanide ions to afford [Ln(dpa-PCL-PEG-OCH3)3](HNEt3)3 (Ln = Eu(3+), Tb(3+)). Micelle-like nanoparticles of dpa-PCL-PEG-OCH3 macroligand and metal-centered polymers were prepared by solvent displacement methods. Dynamic light scattering analysis (DLS) and cryogenic transmission electron microscopy images confirmed the presence of solid sphere (<47 nm in diameter) and vesicle (>47 nm in diameter) morphologies. The viability and stability of the lanthanide complexes in micelle-like nanoparticles was explored by DLS and luminescence spectroscopy, and found to be stable for several weeks.

The biophysical and compositional properties of human basement membranes.

Basement membranes are among the most widespread, non-cellular functional materials in metazoan organisms. Despite this ubiquity, the links between their compositional and biophysical properties are often difficult to establish due to their thin and delicate nature. In this article, we examine these features on a molecular level by combining results from proteomics, elastic, and nanomechanical analyses across a selection of human basement membranes. Comparing results between these different membranes connects certain compositional attributes to distinct nanomechanical signatures and further demonstrates to what extent water defines these properties. In all, these data underline BMs as stiff yet highly elastic connective tissue layers and highlight how the interplay between composition, mechanics and hydration yields such exceptionally adaptable materials.

Material properties of the internal limiting membrane and their significance in chromovitrectomy.

Intraoperative visualization of the internal limiting membrane (ILM), the choice of a point of vantage for lifting an initial flap, the precision with which the ILM is grasped, adhesion between the forceps and the ILM, thickness, stiffness and elasticity of the ILM as well as monitoring of the completeness of ILM removal are all important factors for safety and efficacy of a chromovitrectomy intervention. The understanding of the underlying physical features of the ILM, such as contrast behavior and bioanatomical and biomechanical properties represent, thus, useful prerequisites for successful macular surgery. New analytical tools, such as atomic force microscopy and chromaticity analysis, allow new insights into ILM material characteristics, permitting a systematic approach to refinement of surgical technique. .

Intertidal exposure favors the soft-studded armor of adaptive mussel coatings.

The mussel cuticle, a thin layer that shields byssal threads from environmental exposure, is a model among high-performance coatings for being both hard and hyper-extensible. However, despite avid interest in translating its features into an engineered material, the mechanisms underlying this performance are manifold and incompletely understood. To deepen our understanding of this biomaterial, we explore here the ultrastructural, scratch-resistant, and mechanical features at the submicrometer scale and relate our observations to individual cuticular components. These investigations show that cuticle nanomechanics are governed by granular microinclusions/nanoinclusions, which, contrary to previous interpretations, are three-fold softer than the surrounding matrix. This adaptation, which is found across several related mussel species, is linked to the level of hydration and presumed to maintain bulk performance during tidal exposures. Given the interest in implementing transfer of biological principles to modern materials, these findings may have noteworthy implications for the design of durable synthetic coatings.

Optically responsive supramolecular polymer glasses.

The reversible and dynamic nature of non-covalent interactions between the constituting building blocks renders many supramolecular polymers stimuli-responsive. This was previously exploited to create thermally and optically healable polymers, but it proved challenging to achieve high stiffness and good healability. Here we present a glass-forming supramolecular material that is based on a trifunctional low-molecular-weight monomer ((UPyU)3TMP). Carrying three ureido-4-pyrimidinone (UPy) groups, (UPyU)3TMP forms a dynamic supramolecular polymer network, whose properties are governed by its cross-linked architecture and the large content of the binding motif. This design promotes the formation of a disordered glass, which, in spite of the low molecular weight of the building block, displays typical polymeric behaviour. The material exhibits a high stiffness and offers excellent coating and adhesive properties. On account of reversible dissociation and the formation of a low-viscosity liquid upon irradiation with ultraviolet light, rapid optical healing as well as (de)bonding on demand is possible.

New concepts in basement membrane biology.

Basement membranes (BMs) are thin sheets of extracellular matrix that outline epithelia, muscle fibers, blood vessels and peripheral nerves. The current view of BM structure and functions is based mainly on transmission electron microscopy imaging, in vitro protein binding assays, and phenotype analysis of human patients, mutant mice and invertebrata. Recently, MS-based protein analysis, biomechanical testing and cell adhesion assays with in vivo derived BMs have led to new and unexpected insights. Proteomic analysis combined with ultrastructural studies showed that many BMs undergo compositional and structural changes with advancing age. Atomic force microscopy measurements in combination with phenotype analysis have revealed an altered mechanical stiffness that correlates with specific BM pathologies in mutant mice and human patients. Atomic force microscopy-based height measurements strongly suggest that BMs are more than two-fold thicker than previously estimated, providing greater freedom for modelling the large protein polymers within BMs. In addition, data gathered using BMs extracted from mutant mice showed that laminin has a crucial role in BM stability. Finally, recent evidence demonstrate that BMs are bi-functionally organized, leading to the proposition that BM-sidedness contributes to the alternating epithelial and stromal tissue arrangements that are found in all metazoan species. We propose that BMs are ancient structures with tissue-organizing functions and were essential in the evolution of metazoan species.

Insertion of nanoparticle clusters into vesicle bilayers.

A major contemporary concern in developing effective liposome-nanoparticle hybrids is the present inclusion size limitation of nanoparticles between vesicle bilayers, which is considered to be around 6.5 nm in diameter. In this article, we present experimental observations backed by theoretical considerations which show that greater structures can be incorporated within vesicle membranes by promoting the clustering of nanoparticles before liposome formation. Cryo-transmission electron microscopy and cryo-electron tomography confirm these observations at unprecedented detail and underpin that the liposome membranes can accommodate flexible structures of up to 60 nm in size. These results imply that this material is more versatile in terms of inclusion capabilities and consequently widens the opportunities in developing multivalent vesicles for nanobiotechnology applications.

Nanoscale topographic and biomechanical studies of the human internal limiting membrane.

PURPOSE: The purpose of this article was to create a nanometer scale topographic and biomechanical profile of the human internal limiting membrane (ILM) under native conditions. METHODS: ILMs from the posterior pole of postmortem human eyes were prepared as flat mounts and investigated by atomic force microscopy (AFM) under physiological conditions. Structural analysis was complemented by transmission electron microscopy. RESULTS: Average thickness of the fully hydrated, native ILMs was 3488 ± 460 nm. Thickness variations from 100 nm to 4326 nm characterized the fovea, which displayed a craterlike morphology. Outside the fovea, thickness distribution was uniform. Although mean ILM thicknesses were similar, standard deviation was higher on the retinal than on the vitreal side, indicating greater roughness. Average ILM stiffness was more than fivefold higher on the retinal than on the vitreal side (227 vs. 44 kPa). CONCLUSIONS: A detailed topographical and nanomechanical profile of native human ILM was generated using AFM. Thickness values were significantly higher than in previous studies because of the preservation of native conditions. Both thickness and stiffness showed marked variations around the fovea but were relatively uniform outside the foveal area. Interestingly, the foveal ILM displayed a craterlike morphological appearance with four distinct layers separated by comparatively steep thickness increments. ILM stiffness was considerably higher on the retinal than on the vitreal side. AFM opens new possibilities for investigating native basement membranes under physiological and pathological conditions. Transmission electron microscopy revealed higher extracellular matrix protein density on the retinal than on the vitreal side.

The nanomechanical signature of breast cancer.

Cancer initiation and progression follow complex molecular and structural changes in the extracellular matrix and cellular architecture of living tissue. However, it remains poorly understood how the transformation from health to malignancy alters the mechanical properties of cells within the tumour microenvironment. Here, we show using an indentation-type atomic force microscope (IT-AFM) that unadulterated human breast biopsies display distinct stiffness profiles. Correlative stiffness maps obtained on normal and benign tissues show uniform stiffness profiles that are characterized by a single distinct peak. In contrast, malignant tissues have a broad distribution resulting from tissue heterogeneity, with a prominent low-stiffness peak representative of cancer cells. Similar findings are seen in specific stages of breast cancer in MMTV-PyMT transgenic mice. Further evidence obtained from the lungs of mice with late-stage tumours shows that migration and metastatic spreading is correlated to the low stiffness of hypoxia-associated cancer cells. Overall, nanomechanical profiling by IT-AFM provides quantitative indicators in the clinical diagnostics of breast cancer with translational significance.