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Performing DNA nanotechnology operations on a zebrafish
Nanoscale engineering of surfaces is becoming an indispensable technique to modify membranes and, thus cellular behaviour. Here, such membrane engineering related was explored on the surface of a living animal using DNA nanotechnology. We demonstrate the immobilization of oligonucleotides functionalized with a membrane anchor on 2 day old zebrafish. The protruding single-stranded DNA on the skin of zebrafish served as a handle for complementary DNAs, which allowed the attachment of small molecule cargo, liposomes and dynamic relabeling by DNA hybridization protocols. Robust anchoring of the oligonucleotides was proven as DNA-based amplification processes were successfully performed on the outer membrane of the zebrafish enabling the multiplication of surface functionalities from a single DNA-anchoring unit and the dramatic improvement of fluorescent labeling of these animals. As zebrafish are becoming an alternative to animal models in drug development, toxicology and nanoparticles characterization, we believe the platform presented here allows amalgamation of DNA nanotechnology tools with live animals and this opens up yet unexplored avenues like efficient bio-barcoding as well as in vivo tracking. © The Royal Society of Chemistry.
Cellular responses to beating hydrogels to investigate mechanotransduction
Cells feel the forces exerted on them by the surrounding extracellular matrix (ECM) environment and respond to them. While many cell fate processes are dictated by these forces, which are highly synchronized in space and time, abnormal force transduction is implicated in the progression of many diseases (muscular dystrophy, cancer). However, material platforms that enable transient, cyclic forces in vitro to recreate an in vivo-like scenario remain a challenge. Here, we report a hydrogel system that rapidly beats (actuates) with spatio-temporal control using a near infra-red light trigger. Small, user-defined mechanical forces (~nN) are exerted on cells growing on the hydrogel surface at frequencies up to 10 Hz, revealing insights into the effect of actuation on cell migration and the kinetics of reversible nuclear translocation of the mechanosensor protein myocardin related transcription factor A, depending on the actuation amplitude, duration and frequency.
Engineering biofunctional in vitro vessel models using a multilayer bioprinting technique
Recent advances in the field of bioprinting have led to the development of perfusable complex structures. However, most of the existing printed vascular channels lack the composition or key structural and physiological features of natural blood vessels or they make use of more easily printable but less biocompatible hydrogels. Here, we use a drop-on-demand bioprinting technique to generate in vitro blood vessel models, consisting of a continuous endothelium imitating the tunica intima, an elastic smooth muscle cell layer mimicking the tunica media, and a surrounding fibrous and collagenous matrix of fibroblasts mimicking the tunica adventitia. These vessel models with a wall thickness of up to 425 µm and a diameter of about 1 mm were dynamically cultivated in fluidic bioreactors for up to three weeks under physiological flow conditions. High cell viability (>83%) after printing and the expression of VE-Cadherin, smooth muscle actin, and collagen IV were observed throughout the cultivation period. It can be concluded that the proposed novel technique is suitable to achieve perfusable vessel models with a biofunctional multilayer wall composition. Such structures hold potential for the creation of more physiologically relevant in vitro disease models suitable especially as platforms for the pre-screening of drugs.
Ultra-strong bio-glue from genetically engineered polypeptides
The development of biomedical glues is an important, yet challenging task as seemingly mutually exclusive properties need to be combined in one material, i.e. strong adhesion and adaption to remodeling processes in healing tissue. Here, we report a biocompatible and biodegradable protein-based adhesive with high adhesion strengths. The maximum strength reaches 16.5 ± 2.2 MPa on hard substrates, which is comparable to that of commercial cyanoacrylate superglue and higher than other protein-based adhesives by at least one order of magnitude. Moreover, the strong adhesion on soft tissues qualifies the adhesive as biomedical glue outperforming some commercial products. Robust mechanical properties are realized without covalent bond formation during the adhesion process. A complex consisting of cationic supercharged polypeptides and anionic aromatic surfactants with lysine to surfactant molar ratio of 1:0.9 is driven by multiple supramolecular interactions enabling such strong adhesion. We demonstrate the glue’s robust performance in vitro and in vivo for cosmetic and hemostasis applications and accelerated wound healing by comparison to surgical wound closures.
Gold-DNA nanosunflowers for efficient gene silencing with controllable transformation
The development of an efficient delivery system for enhanced and controlled gene interference–based therapeutics is still facing great challenges. Fortunately, the flourishing field of nanotechnology provides more effective strategies for nucleic acid delivery. Here, the triplex-forming oligonucleotide sequence and its complementary strand were used to mediate self-assembly of ultrasmall gold nanoparticles. The obtained sunflower-like nanostructures exhibited strong near-infrared (NIR) absorption and photothermal conversion ability. Upon NIR irradiation, the large-sized nanostructure could disassemble and generate ultrasmall nanoparticles modified with c-myc oncogene silencing sequence, which could directly target the cell nucleus. Moreover, the controlled gene silencing effect could be realized by synergistically controlling the preincubation time with the self-assembled nanostructure (in vitro and in vivo) and NIR irradiation time point. This study provides a new approach for constructing more efficient and tailorable nanocarriers for gene interference applications
Fibronectin promotes directional persistence in fibroblast migration through interactions with both its cell-binding and heparin-binding domains
The precise mechanisms through which insoluble, cell-adhesive ligands induce and regulate directional cell migration remain obscure. We recently demonstrated that elevated surface density of physically adsorbed plasma fibronectin (FN) promotes high directional persistence in fibroblast migration. While cell-FN association through integrins α5β1 and αvβ3 was necessary, substrates that selectively engaged these integrins did not support the phenotype. We here show that high directional persistence necessitates a combination of the cell-binding and C-terminal heparin-binding domains of FN, but does not require the engagement of syndecan-4 or integrin α4β1. FN treatment with various fixation agents indicated that associated changes in fibroblast motility were due to biochemical changes, rather than alterations in its physical state. The nature of the coating determined the ability of fibroblasts to assemble endogenous or exogenous FN, while FN fibrillogenesis played a minor, but significant, role in regulating directionality. Interestingly, knockdown of cellular FN abolished cell motility altogether, demonstrating a requirement for intracellular processes in enabling fibroblast migration on FN. Lastly, kinase inhibition experiments revealed that regulation of cell speed and directional persistence are decoupled. Hence, we have identified factors that render full-length FN a promoter of directional migration and discuss the possible, relevant mechanisms.
Bio-degradable highly fluorescent conjugated polymer nanoparticles for bio-medical imaging applications
Conjugated polymer nanoparticles exhibit strong fluorescence and have been applied for biological fluorescence imaging in cell culture and in small animals. However, conjugated polymer particles are hydrophobic and often chemically inert materials with diameters ranging from below 50 nm to several microns. As such, conjugated polymer nanoparticles cannot be excreted through the renal system. This drawback has prevented their application for clinical bio-medical imaging. Here, we present fully conjugated polymer nanoparticles based on imidazole units. These nanoparticles can be bio-degraded by activated macrophages. Reactive oxygen species induce scission of the conjugated polymer backbone at the imidazole unit, leading to complete decomposition of the particles into soluble low molecular weight fragments. Furthermore, the nanoparticles can be surface functionalized for directed targeting. The approach opens a wide range of opportunities for conjugated polymer particles in the fields of medical imaging, drug-delivery, and theranostics.
Microfluidic cell sorting: Towards improved biocompatibility of extracorporeal lung assist devices
Extracorporeal lung assist technology is one of the last options in critical care medicine to treat patients suffering from severe oxygenation and decarboxylation disorders. Platelet activation along with the consequent thrombus formation is a potentially life-threatening complication of this technique. To avoid platelet-dependent clot formation, this study aims at developing a microfluidic cell sorting chip that can bypass platelets prior to the membrane oxygenator of the extracorporeal lung assist device. The cell sorting chips were produced by maskless dip-in laser lithography, followed by soft lithography replication using PDMS. Citrated porcine whole blood with a clinically relevant haematocrit of 17% was used for the cell sorting experiments involving three different blood flow rates. The joint effects of flow focusing and hydrodynamic lifting forces within the cell sorting chip resulted in a reduction of up to 57% of the baseline platelet count. This cell sorting strategy is suitable for the continuous and label-free separation of red blood cells and platelets and is potentially applicable for increasing the biocompatibility and lifetime of current extracorporeal lung assist devices.