Scale up of Transmembrane NADH Oxidation in Synthetic Giant Vesicles.

The transfer of electrons across and along biological membranes drives the cellular energetics. In the context of artificial cells, it can be mimicked by minimal means, while using synthetic alternatives of the phospholipid bilayer and the electron-transducing proteins. Furthermore, the scaling up to biologically relevant and optically accessible dimensions may provide further insight and allow assessment of individual events but has been rarely attempted so far. Here, we visualized the mediated transmembrane oxidation of encapsulated NADH in giant unilamellar vesicles via confocal laser scanning and time-correlated single photon counting wide-field microscopy. To this end, we first augmented phospholipid membranes with an amphiphilic copolymer in order to check its influence on the oxidation kinetics spectrophotometrically. Then, we scaled up the compartments and followed the process microscopically.

Compartments for Synthetic Cells: Osmotically Assisted Separation of Oil from Double Emulsions in a Microfluidic Chip.

Liposomes are used in synthetic biology as cell-like compartments and their microfluidic production through double emulsions allows for efficient encapsulation of various components. However, residual oil in the membrane remains a critical bottleneck for creating pristine phospholipid bilayers. It has been discovered that osmotically driven shrinking leads to detachment of the oil drop. Separation inside a microfluidic chip has been realized to automate the procedure, which allows for controlled continuous production of monodisperse liposomes.

Directed Signaling Cascades in Monodisperse Artificial Eukaryotic Cells.

The bottom-up assembly of multicompartment artificial cells that are able to direct biochemical reactions along a specific spatial pathway remains a considerable engineering challenge. In this work, we address this with a microfluidic platform that is able to produce monodisperse multivesicular vesicles (MVVs) to serve as synthetic eukaryotic cells. Using a two-inlet polydimethylsiloxane channel design to co-encapsulate different populations of liposomes we are able to produce lipid-based MVVs in a high-throughput manner and with three separate inner compartments, each containing a different enzyme: α-glucosidase, glucose oxidase, and horseradish peroxidase. We demonstrate the ability of these MVVs to carry out directed chemical communication between the compartments via the reconstitution of size-selective membrane pores. Therefore, the signal transduction, which is triggered externally, follows a specific spatial pathway between the compartments. We use this platform to study the effects of enzyme cascade compartmentalization by direct analytical comparison between bulk, one-, two-, and three-compartment systems. This microfluidic strategy to construct complex hierarchical structures is not only suitable to study compartmentalization effects on biochemical reactions but is also applicable for developing advanced drug delivery systems as well as minimal cells in the field of bottom-up synthetic biology.

Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions.

Self-sustained metabolic pathways in microcompartments are the corner-stone for living systems. From a technological viewpoint, such pathways are a mandatory prerequisite for the reliable design of artificial cells functioning out-of-equilibrium. Here we develop a microfluidic platform for the miniaturization and analysis of metabolic pathways in man-made microcompartments formed of water-in-oil droplets. In a modular approach, we integrate in the microcompartments a nicotinamide adenine dinucleotide (NAD)-dependent enzymatic reaction and a NAD-regeneration module as a minimal metabolism. We show that the microcompartments sustain a metabolically active state until the substrate is fully consumed. Reversibly, the external addition of the substrate reboots the metabolic activity of the microcompartments back to an active state. We therefore control the metabolic state of thousands of independent monodisperse microcompartments, a step of relevance for the construction of large populations of metabolically active artificial cells.

Nanoengineered biomimetic hydrogels for guiding human stem cell osteogenesis in three dimensional microenvironments.

The ability to modulate stem cell differentiation in a three dimensional (3D) microenvironment for bone tissue engineering in absence of exogenous pharmaceutical agents such as bone morphogenic protein (BMP-2) remains a challenge. In this study, we introduce extracellular matrix (ECM)-mimicking nanocomposite hydrogels to induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) for bone regeneration in absence of any osteoinducting factors. In particular, we have reinforced photocrosslinkable collagen-based matrix (gelatin methacryloyl, GelMA) used disk-shaped nanosilicates (nSi), a new class of two-dimensional (2D) nanomaterials. We show that nanoengineered hydrogels supported migration and proliferation of encapsulated hMSCs, with no signs of cell apoptosis or inflammatory cytokine responses. The addition of nSi significantly enhances osteogenic differentiation of encapsulated hMSCs as evident by the increase in alkaline phosphates (ALP) activity and deposition of biomineralized matrix compared to GelMA without nSi. We also show that microfabricated nanoengineered microgels can be used to pattern and control cellular behaviour. Furthermore, we also show that nanoengineered hydrogel have high biocompatibility as determined by in vivo experiments using immunocompetent rat model. Specifically, the hydrogels showed minimum localized immune responses, indicating it ability for tissue engineering applications. Overall, we showed the ability of nanoengineered hydrogels loaded with 2D nanosilicates for osteogenic differentiation of stem cells in vitro, in absence of any growth factors such as BMP-2. Our in vivo studies show high biocompatibility of nanocomposites and show the potential for growth factor free bone regeneration.

Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair.

The objective of this study was to develop an injectable and biocompatible hydrogel which can efficiently deliver a nanocomplex of graphene oxide (GO) and vascular endothelial growth factor-165 (VEGF) pro-angiogenic gene for myocardial therapy. For the study, an efficient nonviral gene delivery system using polyethylenimine (PEI) functionalized GO nanosheets (fGO) complexed with DNAVEGF was formulated and incorporated in the low-modulus methacrylated gelatin (GelMA) hydrogel to promote controlled and localized gene therapy. It was hypothesized that the fGOVEGF/GelMA nanocomposite hydrogels can efficiently transfect myocardial tissues and induce favorable therapeutic effects without invoking cytotoxic effects. To evaluate this hypothesis, a rat model with acute myocardial infarction was used, and the therapeutic hydrogels were injected intramyocardially in the peri-infarct regions. The secreted VEGF from in vitro transfected cardiomyocytes demonstrated profound mitotic activities on endothelial cells. A significant increase in myocardial capillary density at the injected peri-infarct region and reduction in scar area were noted in the infarcted hearts with fGOVEGF/GelMA treatment compared to infarcted hearts treated with untreated sham, GelMA and DNAVEGF/GelMA groups. Furthermore, the fGOVEGF/GelMA group showed significantly higher (p < 0.05, n = 7) cardiac performance in echocardiography compared to other groups, 14 days postinjection. In addition, no significant differences were noticed between GO/GelMA and non-GO groups in the serum cytokine levels and quantitative PCR based inflammatory microRNA (miRNA) marker expressions at the injected sites. Collectively, the current findings suggest the feasibility of a combined hydrogel-based gene therapy system for ischemic heart diseases using nonviral hybrid complex of fGO and DNA.