Enzyme functionalized microgels enable precise regulation of dissolved oxygen and anaerobe culture.

Anaerobes are a major constituent of the gut microbiome and profoundly influence the overall health of humans. However, the lack of a simple, cost-effective, and scalable system that mimics the anaerobic conditions of the human gut is hindering research on the gut microbiome and the development of therapeutics. Here, we address this gap by using glucose oxidase and catalase containing gelatin microparticles (GOx-CAT-GMPs) to precisely regulate dissolved oxygen concentration [O2] via GOx-mediated consumption of oxygen. Fluorescence images generated using conjugated polymer afterglow nanoparticles showed that [O2] can be tuned from 257.9 â€‹± â€‹6.2 to 0.0 â€‹± â€‹4.0 â€‹µM using GOx-CAT-GMPs. Moreover, when the obligate anaerobe Bacteroides thetaiotaomicron was inoculated in media containing GOx-CAT-GMPs, bacterial growth under ambient oxygen was comparable to control conditions in an anaerobic chamber (5.4 â€‹× â€‹105 and 8.8 â€‹× â€‹105 colony forming units mL-1, respectively). Finally, incorporating GOx-CAT-GMPs into a bioreactor that permitted continuous radial diffusion of oxygen and glucose generated a gut-mimetic [O2] gradient of 132.4 â€‹± â€‹2.6 â€‹µM in the outer ring of the reactor to 7.9 â€‹± â€‹1.7 â€‹µM at the core. Collectively, these results indicate that GOx-CAT-GMPs are highly effective oxygen-regulating materials. These materials can potentially be leveraged to advance gut microbiome research and fecal microbiota transplantation, particularly in low-resource settings.

Modification of PEGylated enzyme with glutaraldehyde can enhance stability while avoiding intermolecular crosslinking.

We demonstrate an enzyme stabilization approach whereby a model enzyme is PEGylated, followed by controlled chemical modification with glutaraldehyde. Using this stabilization strategy, size increases and aggregation due to intermolecular crosslinking are avoided. Immediately following synthesis, the PEGylated enzyme with and without glutaraldehyde modification possessed specific activities of 372.9 ± 20.68 U/mg and 373.9 ± 15.14 U/mg, respectively (vs. 317.7 ± 19.31 U/mg for the native enzyme). The glutaraldehyde-modified PEGylated enzyme retains 73% original activity after 4 weeks at 37 °C (vs. 2% retention for control).

In vitro evaluation of chondrosarcoma cells and canine chondrocytes on layer-by-layer (LbL) self-assembled multilayer nanofilms.

Short-term cell-substrate interactions of two secondary chondrocyte cell lines (human chondrosarcoma cells, canine chondrocytes) with layer-by-layer self-assembled multilayer nanofilms were investigated for a better understanding of cellular-behaviour dependence on a number of nanofilm layers. Cell-substrate interactions were studied on polyelectrolyte multilayer nanofilms (PMNs) of eleven different biomaterials. Surface characterization of PMNs performed using AFM showed increasing surface roughness with increasing number of layers for most of the biomaterials. LDH-L and MTT assays were performed on chondrosarcoma cells and canine chondrocytes, respectively. A major observation was that 10-bilayer nanofilms exhibited lesser cytotoxicity towards human chondrosarcoma cells than their 5-bilayer counterparts. In the case of canine chondrocytes, BSA enhanced cell metabolic activity with increasing number of layers, underscoring the importance of the multilayer nanofilm architecture on cellular behaviour.

Polyelectrolyte-coated alginate microspheres as drug delivery carriers for dexamethasone release.

Alginate microspheres loaded with dexamethasone were prepared by the droplet generator technique. Important parameters affecting drug release, including initial drug content, the type of polyelectrolyte coating, and a combination of different ratios of coated and uncoated microspheres were investigated to achieve in vitro dexamethasone delivery with approximately zero order release kinetics, releasing up to 100% of entrapped drug within 1 month, wherein dexamethasone released at a steady rate of 4.83 microg/day after an initial burst release period. These findings imply that these polyelectrolyte-coated alginate microspheres show promise as release systems to improve biocompatibility and prolong lifetime of implantable glucose sensors.

Application of self-assembled ultra-thin film coatings to stabilize macromolecule encapsulation in alginate microspheres.

Alginate-based hydrogels have several unique properties that have enabled them to be used as a matrix for the entrapment of a variety of enzymes, proteins and cells for applications in bioprocessing, drug delivery and chemical sensing. However, control over release rates or, in some cases, stable encapsulation remains a difficult goal, especially for small particles with high surface-area-to-volume ratios. In this work, the potential to limit diffusion of macromolecules embedded in alginate spheres with nanofilm coatings was assessed. Alginate microspheres were fabricated using an emulsification process with high surfactant concentration to form beads in the size range of 2-10 microm. Using calcium chloride for ionotropic gelation, dextran was encapsulated in the gel phase by mixing with the alginate in solution. The exterior surface was then modified with polyelectrolyte coatings using the layer-by-layer self assembly technique. Leaching studies to assess retention of dextran with varying molecular weights confirmed that the application of multi-layer thin films to the alginate microspheres was effective in reducing leaching rate and total loss of the encapsulated material from the microspheres. For the best case, the rate of release for dextran of 2,000,000 Dalton molecular weight decreased from 1% h(-1) in bare microspheres to 0.1% h(-1) in polyelectrolyte-coated microspheres. The effectiveness of nanofilms reducing loss of the encapsulated macromolecules was found to vary between different polycation materials used. These studies support the feasibility of using these microsystems for development of long-term stable encapsulated systems, such as implantable biosensors.

Comparison of selective attachment and growth of smooth muscle cells on gelatin- and fibronectin-coated micropatterns.

Tissue engineering research has been on going for many years, people are making all the effort to explore the cell functions in cellular level and even in molecular level. Making the cells functional in an in vitro environment is a preliminary goal for the implantation and repair of complicated tissues/organs. Fabricating artificial ECM to mimic the in vivo environment is an essential approach in tissue engineering. The work in this paper is to study how rat aorta smooth muscle cells (RASMCs) behave in two engineered cell culture scaffolds: gelatin- and fibronectin (FN)-coated micropatterns. The investigation on the initial attachment and further growth of SMCs cultured on gelatin- and FN-coated micropatterns was addressed. This study focused on both the characterization of gelatin and fibronectin assembly properties and cell responses to these two protein-coated micropatterns. Thin film patterns with gelatin and fibronectin coatings were fabricated on microscope glass slides using photolithography, electrostatic layer-by-layer self-assembly and lift-off (LbL-LO) technologies. In this work, the scaffolds were built up by commonly used polyelectrolyte materials and proteins through LbL process, containing cationic poly(diallyldimethylammonium chloride) (PDDA), poly(allylamine hydrochloride) (PAH), anionic poly(sodium 4-styrenesulfonate) (PSS), gelatin and fibronectin. The resulting polyelectrolyte thin films were characterized by contact angle (CA), quartz crystal microbalance (QCM), atomic force microscopy (AFM), and fluorescence microscopy. CA measurement shows the consistent hydrophylicity of gelatin surfaces in different number of layers with LbL deposition method. Different from our previous QCM measurement of gelatin, fibronectin does not show high electrostatic attraction to either positively or negatively charged polyelectrolytes, although it can be weakly assembled to both polyelectrolyte surfaces. AFM images show Gelatin- and FN-coated micropatterns are around 50-60 nm thick. RASMCs were cultured on these gelatin- and FN-coated micropatterns. It was observed that, for the cells cultured on gelatin-coated micropatterns, they initially landed on the gelatin-coated surface, not on the PDDA-coated surface in between. But further growth of the cells was affected by the shape of the patterns: strip pattern limited cell growth beyond the patterns, but square patterns could not. While, it was found interestingly, for the cells cultured on FN-coated micropatterns, SMCs initially landed on PDDA-coated surface, and then migrated to FN-coated both square and strip patterns. These findings indicate that both gelatin and fibronectin are adhesive proteins, but they have different effects on the initial attachment and later growth for SMCs.

Micropatterning of nanoengineered surfaces to study neuronal cell attachment in vitro.

Methods for producing protein patterns with defined spatial arrangement and micro- and nanoscale features are important for studying cellular-level interactions, including basic cell-cell communications, cell signaling, and mechanisms of drug action. Toward this end, a straightforward, versatile procedure for fabricating micropatterns of bioactive nanofilm coatings as multifunctional biological testbeds is demonstrated. The method, based on a combination of photolithography and layer-by-layer self-assembly (LbL), allows for precise construction of nanocomposite films of potentially complex architecture, and patterning of these films on substrates using a modified lift-off (LO) procedure. As a first step in evaluating nanostructures made with this process, "comparison chips," comprising two coexisting regions of square patterns with relevant proteins/polypeptides on a single substrate, were fabricated with poly(diallyldimethylammonium chloride) (PDDA) as a cell-repellent background. Using neuronal cells as a model biological system, comparison chips were produced with secreted phospholipase A2 (sPLA2), a known membrane-active enzyme for neurons, for direct comparison with gelatin, poly-l-lysine (PLL), or bovine serum albumin (BSA). Fluorescence microscopy, surface profilometry, and atomic force microscopy techniques were used to evaluate the structural properties of the patterns on these chips and show that the patterning technique was successful. Preliminary cell culture studies show that neurons respond and bind specifically to the sPLA2 enzyme embedded in the polyelectrolyte thin films and present as the outermost layer. These findings point to the potential for this method to be applied in developing test substrates for a broad array of studies aimed at identifying important biological structure-function relationships.

RET nanobiosensors using affinity of an apo-enzyme toward its substrate.

Fluorescent biosensors can be highly specific and sensitive, and may be engineered as implantable devices for metabolic monitoring. Commonly-used systems for glucose monitoring based on resonance energy transfer (RET) and competitive binding involve Concanavalin A (Con A), which is known to be toxic, and has problems of aggregation and irreversible binding. This work presents an improved RET assay wherein Con A was replaced by apo-glucose oxidase (apo-GOx). Fluorescence measurements confirm that the apo-GOx/dextran complexes are highly sensitive to glucose, observed as an increase in the donor peak relative to acceptor with the stepwise addition of glucose solution. The assay showed strong signals and excellent repeatability, with a sensitivity of 9x10/sup -4/ (ratio units)/mM over the range of 0-90 mM glucose. If properly encapsulated, these sensors can potentially be used for in vivo sensing without the concern of toxicity associated with Con A.

Using electrostatic self-assembled gradient nanosensor phantoms for calibration of an optical intrinsic signal imaging system.

Wide-field optical intrinsic signal (OIS) imaging has been used in functional cortex mapping for its excellent spatial resolution. To compensate for its low temporal resolution, extrinsic dye signals have been introduced. Fluorescence spectroscopy in the form of nanoengineered sensors has also been used to detect biochemical signals of molecular interactions. In this paper, oxygen-sensitive dye Ru(dpp)/sub 3//sup 2+/ was immobilized into nano-sized spheres by electrostatic layer-by-layer (LbL) self-assembly, and then deposited on glass slides as intensity gradients. By comparing gradient ratios and ratios of function dye Ru(dpp)/sub 3//sup 2+/ and reference dye between epi-fluorescence microscope and an OIS imaging system, the feasibility and efficiency of nano-sized oxygen sensors in OIS imaging were studied.