A method for immobilization of functional proteins by chemical cross-linking of the protein of interest and uncoated iron oxide nanoparticles in the presence of Epichlorohydrin is described. As a ...result of the cross-linking, the proteins form a matrix in which the particles get entrapped. The optimum concentration of Epichlorohydrin that facilitates immobilization of protein without affecting the functional properties of the protein was determined. This method was used to immobilize several functional proteins and the development and functional activity of Protein A-magnetic nanoparticles (MNPs) is described here in detail. The Protein A-MNPs possess high binding capacity due to the increased surface area of uncoated nanoparticles and robust magnetic separation due to the absence of polymeric coating materials. Protein A-MNPs were successfully used for purification of antibodies and also for immunoprecipitation. We also immobilized enzymes such as horse radish peroxidase and esterase and found that by providing the optimum incubation time, temperature and protein to nanoparticle ratio, we can retain the activity and improve the stability of the enzyme. This study is the first demonstration that Epichlorohydrin can be used to entrap nanoparticles in a cross-linked matrix of protein without impairing the activity of immobilized protein.
We demonstrate through the use of a unique acoustically driven microfluidic extensional rheometry platform (ADMiER) that a single measurement – i.e. the time required for a liquid bridge filament ...comprising a microlitre semen sample to thin and break up under elastocapillary stresses – constitutes an appropriate proxy for quantifying the motile sperm concentration of the sample in place of computer-assisted sperm analysis (CASA) and haemocytometer measurements used in conventional semen assessment – without the need to separately resolve for individual dependencies on each sperm parameter. By benchmarking diagnostic test accuracy results of blind random bull semen samples (
$n=35$
) against OpenCASA measurements of these parameters, ADMiER is capable of predicting sperm quality to 93.7 % accuracy, 91.4 % sensitivity and 97.5 % specificity, with respect to commonly adopted veterinary industry minimum values for fertility. These results therefore highlight the potential diagnostic capability of the platform as a conceptual first step towards the development of a rapid, low-cost and portable alternative for veterinary male bovine fertility assessment.
Encapsulating enzymes within metal–organic frameworks (MOFs) constitute a promising strategy to circumvent their fragile nature in harsh environments, although deleterious molecular conformational ...changes and decreased substrate accessibility within the pores of the MOF during enzyme loading can often result in decreased catalytic activity. Herein, we report a rapid (within seconds) and facile one-pot aerosol-based method for simultaneous MOF crystallisation and enzyme encapsulation for the synthesis of ZIF-8/horseradish peroxidase (HRP) biocomposites using a high frequency acoustomicrofluidic nebulisation platform. As a consequence of the enhanced mixing and fast crystallisation afforded by the acoustic coupling into the precursors, the resultant combination of enhanced porosity, missing-linker/metal-node defect creation, pore activation and favourable conformational changes to the enzyme secondary structure allows for more efficient enzyme loading and greater enzyme–substrate interactions, and hence stronger enzyme binding within the MOF structure. Together, these culminate in a biocomposite that possesses four times greater enzymatic activity compared to that for the same material synthesized using conventional bulk-solution techniques, and endows the enzyme with increased protective effect (by approximately three to seven times) against high temperature and organic solvents, even after several exposure cycles. Such a possibility facilitates better potential for the enzyme to be recycled—an important factor given their typically high costs and the difficulty in recovering free enzymes in solution.
•A new facile and rapid one-pot acoustic method for simultaneously synthesizing metal–organic frameworks (ZIF-8) and loading horseradish peroxidase into it.•Four times greater enzymatic activity compared to the same material synthesized and loaded via convectional bulk-solution methods.•Enzyme encapsulation within the acoustically-synthesized metal–organic framework affords better protection from degradation due to high temperature and organic solvents.
Stem cell fate can be directed through the application of various external physical stimuli, enabling a controlled approach to targeted differentiation. Studies involving the use of dynamic ...mechanical cues driven by vibrational excitation to date have, however, been limited to low frequency (Hz to kHz) forcing over extended durations (typically continuous treatment for >7 days). Contrary to previous assertions that there is little benefit in applying frequencies beyond 1 kHz, we show here that high frequency MHz‐order mechanostimulation in the form of nanoscale amplitude surface reflected bulk waves are capable of triggering differentiation of human mesenchymal stem cells from various donor sources toward an osteoblast lineage, with early, short time stimuli inducing long‐term osteogenic commitment. More specifically, rapid treatments (10 min daily over 5 days) of the high frequency (10 MHz) mechanostimulation are shown to trigger significant upregulation in early osteogenic markers (RUNX2, COL1A1) and sustained increase in late markers (osteocalcin, osteopontin) through a mechanistic pathway involving piezo channel activation and Rho‐associated protein kinase signaling. Given the miniaturizability and low cost of the devices, the possibility for upscaling the platform toward practical bioreactors, to address a pressing need for more efficient stem cell differentiation technologies in the pursuit of translatable regenerative medicine strategies, is ensivaged.
Although it has previously been claimed that 1 kHz mechanostimulation is optimal for inducing osteogenic differentiation in stem cells, with little further benefit to be derived from the use of higher frequencies, it is shown and explicated here how mechanostimulation at 10 MHz not only triggers comparable levels of osteogenic differentiation, but does so much earlier, with substantially shorter treatment regimens.
Osteogenic Differentiation
In article number 2106823, Lizebona August Ambattu, Amy Gelmi, and Leslie Y. Yeo, expose mesenchymal stem cells to minutes of high frequency (10 MHz) mechanostimulation ...daily in the form of surface reflected bulk waves generated on a piezoelectric substrate, leading to their early but long‐term differentiation into osteoblasts.
Exosomes are promising disease diagnostic markers and drug delivery vehicles, although their use in practice is limited by insufficient homogeneous quantities that can be produced. We reveal that ...exposing cells to high frequency acoustic irradiation stimulates their generation without detriment to cell viability by exploiting their innate membrane repair mechanism, wherein the enhanced recruitment of calcium ions from the extracellular milieu into the cells triggers an ESCRT pathway known to orchestrate exosomal production. Given the high post-irradiation cell viabilities (≈95%), we are able to recycle the cells through iterative irradiation and post-excitation incubation steps, which facilitate high throughput production of a homogeneous population of exosomes-a particular challenge for translating exosome therapy into clinical practice. In particular, we show that approximately eight- to ten-fold enrichment in the number of exosomes produced can be achieved with just 7 cycles over 280 mins, equivalent to a yield of around 1.7-2.1-fold/h.
The endothelial junction plays a central role in regulating intravascular and interstitial tissue permeability. The ability to manipulate its integrity therefore not only facilitates an improved ...understanding of its underlying molecular mechanisms but also provides insight into potential therapeutic solutions. Herein, we explore the effects of short-duration nanometer-amplitude MHz-order mechanostimulation on interendothelial junction stability and hence the barrier capacity of endothelial monolayers. Following an initial transient in which the endothelial barrier is permeabilised due to Rho–ROCK-activated actin stress fibre formation and junction disruption typical of a cell's response to insults, we observe, quite uniquely, the integrity of the endothelial barrier to not only spontaneously recover but also to be enhanced considerably—without the need for additional stimuli or intervention. Central to this peculiar biphasic response, which has not been observed with other stimuli to date, is the role of second messenger calcium and cyclic adenosine monophosphate (cAMP) signalling. We show that intracellular Ca2+, modulated by the high frequency excitation, is responsible for activating reorganisation of the actin cytoskeleton in the barrier recovery phase, in which circumferential actin bundles are formed to stabilise the adherens junctions via a cAMP-mediated Epac1–Rap1 pathway. Despite the short-duration stimulation (8 min), the approximate 4-fold enhancement in the transendothelial electrical resistance (TEER) of endothelial cells from different tissue sources, and the corresponding reduction in paracellular permeability, was found to persist over hours. The effect can further be extended through multiple treatments without resulting in hyperpermeabilisation of the barrier, as found with prolonged use of chemical stimuli, through which only 1.1- to 1.2-fold improvement in TEER has been reported. Such an ability to regulate and enhance endothelial barrier capacity is particularly useful in the development of in vitro barrier models that more closely resemble their in vivo counterparts.
All cells possess an innate ability to respond to a range of mechanical stimuli through their complex internal machinery. This comprises various mechanosensory elements that detect these mechanical ...cues and diverse cytoskeletal structures that transmit the force to different parts of the cell, where they are transcribed into complex transcriptomic and signaling events that determine their response and fate. In contrast to
(or
) mechanostimuli primarily involving constant-force loading such as compression, tension, and shear (or forces applied at very low oscillatory frequencies (
Hz) that essentially render their effects quasi-static),
mechanostimuli comprising more complex vibrational forms (e.g., time-dependent, i.e., periodic, forcing) at higher frequencies are less well understood in comparison. We review the mechanotransductive processes associated with such acoustic forcing, typically at ultrasonic frequencies (
kHz), and discuss the various applications that arise from the cellular responses that are generated, particularly for regenerative therapeutics, such as exosome biogenesis, stem cell differentiation, and endothelial barrier modulation. Finally, we offer perspectives on the possible existence of a universal mechanism that is common across all forms of acoustically driven mechanostimuli that underscores the central role of the cell membrane as the key effector, and calcium as the dominant second messenger, in the mechanotransduction process.
•Pullulan–PEI–ascorbic acid (PPAA) was synthesized toward anti-cancer gene delivery.•PPAA was found to have collagen synthesis promoting property.•The transfection efficiency of the nanoplexes was ...unaffected in presence of serum.•Cell death by PPAA mediated p53 transfection in C6 glioma cells was 96% by FACS.
Cationized pullulan (pullulan–PEI; PP) was synthesized and further modified with an anti-oxidant molecule, ascorbic acid (PPAA) at various ratios. The nanoplexes formed at an optimum ratio of 4:1 was within a size of 150nm and had a zeta potential of 9–14mV. The nanoplexes at this ratio was used for further investigations. The cell internalization and transfection efficiency of these nanoplexes were determined in presence of serum. The internalization and transfection efficiency were found to be unaffected by the presence of fetal bovine serum. Another interesting observation was that this polymer was found to have collagen synthesis promoting property. The collagen synthesis effect of these polymers was quantified and observed that PPAA3 promoted the highest. Transfection efficiency was evaluated by assessing the p53 gene expression in C6 rat glioma cells and cell death was quantified to be 96% by flow cytometry, thus establishing the high efficacy of this polymer.
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