1,249 research outputs found
Microfluidics for Production of Particles : Mechanism, Methodology, and Applications
In the past two decades, microfluidics-based particle production is widely applied for multiple biological usages. Compared to conventional bulk methods, microfluidic-assisted particle production shows significant advantages, such as narrower particle size distribution, higher reproducibility, improved encapsulation efficiency, and enhanced scaling-up potency. Herein, an overview of the recent progress of the microfluidics technology for nano-, microparticles or droplet fabrication, and their biological applications is provided. For both nano-, microparticles/droplets, the previously established mechanisms behind particle production via microfluidics and some typical examples during the past five years are discussed. The emerging interdisciplinary technologies based on microfluidics that have produced microparticles or droplets for cellular analysis and artificial cells fabrication are summarized. The potential drawbacks and future perspectives are also briefly discussed.Peer reviewe
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Submicroscopic characterization of biopolymer networks in solution by Thermal Noise Imaging
Biopolymer networks display a wide range of interesting mechanical properties that are essential for living organisms. For example, a highly nonlinear elastic response to strain gives biopolymer networks the ability to comply with small stresses but to resist large ones. These macroscopic mechanical properties have their origin in the properties of the individual filaments and their connectedness, like cross-linking geometry and pore size distribution. While the macroscopic properties of biopolymer networks have been extensively studied, there has been a lack of experimental techniques that can simultaneously determine mechanical and architectural properties of networks in situ with single filament resolution. This work introduces Thermal Noise Imaging (TNI) as a novel quantitative method to address these issues. TNI is a three-dimensional scanning probe technique that utilizes the confined thermal motion of an optically trapped particle as a three-dimensional, noninvasive scanner for soft, biological material. Using a photonic force microscope (PFM) custom built for this research, the position of the probe can be detected with nanometer precision and megahertz bandwidth. Two sets of single molecule experiments are described that demonstrate the microscope's exceptional precision and stability. Micrometer scale thermal noise images inside a collagen network are shown and quantitative information about cross-linking geometry is extracted from the data. Further, by imaging microtubules grafted to a support it is shown that the acquired data yield information about the transversal fluctuations of the imaged fibers and about fiber elasticity. These results pave the way for an investigation of force distributions inside biopolymer networks on the single filament level.Physic
Evaluating cell-mimicking giant unilamellar vesicles as simplified biological models using single molecule methods
One of the main drivers within the field of bottom-up synthetic biology is to develop artificial chemical machines, perhaps even living systems, that have programmable functionality. Bottom-up methods take an engineering approach to biology, with multiple downstream applications. Such applications that require very specific quantities of product e.g., antibodies, drug, or vaccines, necessitate the design of
vesicles with total and precise control of a vesicle’s molecular machinery.
There exists a wide range of toolkits to produce and engineer artificial cells with an ever-increasing array of functionality and capability. However, little attention has been given to the quality control of vesicle production; so, to date, there is a paucity of techniques that are able to measure their molecular constituents precisely upon formation and with absolute quantification. The work outlined here presents the
development of a microfluidic-based methodology that is able to characterise artificial cells at the single vesicle level with single-molecule resolution.
High content fluorescence microscopy was used to assess the properties of Giant unilamellar vesicles (GUVs) and their statistics at the population level. Since this technique is semi-quantitative, the relative concentration of protein across the GUV population was determined however the absolute concentration of protein within each GUV was not.
To overcome this, a lab-on-a-chip approach was taken to determine the precise number of biomolecules within each GUV and across the population of GUVs. The pulldown-5
array single-molecule high-throughput (PASH) chip was developed and capable of determining the encapsulation efficiency of protein within GUVs produced by phase transfer of an inverted emulsion. Using this approach, it was possible to determine the variability of the encapsulation efficiency between GUVs across a population as well as between different populations while testing batch-to-batch variation. The encapsulation efficiency measured to be 11.4 ± 6.2% across all tested parameters. This has consequences for the use of GUVs as precise biological models as well as for their development in a variety of biotherapeutic applications.
To determine whether GUVs with specific concentrations of biomolecules could be produced by phase transfer, changes in the concentration of the seeding materials and reagents were investigated;
while inefficiencies in encapsulation could be overcome, the variation in concentration could not. The results presented in this thesis help to understand the limitation of the phase transfer technique applied to systems biology research. In biological systems, measuring changes in gene expression at the transcriptomic and proteomic level is important.
When used to develop simplified models of protein expression, these results indicate that vesicles produced using phase transfer may only be confidently used to produce 10-fold changes in encapsulant protein. It is possible to distinguish different GUV populations with a precision down to a two-fold change in encapsulant protein but with significant population overlap. The understanding of the limitations of encapsulation efficiency provides insight into the potential relevance of such systems for a variety of therapeutic applications such as smart drug delivery.Open Acces
Impact of substrate topology, chemical stimuli and Janus nanoparticles on cellular properties
Cellular behavior is influenced by many biochemical but also physical factors in the direct cellular environment. Thereby, cells not only react to external cues, the interaction between cells and their environment is also dependent on the properties of the cell itself. The endocytosis of nanoparticles for example depends on the intermolecular forces between plasma membrane and particle as well as on the mechanical properties of the membrane. In the first part of this thesis I focus on the interaction between inorganic Janus nanoparticles, a new type of nanomaterials, which possess amphiphilic properties, and model membranes. In coarse grain simulations it has been demostrated that incubation of membranes with these particles lead either to pore formation in the lipid bilayer or to tubulation and vesiculation by long-range attractive interaction between particles bound to the membrane. Conducting surface plasmon resonance spectroscopy experiments I show that the binding energy of the used inorganic Janus particles to a solid supported monolayer could be sufficient to induce tubulation of tension-free membranes but is to small to provide the energy necessary to form a vesicle. This result is confirmed by fluorescence microscopic examination of giant unilamellar vesicles serving as a model system for the plasmamembrane, which were treated with Janus particles. Vesicles incubated with Janus particles show inwards directed membrane tubes, while incubation of vesicles with isotropic control particles had no effect on the membrane or could be attributed to an osmotic gradient. However, uptake experiments into living cells and cytotoxicity assays show no obvious difference between spherical particles and Janus particles, which hints for a negligible contribution of nanoparticle-induced tubulation or vesiculation to cellular uptake of nanoparticles and cytotoxicity.
On the one hand mechanical properties of the cell influence the interaction between the cell and its environment. On the other hand, mechanical properties of cells change in response to environmental cues. Therefore, in the next part, atomic force microscopy-based microrheology is used to measure frequency-dependent mechanical properties of cells in different conditions. Fixation of cells with different chemical fixatives and transformation of epithelial cells to mesenchymal cells lead to more solid-like mechanical properties, while interaction with the actin cytoskeleton lead to more fluid-like properties. A comparison between malignant cells and non-malignant cells shows that malignant cells are more fluid-like compared to their non-malignant counterparts. Furthermore, the influence of substrate topology on cellular mechanics and cytoskeletal arrangement is examined. Changing physical properties of the substrate such as stiffness or topography has been shown to affect plenty of cellular processes like migration, proliferation, morphology or differentiation. Here, I investigate the impact of porous substrates on cellular morphology, cytoskeletal organization and elasticity in the context of confluent epithelial monolayers. I found that cells eventually self-organize to match the geometry of the pore pattern and remodel their actin cytoskeleton to reinforce their adhesion zone. Cells fluidize with increasing pore size up to 2 µm but eventually become stiffer if grown on very large pores up to 5 µm.
The adhesion of cells to substrates is further researched by application of metal-induced energy transfer fluorescence lifetime imaging, which is used for the first time for this purpose. The fluorescence lifetime of a fluorophore in proximity to a metal layer is a function of the distance between fluorophore and metal layer. Applying a quantitative model of this interaction facilitates locating the fluorophore with nanometer precision in the axial direction up to 200 nm above the metal layer. By staining of the plasmamembrane I was able to image to basal membrane of three different cell lines and follow spreading of cells with high axial resolution. The introduced method is not restricted to measurement of cell/substrate distance and can be used for applications, which necessitate axial nanometer resolution in a range up to 200 nm
Multimodal nanoparticles for quantitative imaging
The scope of this thesis is research related to applications of nanoparticles in quantitative preclinical imaging. Nanoparticles are a versatile platform that can interact with biological systems at many different length scales and can furthermore be rendered visible for basically any medical imaging technique by modification with appropriate contrast providing moieties. Thus, nanoparticles can be used as a new class of contrast agents for basically all imaging modalities, e.g. as long circulating blood pool agents in CT, or as MRI contrast agents. Vice versa, non-invasive imaging techniques can be used to for example follow the biodistribution of nanoparticles in vivo and apply nanoparticles as a tool to investigate biological processes related to disease processes. Dual modal imaging applying multifunctional and dual-labeled nanoparticles offer new approaches to quantitative imaging, giving new insights into technology development on one side and biological read-outs on the other. For instance, quantification of biological processes that lie at the basis in the development of disease may lead to earlier detection and better disease diagnosis and treatment. Results and concepts presented in this thesis have high impact on therapeutic application of nanoparticles, for example when they are used as drug delivery systems. Imaging can provide valuable information on drug delivery and biodistribution in a quantitative manner, which may help in development of new therapeutic strategies. Nanoparticles are promising structures for quantitative imaging. Its surface can be utilized to attach almost any desirable molecule. Nanoparticles are relatively large in size (typically 10-200 nm) and can for instance accommodate a high payload of contrast agent per particle on its surface or inside the particle, thereby increasing the signal/particle by five orders of magnitude. In addition, also multiple imaging probes for different imaging modalities can be incorporated providing a double read-out. For the understanding of biological processes, targeting ligands such as antibodies, proteins and peptides can be attached to its surface. Despite the wide variety of possibilities with nanoparticles, they have hardly been studies for quantitative imaging purposes. Therefore, the aim of the research described in this thesis was to explore and develop several nanoparticles for quantitative imaging by using existing or newly developed imaging techniques. Chapter 1 gives a general introduction in the field of nanoparticles for quantitative imaging. Several imaging techniques are described such as CT, Spectral CT, SPECT and MRI, and how nanoparticles can play an important role in research. Chapter 2 describes the development of a novel nanoparticulate CT contrast agent. Several amphiphilic molecules were investigated in this chapter in the combination with different iodinated oils for their influence on the size stability of the nanoparticles. In Chapter 3, the dose dependent biodistribution of the nanoparticles is investigated as well as strategies to vary the biodistribution. The effect of a co-injection with liposomes and soy bean oil emulsions was investigated using CT, SPECT and ¿-counting. The final optimized blood pool CT contrast agent from chapter 2 and 3 can be used for qualitative imaging in CT as well as in quantitative imaging in Spectral CT. Chapter 4 describes the very first use of this novel imaging technique Spectral CT in quantitative imaging. For this, the nanoparticles of chapter 2 were extended to a multimodal nanoparticulate contrast agent for CT, Spectral CT and SPECT. Spectral CT quantification was compared to quantification using SPECT and ICP-MS to demonstrate the correlations and accuracy of the techniques. In Chapter 5, the development is described of a dual-isotope SPECT imaging protocol as a tool for pre-clinical testing of new molecular imaging tracers. New molecular targeting probes are consistently investigated as a tool to enable target specific binding of nanoparticles to cellular surfaces of interest. Dual-isotope SPECT can be used in which the biodistribution of two different ligands labelled with two different radionuclides can be studied in the same animal, thereby excluding experimental and physiological inter-animal variations. The developed dual-isotope protocol was tested using a known angiogenesis specific ligand (cRGD peptide) in comparison to a potential non-specific control (cRAD peptide). Chapter 6 describes the use of a multimodal radiolabeled paramagnetic liposomal contrast agent that allows simultaneous imaging with SPECT and MRI. A double read-out is then possible and demonstrates the additional advantages of the combination of the two techniques. SPECT can for instance quantify the nanoparticle concentration and MRI can spatially localize the nanoparticle. The combination however gives an indirect read-out of the water exchange, which in return reveals insights in biological processes and environments. Chapter 7 describes a study that investigates the use of nanoparticles in the quantitative imaging technique fluorine MRI. The use of gadolinium-complexes as signal modulating ingredients into the nanoparticle formulation has emerged as a promising approach towards improvement of the fluorine signal. Paramagnetic lipids based on gadolinium complexes can be incorporated to increase the 19F MR signal per particle. Here, 3 different paramagnetic lipids were investigated on its influence at five different field strengths. This furthermore also provides important insights in the dependency of the magnetic field on fluorine signal intensity. The final Chapter 8 describes the future perspectives of the use of multimodal nanoparticles for quantitative imaging
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