Bioengineering of Artificial Antigen Presenting Cells and Lymphoid Organs

The immune system protects the body against a wide range of infectious diseases and cancer by leveraging the efficiency of immune cells and lymphoid organs. Over the past decade, immune cell/organ therapies based on the manipulation, infusion, and implantation of autologous or allogeneic immune cells/organs into patients have been widely tested and have made great progress in clinical applications. Despite these advances, therapy with natural immune cells or lymphoid organs is relatively expensive and time-consuming. Alternatively, biomimetic materials and strategies have been applied to develop artificial immune cells and lymphoid organs, which have attracted considerable attentions. In this review, we survey the latest studies on engineering biomimetic materials for immunotherapy, focusing on the perspectives of bioengineering artificial antigen presenting cells and lymphoid organs. The opportunities and challenges of this field are also discussed

Methods for immobilizing receptors in microfluidic devices: A review

In this review article, we discuss state-of-the-art methods for immobilizing functional receptors in microfluidic devices. Strategies used to immobilize receptors in such devices are essential for the development of specific, sensitive (bio)chemical assays that can be used for a wide range of applications. In the first section, we review the principles and the chemistry of immobilization techniques that are the most commonly used in microfluidics. We afterward describe immobilization methods on static surfaces from microchannel surfaces to electrode surfaces with a particular attention to opportunities offered by hydrogel surfaces. Finally, we discuss immobilization methods on mobile surfaces with an emphasis on both magnetic and non-magnetic microbeads, and finally, we highlight recent developments of new types of mobile supports

Bio-sample environment manipulation using advanced microscopy techniques

2010/2011Under physiological conditions in the brain, molecules are released with high spatial and temporal resolution. A lot of efforts have been done in the last years in order to develop techniques that mimic this situation. Among them, we mention the use of micropipettes for the ejection of fluids, the use of AFM (Atomic Force Microscopy), microfluidic devices and optical manipulation. The latter approach exploits light to manipulate the samples, e.g. to create transient pores in the cell membrane or to move small objects carrying a stimulus. This Thesis concerns with the development of new techniques for the local delivery of molecules based on optical manipulation technologies, and in particular on optical tweezers. Sub-micrometer particles in a compact trap, such as the single-beam gradient or optical tweezers, can be localized within a small fraction of a wavelength of light or moved over long distances of many centimeters without any mechanical contact. A three-dimensional trap is simply created by focusing a laser beam through a microscope objective with high numerical aperture. We studied three types of vectors for local delivery of molecules, which can be optically manipulated: microbeads, micron-sized liposomes and Quantum dots (Qdots). Silica microbeads can be covalently functionalized on their surface with the protein of interest and placed in contact with the desired part of a cell. In order to validate the technique, we functionalized beads with a secretory molecule, the neurotrophin Brain-derived neurotrophic factor (BDNF). BDNF is a key regulator of neuronal development and plasticity. We showed that single BDNF-coated microbeads can be extracted with optical tweezers from small reservoirs and positioned with submicrometric precision to specific sites on the dendrites of cultured hippocampal neurons. Localized contact of microbeads functionalized with BDNF induced focal increase of Calcium signaling in the stimulated dendrite, specific activation of the TrkB receptor pathway and influenced the development of growth cones. Remarkably, a single BDNF-coated bead positioned on a dendrite was found to be enough for TrkB phosphorylation, an efficient and long-lasting activation of Calcium signaling in the soma, and c-Fos signaling in the nucleus, comparable to bath stimulation conditions. Moreover, since BDNF is covalently cross-linked to the bead surface we could demonstrate that activation of some of the TrkB receptor pathway does not necessarily require BDNF endocytosis. In the case of liposomes, the molecules of interest were encapsulated within their lumen. Single liposomes were trapped and transported by means of optical tweezers to the site of stimulation on cultured neurons. Finally, the release of liposome content was induced by application of UV-pulses that broke the liposome membrane. In order to test the effect of the UV-induced release, liposomes with a diameter ranging from 1 to 10 μm (fL to pL volumes), were filled with KCl and tested on neuronal cells. Neuronal cultures, loaded with Ca2+ dye, were monitored by imaging intracellular Ca2+. An efficient release from the liposomes was demonstrated by detectable Calcium signals, indicating induced depolarization of the neuronal cells by KCl. Afterwards, this technique was used to address a biological issue, that is the effect of two proteins (Semaphorin 3A and Netrin-1) on growth cones. The growth cone is an intracellular apparatus located at the tip of the neurite of developing neurons. Its motility governs axonal path-finding and the construction of neuronal networks. Growth cones are highly dynamic structures that respond to external stimuli turning towards or away from the chemical gradient. We were able to demonstrate an attractive effect of Netrin-1 on the growth cones of primary hippocampal neurons. On the contrary, Semaphorin 3A showed a repellant behavior. To correlate the high resolution of vector manipulation with high resolution of imaging we used STimulated Emission Depletion (STED) to investigate the intimate organization of two main cytoskeleton components: actin and tubulin filaments. STED microscopy allowed imaging of actin bundles in the filopodia and organized network in lamellipodia with un-precedent resolution, beyond the diffraction barrier. Lastly, we used liposomes to encapsulate Quantum dots. Qdots are bright and photostable nanocrystals. Due to their small size, similar to that of proteins, Qdots may be endocyted along the receptor-mediated endocytosis pathway, when they are functionalized with the appropriate ligand. As case study we considered the BDNF-TrkB endocytotic pathway. We optimized the protocol for the direct binding of BDNF to Qdots and we demonstrated the possibility of encapsulating and releasing them from liposomes. Concluding, two different approaches for local stimulation of neurons, based on optical manipulation of microvectors, were presented and validated in this thesis. Indirect optical manipulation of nanovectors (Qdots) encapsulated in liposomes has been demonstrated as well. The techniques were then successfully applied to address some biological issues, that in turn required the optimization of other imaging tools (super resolution microscopy and Qdots).XXIV Ciclo198

Biosensors to Monitor Cell Activity in 3D Hydrogel-Based Tissue Models

Three-dimensional (3D) culture models have gained relevant interest in tissue engineering and drug discovery owing to their suitability to reproduce in vitro some key aspects of human tissues and to provide predictive information for in vivo tests. In this context, the use of hydrogels as artificial extracellular matrices is of paramount relevance, since they allow closer recapitulation of (patho)physiological features of human tissues. However, most of the analyses aimed at characterizing these models are based on time-consuming and endpoint assays, which can provide only static and limited data on cellular behavior. On the other hand, biosensing systems could be adopted to measure on-line cellular activity, as currently performed in bi-dimensional, i.e., monolayer, cell culture systems; however, their translation and integration within 3D hydrogel-based systems is not straight forward, due to the geometry and materials properties of these advanced cell culturing approaches. Therefore, researchers have adopted different strategies, through the development of biochemical, electrochemical and optical sensors, but challenges still remain in employing these devices. In this review, after examining recent advances in adapting existing biosensors from traditional cell monolayers to polymeric 3D cells cultures, we will focus on novel designs and outcomes of a range of biosensors specifically developed to provide real-time analysis of hydrogel-based cultures

Ultrasonic assisted layer-by-layer assembly for stable nanocolloids of Curcumin and Paclitaxel

Researchers have been trying to fight cancer with synthesis of new bioactive compounds but many of these novel drugs have low solubility in water and it is difficult to deliver them into a patient\u27s body. One way of solving this particular problem is to use nanoscale drug delivery systems. In this dissertation, we describe using an ultrasonic assisted layer-by-layer encapsulation process to prepare anti-cancer drugs with 50∼200 nm particle size with designed coating to achieve sustained release and target delivery. Two methods for systematic manufacture of low solubility anti-cancer drug nanoparticles were proposed: I) Top-down approach to breakdown larger drug crystals into nano-size particles. 2) Bottom-up approach to fabricate nano size drug crystals from a drug solution

Concentration of Phosphorylated Proteins Using Modified PMMA Microanalytical Devices

This work describes the application of PMMA-based microanalytical devices for the affinity-type preconcentration of posttranslational modified proteins (PTMs). The choice of poly(methyl methacrylate), PMMA, is based on its biocompatibility, its functional methyl ester group for potential modification, and its extensive applications to create biological microelectromechanical systems (BioMEMS). Developing methodologies for preconcentration of PTMs is important for cancer diagnosis due to PTMs’ influence in the regulatory mechanism underlying the early stage of apoptosis or regulated cell death. Towards this goal, nitroavidin which can reversibly binds to biotin (and biotinylated proteins), was prepared using reported procedure and was characterized using several techniques such as UV-Visible spectroscopy, sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), and Western blot experiments. UV-Visible spectroscopy experiments showed reversible binding of nitroavidin towards the biotin analogue 2-(4’-hydroxyazobenzene) benzoic acid, HABA. From mass spectrometry studies, nitrotyrosine was confirmed to be present in the prepared nitroavidin through an observed photoinduced chemical fragmentation. SPR experiments revealed decrease in binding of nitroavidin towards biotinylated proteins (the equilibrium dissociation constant obtained for the biotin−nitroavidin interactions is higher, KD = 4 x 10–6 M, than biotin-avidin interactions, KD = 1 x 10–13 M). Also, there was an observed efficiency of 23 ± 1% for the capture process of biotinylated proteins on nitroavidin−functionalized PMMA open microchannels, while high capture efficiency (96 ± 0.5%) for bound biotinylated proteins were observed on PMMA microchannels with fabricated microposts. To further improve the efficiency of capture and release processes, PMMA ultra-high-aspect-ratio nanostructures (UHRANs) were employed to provide higher surface-to-volume reactor bed. These PMMA UHRANs were fabricated in our group using previously reported template-based anodization. PMMA nanopillars or nanoposts were developed using photopolymerization between the methyl methacrylate monomer and initiator, while PMMA nanotubes were fabricated using PMMA melt. These nanostructures were UV-modified to promote formation of surface carboxylic acids (pendant −COOH). The confirmation of surface –COOH functionalization on these surfaces was achieved using different surface labeling techniques such as thallium (I) ethoxide and sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate (sulfo-SDTB) and were determined using several techniques such as confocal fluorescence microscopy, UV-Visible spectroscopy, AFM, SEM, and XPS

Theranostic Nanoparticles and Their Spectrum in Cancer

Nanoparticles offer a lot of advantageous backgrounds for many applications due to their physical, chemical and biological properties. Their different composition (metals, lipids, polymers, peptides) and shapes (spheres, rods, pyramids, flowers and so on) are influenced by the synthesis methods and functionalization procedures. However, in the medical field, researchers focus on the biocompatibility and biodegradability of the nanoparticles in their attempts for a targeted therapy in which the nanocarriers need to bypass certain biological barriers. Moreover, the increased interest in molecular imaging has brought nanoparticles in the spotlight for their applications in two distinct directions: therapy and diagnosis. Furthermore, recent advances in nanoparticle designs have introduced novel nano-objects suitable as both detection and delivery systems at the same time, thus providing theranostic applications

RECENT ADVANCES IN MOLECULAR MEDICINE AND TRANSLATIONAL RESEARCH

ABSTRACT BOO

Miniaturized single-cell analyses for biomedical applications

Numerous diseases affecting living beings have as a cause a modification of gene expression resulting in anomalous expression of proteins in up- or down-regulated fashion, with altered functions or expression of proteins that are not present in the cell in a normal situation. Among the well-known examples is cancer, where a series of DNA damages turn cells out of control of the rest of the body, from which they do not receive or follow control signals. To have a better understanding of the mechanism of these diseases, in particular the genetic diversity existing in a single affected tissue, it is necessary to be able to perform single-cell analyses that unveil the subpopulations of diseased cells leading to adequate medical treatment. In the course of this thesis, we report on the development of single-cell analysis methods which are of interest for medical applications. The first part focuses on the investigation of the viscoelastic properties of cell membranes by observing the back-relaxation of plasma membrane nanotubes which have been pulled out of a cell by an optical tweezer. Applied to the investigation of the viscoelastic properties of individual tumor cells taken from patients, we could show that this method can distinguish the state of progress of skin melanoma. In the second part, we used beads comprising a chemically modified surface to capture specifically one or several proteins inside single-cells. After extraction out of the cell, the affinity bead is transferred in a microfluidic stream of a fluorescently labeled antibody to detect and quantify the protein(s) of interest. The extraction and detection procedures occur inside a microfluidic platform to allow future automatization of the process. The last chapter focuses on the use of cell-derived extracellular vesicles (EVs) as diagnostic and therapeutic agents. With this goal in mind, we explore the potential of EVs as carriers to transfer genetic material into cells. To demonstrate the feasibility of this approach, we encapsulate EVs inside a giant unilamellar vesicle and release their cargo in a time- and space-controlled manner. This method could have therapeutic applications using a patient's self-EVs for gene therapy

Nanoparticles in polyelectrolyte multilayer layer-by-layer (LbL) films and capsules : key enabling components of hybrid coatings

Originally regarded as auxiliary additives, nanoparticles have become important constituents of polyelectrolyte multilayers. They represent the key components to enhance mechanical properties, enable activation by laser light or ultrasound, construct anisotropic and multicompartment structures, and facilitate the development of novel sensors and movable particles. Here, we discuss an increasingly important role of inorganic nanoparticles in the layer-by-layer assembly—effectively leading to the construction of the so-called hybrid coatings. The principles of assembly are discussed together with the properties of nanoparticles and layer-by-layer polymeric assembly essential in building hybrid coatings. Applications and emerging trends in development of such novel materials are also identified