Multi-mode atomic force microscope as a versatile tool for bionanotechnology

The kernel of this dissertation is multi-mode atomic force microscopy (AFM) which is a useful and powerful tool for characterizing and analyzing samples of nano- or micro size. Various modes can satisfy specified requirements according to different samples, i.e., topography, surface electrostatic potential, magnetic domain visual observation, single molecular force analysis and a novel real-time monitoring cell viability system based on modification of AFM. No matter whether samples are in air or in liquid, topological image can be realized. Hence, the flexibility makes AFM a universal tool for exploring the biological nano-world. The subjects consist of different working modes towards biological applications. Firstly, topography is aimed at quantitative analysis of cellular morphology and surface changes, which are effected by uptake of nanoparticles. In the case of concentration-dependent experiments, the volume and number of filopodia is calculated by analyzing topological images of AFM. It is verified that cellular morphology plays an important role for quantitative indicating of harmful effects of NPs to cells. In addition, the roughness of the cellular surface which derives from disruption of cell membrane integrity, when the cells internalized magnetic NPs subjected to a rotating magnetic field, is evaluated for exploring magneto-cell-poration and magneto-cellanalysis. Secondly, single molecule force microscopy is aimed at quantitative analysis of elasticity of gold nanoparticles (Au NPs), which are coated with polyethylene glycol (PEG), whereby the diameter of the gold cores as well as the thickness of the shell of PEG was varied. A conical tip indent into single NP and then Sneddon’s equation is employed for calculating the elasticity, which serves as one of the basic physicochemical parameters having effect on structural and functional cell parameters. Thirdly, magnetic force microscopy is aimed at qualitative visual observation of magnetic domains of the sample, which is a multifunctional co-loading NP with anti-drug tetradine and superparamagnetic iron dioxide (Fe3O4) NPs. The magnetic domains of co-loading NPs, which is reflected in phase section, can present magnetic profile which is attributed to the Fe3O4 NPs. Thus such multifunctional co-loading NPs are further used for magnetic ablation to tumor cells, so that a dual enhanced anti-cancer NP can be successfully realized. Fourthly, electrostatic force microscopy (EFM) is aimed at qualitative visual observation of electrostatic potential on surface of the sample, which is a mutant purple membrane (PM) modified by functional NPs. A bias voltage between a conductive tip and the modified PM is applied in an oscillating mode. The tip is lifted such that it can induce a long term electrostatic force without effect of molecular repulsive force. Thus electric gradient dependent on surface of the PM makes phase shift in a given frequency and then the EFM signal is extracted. Therefore, the electric property of such a novel biomembrane is characterized. Fifthly, a generally applicable quantitative real-time cell viability monitoring system which uses cell adhesion property is successfully setup based on the oscillation system of AFM. The amplitude of an oscillating cantilever at a given frequency is highly dependent on the mass of the cantilever, in this situation, the mass of attached cells on the cantilever. In our method, the dynamic toxic process can be observed and recorded, and can be analyzed even at an early stage of intoxication. Therefore, this will be a greatly promising method for real-time exploring and quantitatively analyzing of cellular toxicity

Desarrollo de nanocápsulas lipídicas como estrategia para facilitar el paso a través de la barrera hematoencefálica de fármacos que actúan a nivel del sistema nervioso central

Tesis de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Farmacia Galénica y Tecnología Alimentaria, leída el 17/09/2018. Tesis formato europeo (compendio de artículos)Diseases affecting the central nervous system (CNS) should be regarded as a major health challenge due to their steadily rising incidences and to the current lack of effective treatments given the hindrance to brain drug delivery imposed by the blood-brain barrier (BBB). Some of the described delivery strategies to circumvent the BBB such as the direct intracerebral administration and the artificial disruption of the tight junctions involve high risk of neurological damage. Hence, every effort is currently being devoted to achieving efficient transport across the brain endothelium with targeted drug carriers following minimally-invasive intravenous injection. In particular, nanomedicine is chiefly germane to the field of chemotherapy wherein dose availability at the target site cannot be enhanced by dose increase for fear of severe side effects. Since efficient brain targeting should not solely rely on passive targeting, brain active targeting of nanomedicines into the CNS is being explored...Las patologías que afectan al sistema nervioso central representan un desafío terapéutico por su incidencia creciente y la limitación del acceso a sistema nervioso central de la mayoría de fármacos administrados por vía sistémica por parte de la barrera hematoencefálica. Algunas de las estrategias para sortear esta barrera, incluyendo la administración intracerebral o la disrupción artificial de sus uniones estrechas, suponen un elevado riesgo de daño neurológico. Por ello, actualmente se persigue diseñar transportadores de fármacos capaces de atravesar de manera eficiente el endotelio cerebral tras su administración intravenosa. En concreto, la vectorización de antineoplásicos en nanotransportadores para el tratamiento de tumores cerebrales supondría un sustancial avance en terapéutica por la reducción de efectos secundarios derivados de su distribución sistémica. Dado que la distribución de transportadores a sistema nervioso central no puede depender en exclusiva de la vectorización pasiva, a fin de favorecer su paso a través de la barrera hematoencefálica, se está investigando la incorporación de distintos ligandos a estos sistemas. El objetivo global de esta tesis doctoral es diseñar, desarrollar y evaluar a nivel preclínico un nanotransportador lipídico capaz de atravesar la barrera hematoencefálica para vectorizar fármacos a nivel del sistema nervioso central tras una administración intravenosa. Este objetivo general se desglosa en tres objetivos específicos Estudiar los parámetros experimentales determinantes en la obtención de nanocápsulas lipídicas mediante el método térmico de inversión de fases para habilitar la producción de nanocápsulas lipídicas bajo demanda. Desarrollar una novedosa estrategia de funcionalización de nanocápsulas lipídicas con cannabidiol para favorecer su distribución a sistema nervioso central y evaluar su potencial in vitro e in vivo. Encapsular cannabidiol en el núcleo oleoso de las nanocápsulas y evaluar in vitro su eficacia como sistemas de liberación prolongada con actividad frente a la línea celular U373MG de glioblastoma humano. Asimismo, se persigue evaluar la estrategia de funcionalización con cannabidiol para potenciar la captación por células de glioma. En cuanto a los resultados, la obtención de nanocápsulas lipídicas con tamaños de partícula predeterminados para aumentar las posibilidades de éxito de tratamientos de patologías del sistema nervioso central puede conseguirse mediante el método térmico de inversión de fases, pues el diámetro volumen se ajusta a un modelo matemático en una variable (el cociente másico entre la fase interna oleosa y el tensioactivo). Este modelo es válido para nanocápsulas blancas y cargadas con fármaco. Además, una disminución de tamaño de las nanocápsulas lipídicas en el intervalo 20-60 nm incrementa en 2,5 y 1,6-2,5 veces su paso a través de la barrera hematoencefálica in vitro e in vivo, respectivamente. Por otra parte, la funcionalización de las nanocápsulas lipídicas con el fitocannabinoide cannabidiol aumenta el paso a través de barrera hematoencefálica 4,3 y 2,5 veces in vitro e in vivo, respectivamente. Una disminución de tamaño de las nanocápsulas lipídicas incrementa 3 veces la captación por células de glioma. Asimismo, el tamaño de las nanocápsulas lipídicas condiciona la liberación de fármacos: nanocápsulas de 20 nm cargadas con cannabidiol reducen invariablemente 3 veces los valores de concentración inhibitoria 50 en comparación con sus homólogas de 50 nm. Además, la funcionalización de nanocápsulas lipídicas con cannabidiol aumenta la captación por células de glioma 3,4 veces. Como conclusión, las nanocápsulas lipídicas, cargadas y funcionalizadas con cannabidiol, son prometedores candidatos para el tratamiento de gliomas con capacidad de vectorización a través de barrera hematoencefálica y de células de glioma. Su potencial terapéutico debe ser evaluado en modelos animales de glioma.Depto. de Farmacia Galénica y Tecnología AlimentariaFac. de FarmaciaTRUEunpu

Biocomposite Inks for 3D Printing

Three-dimensional (3D) printing has evolved massively during the last years. The 3D printing technologies offer various advantages, including: i) tailor-made design, ii) rapid prototyping, and iii) manufacturing of complex structures. Importantly, 3D printing is currently finding its potential in tissue engineering, wound dressings, tissue models for drug testing, prosthesis, and biosensors, to name a few. One important factor is the optimized composition of inks that can facilitate the deposition of cells, fabrication of vascularized tissue and the structuring of complex constructs that are similar to functional organs. Biocomposite inks can include synthetic and natural polymers, such as poly (ε-caprolactone), polylactic acid, collagen, hyaluronic acid, alginate, nanocellulose, and may be complemented with cross-linkers to stabilize the constructs and with bioactive molecules to add functionality. Inks that contain living cells are referred to as bioinks and the process as 3D bioprinting. Some of the key aspects of the formulation of bioinks are, e.g., the tailoring of mechanical properties, biocompatibility and the rheological behavior of the ink which may affect the cell viability, proliferation, and cell differentiation.The current Special Issue emphasizes the bio-technological engineering of novel biocomposite inks for various 3D printing technologies, also considering important aspects in the production and use of bioinks

Biofabricating the vascular tree in engineered bone tissue

The development of tissue engineering strategies for treatment of large bone defects has become increasingly relevant, given the growing demand for bone substitutes. Native bone is composed of a dense vascular network necessary for the regulation of bone development, regeneration and homeostasis. A major obstacle in fabricating living, clinically relevant-sized bone mimics (1-10 cm3) is the limited supply of nutrients, including oxygen to the core of the construct. Therefore, strategies to support vascularization are pivotal for the development of tissue engineered bone constructs. Creating a functional bone construct integrated with a vascular network, capable of delivering the necessary nutrients for optimal tissue development is imperative for translation into the clinics. The vascular system is composed of a complex network that runs throughout the body in a tree-like hierarchical branching fashion. A significant challenge for tissue engineering approaches lies in mimicking the intricate, multi-scale structures consisting of larger vessels (macro-vessels) which interconnect with multiple sprouting vessels (microvessels) in a closed network. The advent of biofabrication has enabled complex, out of plane channels to be generated and has laid the groundwork for the creation of multi-scale vasculature in recent years. This review highlights the key state-of-the-art achievements for the development of vascular networks of varying scales in the field of biofabrication with a particular focus for its application in developing a functional tissue engineered bone construct. STATEMENT OF SIGNIFICANCE: There is a growing need for bone substitutes to overcome the limited supply of patient-derived bone. Bone tissue engineering aims to overcome this by combining stem cells with scaffolds to restore missing bone. The current bottleneck in upscaling is the lack of an integrated vascular network, required for the delivery of nutrients to cells. 3D bioprinting techniques has enabled the creation of complex hollow structures of varying dimensions that resemble native blood vessels. The convergence of multiple materials, cell types and fabrication approaches, opens the possibility of developing clinically-relevant sized vascularized bone constructs. This review provides an up-to-date insight of the technologies currently available for the generation of complex vascular networks, with a focus on their application in bone tissue engineering

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Development of a vascularized, induced pluripotent stem cell-derived liver-tissue mimic for therapeutic applications.

This dissertation describes the incorporation of several technologies (stem cells, gene therapy, tissue engineering and regenerative medicine) into a single project that aims to produce a liver-tissue mimic for therapeutic applications. The liver is arguably one of the most complex organs in the body. In addition to its remarkable capacity to regenerate, it performs a host of vital functions. As a result, its impairment has widespread systemic consequences. The work described herein focused on the liver in the context of cardiovascular disease and used the heritable disorder Familial Hypercholesterolemia (FH) as a clinical disease model. As (a) the only definitive cure for FH is currently liver transplant and (b) the availability of quality liver organs for transplant is critically low, these studies seek to develop a liver-tissue mimic comprised of two parts: functional hepatocyte-like cells (derived from induced pluripotent stem cells, or iPSC) and vascular support (provided by adipose-derived stromal vascular fraction (SVF)). The dissertation is divided into six sections. Chapter I provides an introductory overview and lists the aims and hypotheses for the dissertation. Chapter II provides a four-part background discussing cholesterol metabolism, the liver organ, stem cells, and the vasculature. Chapter III describes our efforts to generate a proof-of-concept liver-tissue mimic, using HepG2 as a hepatocyte model cell source and SVF cells as the vascular support system. As vascular support is critical for parenchymal survival and function, Chapter IV examines the mechanisms of spontaneous SVF vascular self-assembly. Chapter V discusses development of a patient-specific, therapeutic cell system. FH-patient dermal fibroblasts were programmed into iPSC using modRNA technology, and eventually subjected the iPSC to directed differentiation into hepatocyte-like cells. Yet, as the iPSC were derived from an FH patient, the cells required functional restoration of their LDL-R in order to impart any therapeutic benefit. To accomplish this, a novel episomal LDL-R plasmid containing (a) upstream regulatory control sequences that confer physiological feedback control of LDL-R expression and (b) Epstein-Barr sequences for episomal retention and replication was used. To mitigate any potential concerns associated with viral vectors, the iPSC were derived and corrected using non-viral modalities. These cells were combined with an SVF derived vascular support system to assess iPSC-HLC survival characteristics in vivo. Chapter VI provides a comprehensive discussion regarding our experimental efforts. These experiments demonstrate the development of a vascularized, iPSC-derived hepatocyte liver-tissue mimic that could potentially be used for therapeutic applications, such as for the treatment of FH. Efforts to create this tissue engineered liver construct were guided by three aims: (1) Assess the role of adipose SVF in providing vascular support to implanted parenchymal cells, (2) Evaluate and define the mechanism(s) of SVF vascular self-assembly, and (3) Restore the functionality of monogenic-deficient FH cells. These studies provide several proofs-of-principle towards the development of effective cell-based treatments, not only for FH, but also for other diseases classically requiring whole-organ transplantation

The Assessment of Effects of Carbon Quantum Dots on Immune System Biomarkers Using RAW 264.7 Macrophage Cells

>Magister Scientiae - MScNanotechnology is a rapidly growing field of research. Due to major innovations brought about by developments in nanotech, several consumer products are currently available containing nanomaterials. The increase of nanomaterial production and use is accompanied by the increased potential of human, plant and animal exposure to these nanomaterials. As a relatively new nanomaterial, carbon quantum dots (CQDs) are being extensively used and researched due to its unique properties. Although many studies have assessed the toxic potential of CQDs, and found them to exhibit low toxicity, there is lack of work assessing the effects on the immune system. In the present study, RAW 264.7 murine macrophages were used as model to assess the immunomodulatory potential of CQDs. RAW cells exposed to varying concentrations of CQDs (0-500μg/ml), showed that CQDs caused a reduction at cell viability. In the absence of a mitogen CQDs, induced an inflammatory response by stimulating the release of various cytokines and chemokines such as, TNFα, MIP-1α, MIP-1β, MIP-2, IP-10, G-CSF, GM-CSF, and JE