Dynamics of protein interactions with new biomimetic interfaces: toward blood-compatible biomaterials

2019 Fall.Includes bibliographical references.Nonspecific blood protein adsorption on the surfaces is the first event that occurs within seconds when a biomaterial comes into contact with blood. This phenomenon may ultimately lead to significant adverse biological responses. Therefore, preventing blood protein adsorption on biomaterial surfaces is a prerequisite towards designing blood-compatible artificial surfaces. This project aims to address this problem by engineering surfaces that mimic the inside surface of blood vessels, which is the only known material that is completely blood-compatible. The inside surface of blood vessels presents a carbohydrate-rich, gel-like, dynamic surface layer called the endothelial glycocalyx. The polysaccharides in the glycocalyx include polyanionic glycosaminoglycans (GAGs). This polysaccharide-rich surface has excellent and unique blood compatibility. We developed a technique for preparing and characterizing dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. The glycocalyx-mimetic surfaces were prepared by adsorbing heparin- or chondroitin sulfate-containing polyelectrolyte complex nanoparticles (PCNs) to chitosan-hyaluronan polyelectrolyte multilayers (PEMs). We then studied in detail the interactions of two important blood proteins (albumin and fibrinogen) with these glycocalyx mimics. Surface plasmon resonance (SPR) is a common ensemble averaging technique for detection of biomolecular interactions. SPR was used to quantify the amount of protein adsorption on these surfaces. Moreover, single-molecule microscopy along with advanced particle tracking were used to directly study the interaction of single-molecule proteins with synthetic surfaces. Finally, we developed a groundwork for a kinetic model of long-term protein adsorption on biomaterial surfaces. In the first chapter, we thoroughly summarize the important blood-material interactions that regulate blood compatibility, organize recent developments in this field from a materials perspective, and recommend areas for future research. In the second chapter, we report the preparation and characterization of dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. In the third chapter, we investigate how combining surface plasmon resonance, X-ray spectroscopy, atomic force microscopy, and single-molecule total internal reflection fluorescence microscopy provides a more complete picture of protein adsorption on ultralow fouling polyelectrolyte multilayer and polymer brush surfaces, over different regimes of protein concentration. In the fourth chapter, the interactions of two important proteins from the blood (albumin and fibrinogen) with glycocalyx-mimetic surfaces are revealed in detail using surface plasmon resonance and single-molecule microscopy. Finally, in the fifth chapter, the long-term protein interactions with different biomaterial surfaces are studied with single-molecule microscopy an

Smart and Functional Polymers

This book is based on the Special Issue of the journal Molecules on “Smart and Functional Polymers”. The collected research and review articles focus on the synthesis and characterization of advanced functional polymers, polymers with specific structures and performances, current improvements in advanced polymer-based materials for various applications, and the opportunities and challenges in the future. The topics cover the emerging synthesis and characterization technology of smart polymers, core?shell structure polymers, stimuli-responsive polymers, anhydrous electrorheological materials fabricated from conducting polymers, reversible polymerization systems, and biomedical polymers for drug delivery and disease theranostics. In summary, this book provides a comprehensive overview of the latest synthesis approaches, representative structures and performances, and various applications of smart and functional polymers. It will serve as a useful reference for all researchers and readers interested in polymer sciences and technologies

Application of Nanomaterials in Biomedical Imaging and Cancer Therapy

To mark the recent advances in nanomaterials and nanotechnology in biomedical imaging and cancer therapy, this book, entitled Application of Nanomaterials in Biomedical Imaging and Cancer Therapy includes a collection of important nanomaterial studies on medical imaging and therapy. The book covers recent works on hyperthermia, external beam radiotherapy, MRI-guided radiotherapy, immunotherapy, photothermal therapy, and photodynamic therapy, as well as medical imaging, including high-contrast and deep-tissue imaging, quantum sensing, super-resolution microscopy, and three-dimensional correlative light and electron microscopy. The significant research results and findings explored in this work are expected to help students, researchers and teachers working in the field of nanomaterials and nanotechnology in biomedical physics, to keep pace with the rapid development and the applications of nanomaterials in precise imaging and targeted therapy

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

Sewage sludge heavy metal analysis and agricultural prospects for Fiji

Insoluble residues produced in Waste Water Treatment Plants (WWTP) as by products are known as sewage sludge (SS). Land application of SS, particularly in agricultural lands, is becoming an alternative disposal method in Fiji. However, currently there is no legislative framework governing its use. SS together with its high nutrient and organic matter contents, constitutes some undesired pollutants such as heavy metals, which may limit its extensive use. The focus of this study therefore was to determine the total concentrations of Pb, Zn, Cd, Cu, Cr, Ni and Mn in the SS produced at the Kinoya WWTP (Fiji) and in the non-fertile soil amended with the SS at 20, 40, 60, 80% application rates and in the control (100% Soil). The bioavailable heavy metals were also determined as it depicts the true extent of metal contamination. The treatment mixtures were then used to cultivate cabbage plants in which the total heavy metal uptake was investigated. Total Zn (695.6 mg/kg) was present in the highest amounts in the 100% SS (control), followed by Pb (370.9 mg/kg), Mn (35.0 mg/kg), Cu (65.5 mg/kg), Cr (20.5 mg/kg) and finally Cd (13.5 mg/kg) and hence a similar trend was seen in all treatment mixtures. The potential mobility of sludgeborne heavy metals can be classified as Ni > Cu > Cd > Zn > Mn > Cr > Pb. Total metal uptake in plant leaves and stems showed only the bioavailable metals Cu, Cd, Zn and Mn, with maximum uptake occurring in the leaves. Ni, despite being highly mobile was not detected, due to minute concentrations in the SS treatments. Optimum growth occurred in the 20 and 40% SS treatments. However maximum Cu and Mn uptake occurred in the 40% SS treatment thereby making the 20% treatment the most feasible. Furthermore the total and bioavailable metal concentrations observed were within the safe and permitted limits of the EEC and USEPA legislations

Well-defined polyglutamates as carriers for the treatment of neurodegenerative diseases

Alzheimer’s disease (AD) is a neurodegenerative multiple process of the central nervous system, which currently represents the most common cost of Dementia. The already high incidence of AD is predicted to dramatically increase over the years. In fact, the experts claim that it will become a global epidemy by 2050. Consequently, direct and indirect costs related to AD are doomed to dramatically increase. For instance, only in America, AD related burden will overcome the trillion of dollars by 2050. Moreover, available medication (Exelon®, Namenda®, Aricept®, and Razadyne®) produce moderate symptomatic benefits, but do not stop disease progression. Hence, AD, among other neurodegenerative disorders, can be considered an unmet medical need. Neuroprotective drugs, such as, curcuminoids are been taking in high consideration in order to approach these fatal disorders from a protective and preventive point of view. In this context, nanomedicine and, in particular, Polymer Therapeutics (PT) emerge as a powerful alternative to overcome the limitations of low MW drugs including their poor pharmacokinetic and pharmacodynamic profiles and low solubility in aqueous solvents, required for i.v. administration. Nonetheless, in the PT field, there is a need to develop new and innovative polymer carriers to be used as drug delivery systems and/or imaging agents owing to the fact that there is no universal polymeric system that can be used in the treatment of all diseases. Apart from biodegradability, the development of novel well-defined architectures with higher MW (in order to increase passive targeting provided by the EPR effect), predictable structure and conformation (defined three-dimensional architecture in solution), higher homogeneity, greater drug loading capacity and increased multivalency is considered crucial. To this respect, polypeptides are envisaged to achieve a major impact on a number of different relevant areas including nanomedicine. Thus, new PT based on amino acids are excellent candidates for drug delivery, as they do not suffer from the previously mentioned limitations. Concretely, polyglutamates constitute a versatile platform, which has been effectively used as building blocks in polymer drug conjugates and polymeric micelles for various medical applications ranging from cancer to regenerative medicine. Moreover, it is expected its FDA approval after approval of PGA-paclitaxel conjugate, OpaxioTM for the treatment of various cancers alone or in combination (OpaxioTM has been recently designated as orphan drug in combination with radiotherapy and temozolomide for the treatment of glioblastoma multiforme). Nevertheless, control on polymer chain length, polydispersities and stereochemistry has been the major challenge in the development of synthetic polypeptides over the past years. Besides, the use of branched polymers is emerging in order to accomplish the previously described requisites. They exhibit special properties when compared to their linear counterparts. As a result of their different architectures, solution conformation, size and shape as well as greater multivalency, different therapeutic outputs could be gained. Due to their compact and globular shapes they are postulated to perform better regarding to overcome biological barriers, a pre-requisite in neurodegenerative disorders treatment as well as diagnostics due to the presence of the blood-brain barrier (BBB), one of the most challenging to surpass. Therefore, the main aim of this thesis was the design of new versatile polyglutamate-based nanotherapeutics to be used in the treatment and/or diagnosis of devastating neurodegenerative pathologies such as AD. In order to accomplish our final goal, firstly, we report the development of synthetic pathways to a plethora of functional polyglutamates with well-defined structure, adjustable MW and low polydispersities (Đ <1.2) applying the ring opening polymerization (ROP) of N-Carboxyanhydrides (NCA) with novel initiators. Furthermore, this methodology has been extended to reach a number of architectures based on PGA, including stars, grafts, and hybrid di-block copolymers. In addition, a versatile post-polymerization modification method to introduce a variety of functionalities such as alkyne, azides, reactive disulfides, maleimide groups or protected amines has been developed, yielding a set of orthogonal reactive attachment sites suitable for further bioconjugations. The physico-chemical properties of the obtained polyglutamates have been exhaustively investigated, in terms of size and solution conformation by the use of a battery of complex techniques including DLS, DOSY-NMR, CD, TEM and SANS. Furthermore, we have developed a novel PGA-based family of systems that, according to their physico-chemical characterization, underwent a self-assembly process where it did exist a structure/conformation-concentration dependency encountering at low concentrations “unimers” of 5-10 nm size, whereas bigger structures of around 100-180 nm were formed at high concentrations. After covalent entrapment of these bigger structures by means of click chemistry, the concentration dependence conformation was clearly eliminated. We have taken profit from that special behavior to develop a strategy in order to reach complex polypeptide based architectures through bottom-up approaches Preliminary in vitro evaluation in selected cell models in terms of biodegradability, biocompatibility and cellular uptake is presented. Furthermore, after an adequate labeling with fluorescence/NIR probes or/and cation complexing moieties towards the use of MRI and/or PET techniques, the in vivo fate (pharmacokinetics and biodistribution) of our polyglutamates is also described. Preliminary results suggest that they were non-toxic entities, validating them as possible carriers for drug delivery. The covalently entrapped unique architectures have been ultimately used to reach carriers for BBB crossing by means of surface modifications with targeting units and imaging agents. Their BBB crossing properties have being explored in vivo, reaching at least 1.2 % of injected dose in the brain. Thus, those results make them optimal candidates to be used in AD treatment. Among all the biological hallmarks of AD, we are centering our efforts in the amyloid pathway, by the use of curcuminoids and with a neuroprotective approach by combining them with the presence of propagyl moieties within the construct. Their biological output regarding cellular uptake, cell viability, drug release profile and biodistribution has been investigated. Moreover, proof of concept of their activity was achieved in vitro, in organotypic hippocampal cultures and is currently being validated in vivo. Finally, the potential of PGA-based conjugates as tissue-specific smart imaging probes is also explored within the frame of the European consortium LIVIMODE. The combination of NIRF enzyme specific smart probes together with the tissue specificity provided by PGA as carrier is explored to be applied in the early detection of disease-related events in vitro as well as in vivo. This strategy could be used for the development of theranostics towards the early detection and treatment of neurodegenerative disorder

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