Design of protein-nanomaterial hybrids as tools for sensing, imaging and bioelectronics

217 p.El diseño de proteínas permite construir herramientas nanotecnológicas adaptadas para su uso en campos como la biomedicina o la industria. Las proteínas de repetición CTPR son una buena opción para desarrollar nano-herramientas dada su estructura modular y tolerancia a mutaciones, lo que permite combinar módulos funcionalizados sin comprometer la estabilidad de la proteína. Además, las proteínas CTPR pueden modificarse para desarrollar módulos que coordinan metales, lo que permite la unión de nanomateriales metálicos con propiedades interesantes como las nanopartículas de oro, o la síntesis de nanocristales metálicos in situ. En la presente tesis doctoral se propone un sistema modular de proteínas CTPR funcionalizadas con nanomateriales metálicos para su aplicación como herramientas nanotecnológicas en sensórica, imagen y bioelectrónica. Para ello, primero se establece un diseño de CTPR con residuos de coordinación de metales y se estudia en profundidad las propiedades fotoluminiscentes que emergen de nanocristales de oro coordinados a dichas CTPR. A continuación, se elaboran diseños de CTPR coordinando nanomateriales metálicos y se aplican como sensores de parámetros ambientales, como la temperatura o la presencia de iones metálicos; como sondas fluorescentes para detección correlativa de orgánulos celulares usando microscopía de fluorescencia y fluorescencia de rayos-X; y como bloques de construcción para elaborar biomateriales conductores

Advanced Electrochemical and Opto-Electrochemical Biosensors for Quantitative Analysis of Disease Markers and Viruses

The recent global events of the SARS-CoV-2 pandemic in 2020 have alerted the world to the urgent need to develop fast, sensitive, simple, and inexpensive analytical tools that are capable of carrying out a large number of quantitative analyses, not only in centralized laboratories and core facilities but also on site and for point-of-care applications. In particular, in the case of immunological tests, the required sensitivity and specificity is often lacking when carrying out large-scale screening using decentralized methods, while a centralized laboratory with qualified personnel is required for providing quantitative and reliable responses. The advantages typical of electrochemical and optical biosensors (low cost and easy transduction) can nowadays be complemented in terms of improved sensitivity by combining electrochemistry (EC) with optical techniques such as electrochemiluminescence (ECL), EC/surface-enhanced Raman spectroscopy (SERS), and EC/surface plasmon resonance (SPR). This Special Issue addresses existing knowledge gaps and aids in exploring new approaches, solutions, and applications for opto-electrochemical biosensors in the quantitative detection of disease markers, such as cancer biomarkers proteins and allergens, and pathogenic agents such as viruses. Included are seven peer-reviewed papers that cover a range of subjects and applications related to the strategies developed for early diagnosis

Protein Adsorption and Conformational Changes

Protein adsorption to solids, nanomaterials, and biological surfaces is of central interest in many fields, including biomedicine, bioanalytical chemistry, materials engineering, bio-nanotechnology, and basic biomolecular research. Although protein adsorption may sometimes occur with little consequence on molecular structure, interactions with surfaces frequently cause changes in local or global conformations and dynamics, perturbations to secondary structures or tertiary folds, eventually resulting in dramatically altered protein function. Importantly, surfaces may trigger protein misfolding and self-aggregation, or, conversely, promote protein structure formation. The use of nanoscale surfaces to remodel the conformational landscape and the aggregation pathways of amyloidogenic peptides and proteins has been proposed as a promising strategy against several severe human diseases. The rapid growth of applications and technological innovation which is based on or concerned with protein adsorption necessitates renewed efforts to provide molecular-level insights into adsorption-induced protein structural perturbations. In this Special Issue, we gathered the recent findings of experimental and computational investigations that contributed novel insights into protein adsorption with a focus on the structural and dynamic aspects of proteins

NanoJanus and Nanosatellite Assembly for Biomolecular Delivery and Cancer Therapeutics

Nanotechnology has been utilized widely in medical fields to improve the treatment and diagnosis of several diseases. One of the key players to drive medical nanotechnology forward is nanoparticles, which have been intensively studied and used as a tool for imaging, drug delivery, and disease treatments. Gold and iron oxide, undoubtedly, are on the short list of the nanoparticles used in medical nanotechnology due to their biocompatibility, tunable surface, and unique physico-chemical properties. In this dissertation, we developed novel nanostructures using gold, iron oxide nanoparticles and polymers for various applications including Janus motors, vaccine delivery, and controlled drug release. We generated an asymmetrical Janus nanostructure using thermo-cleavable polymer, gold, and iron oxide nanoparticles for photothermal enhancement and nano motors through an active rotational motion. Gold/iron oxide Janus nanoparticles (JNS) are developed by a seed-mediated self-assembly using a thermo-cleavable polymer facilitating the process. The formed JNS strongly displays an asymmetrical photothermal effect to activate a rotational motion and enhances photothermia resulting in significant cell killing effects under weak near-infrared (NIR) light exposure. In addition, the JNS displays distinct active rotational motion under NIR laser light due to the temperature gradient at its surface, which can be used potentially as Janus motors for drug delivery in the future. We next harnessed the same thermo-cleavable polymer used in JNS formation for controlled drug release under NIR laser light irradiation. The iron oxide nanoparticles (IONP) were first encapsulated in the thermo-cleavable polymeric micelles with doxorubicin (Dox), a chemotherapeutic drug. After NIR trigger, the polymer is cleaved due to heat transfer from the IONP resulting in the release of doxorubicin from the micelles. This study demonstrated that the thermo-cleavable polymer could be used as a smart material for controlled drug release. We also generated another type of secondary structure, a “gold/iron oxide nanosatellite”, using poly (- methacryloxypropyl trimethoxysilane) -b- poly (ethylene oxide) polymer (MPS-b-PEO). This nanosatellite structure, in which IONP is a central core and surrounded by multiple gold nanoparticles as satellites, is used for delivering antigens and an adjuvant for HPV+ head and neck cancer treatment. These nanosatellites deliver high surface density of E7/E6 oncogenic peptides and cyclic- guanosine-adenosine monophosphate (cGAMP) adjuvant to antigen presenting cells (APC) and further activate type I interferon (IFN-I) response. The nanosatellite vaccine also promotes antigen specific CD8+ T cells to infiltrate the tumors and inhibits tumor growth in an HPV+ head and neck tumor model when used as a single therapy or in combination therapy with an anti PD-L1 antibody. Nanosatellites offer many advantages for antigenic peptide and adjuvant delivery such as having a larger surface area, higher antigenic peptide density, higher cell uptake, and lower systemic elimination. This thesis presents the versatile developments and applications of gold/iron oxide nanostructures (Janus and Nanosatellite) which have advantages for drug and vaccine delivery in the future.PHDPharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140893/1/kanokwas_1.pd

Breaking Barriers: Bioinspired Strategies for Targeted Neuronal Delivery to the Central Nervous System

Central nervous system (CNS) disorders encompass a vast spectrum of pathological conditions and represent a growing concern worldwide. Despite the high social and clinical interest in trying to solve these pathologies, there are many challenges to bridge in order to achieve an effective therapy. One of the main obstacles to advancements in this field that has hampered many of the therapeutic strategies proposed to date is the presence of the CNS barriers that restrict the access to the brain. However, adequate brain biodistribution and neuronal cells specific accumulation in the targeted site also represent major hurdles to the attainment of a successful CNS treatment. Over the last few years, nanotechnology has taken a step forward towards the development of therapeutics in neurologic diseases and different approaches have been developed to surpass these obstacles. The versatility of the designed nanocarriers in terms of physical and chemical properties, and the possibility to functionalize them with specific moieties, have resulted in improved neurotargeted delivery profiles. With the concomitant progress in biology research, many of these strategies have been inspired by nature and have taken advantage of physiological processes to achieve brain delivery. Here, the different nanosystems and targeting moieties used to achieve a neuronal delivery reported in the open literature are comprehensively reviewed and critically discussed, with emphasis on the most recent bioinspired advances in the field. Finally, we express our view on the paramount challenges in targeted neuronal delivery that need to be overcome for these promising therapeutics to move from the bench to the bedside.This work was financially supported by the project PTDC/CTM-NAN/3547/2014 (POCI-01-0145-FEDER-016639) funded by FEDER funds through the Programa Operacional Competitividade e Internacionalização-COMPETE 2020 and Portuguese funds through FCT–Fundação para a Ciência e a Tecnologia. A.P.S., B.C. and S.D.S. acknowledge FCT for the Ph.D. scholarships (SFRH/BD/137073/2018 and SFRH/BD/145652/2019) and the contract under the Norma Transitória–DL57/2016/CP/CP1360/CT0013, respectively. V.L. acknowledges her contract in the framework of the project NORTE-01-0247-FEDER-033399, funded by FEDER funds through the Sistema de Incentivos à Investigação e Desenvolvimento Tecnológico (SI I&DT), Aviso nº 03/SI/2017, Projetos em Co-promoção do Programa Interfac

Systematic investigations on the protein corona of different nanocarriers and its role in cellular uptake

In the field of nanomedicine, nanomaterials are becoming increasingly important as nanotransporters, e.g. to better formulate active substances, protect them from degradation and transport them in a targeted manner. However, as soon as nanoparticles are exposed to a complex biological environment (e.g. blood), they interact with various biomolecules and bind them as a corona. The protein corona is the key factor that significantly influences both the biological half-life as well as the transport and uptake of nanoparticles in vivo. In order to better understand and eventually predict the behaviour of nanoparticles in vivo, it is essential to decipher the interaction of nanoparticles and proteins. In this work, the influence of different physicochemical properties (size, surface charge, structure and hydrophobicity) of nanoparticles on the formation of a protein corona was investigated. In terms of future utilization as nanocarriers, the connection between protein corona and cellular uptake was also analysed in more detail. The aim was to identify interaction partners, i.e. docking sites, for corona proteins on the cell membrane and thus possible cellular uptake pathways. The nanoparticles used in this dissertation were extensively characterised with regard to their physicochemical properties. Subsequently, the protein corona in human serum was determined using various methods such as 2D electrophoresis in combination with MALDI TOF/TOF mass spectrometry. The cellular uptake of the nanoparticles into human THP-1 monocytes was investigated by flow cytometry and fluorescence microscopy. In addition, cellular interaction partners were identified via pull-down experiments. Within this work it could be shown that all investigated physicochemical properties have an influence on the formation and composition of the protein corona. The most pronounced influence was exerted by the surface charge and the hydrophobicity. Increasing hydrophobicity led to the binding of more proteins. Similarly, a negative charge led to a higher number of bound proteins compared to a positively charged nanoparticle surface. For the selected particle systems, changes in the composition of the protein corona led to differences in cellular uptake in vitro. Thus, the amount of nanoparticles taken up in human THP-1 monocytes correlated with changes in the composition of the protein corona. In addition, for the dendritic polyglycerols it was possible to identify potential initial docking sites on human THP-1 monocytes and to obtain indications of a possible uptake mechanism. For example, the identification of serotransferin indicates clathrin-mediated endocytosis. The results obtained confirm that the protein corona has a significant influence on cellular uptake. Certain proteins (opsonins) can mediate the uptake of nanoparticles by the mononuclear-phagocytic system and thus shorten the biological half-life and systemic circulation time. These results provide an important contribution to the optimised design of future nanocarriers.Im Bereich der Nanomedizin gewinnen Nanomaterialien als Nanotransporter mehr und mehr an Wichtigkeit, z.B. um Wirkstoffe besser zu formulieren, vor Abbau zu schützen und zielgerichtet zu transportieren. Allerdings interagieren Nanopartikel, sobald diese einer komplexen biologischen Umgebung (beispielsweise Blut) ausgesetzt sind, mit diversen Biomolekülen und binden diese als Korona. Die Proteinkorona ist der Schlüsselfaktor, welcher maßgeblich sowohl die biologische Halbwertszeit als auch den Transport und die Aufnahme von Nanopartikeln in vivo beeinflusst. Um das Verhalten von Nanopartikeln in vivo besser zu verstehen und gegebenenfalls auch vorhersagen zu können, ist es von zentraler Bedeutung, die Interaktion von Nanopartikeln und Proteinen zu entschlüsseln. Im Rahmen dieser Arbeit untersuchte ich den Einfluss verschiedener physiko-chemischer Eigenschaften (Größe, Oberflächenladung, Struktur und Hydrophobizität) von Nanopartikeln auf die Bildung einer Proteinkorona. In Hinblick auf die spätere Anwendung als Nanocarrier wurde zudem der Zusammenhang zwischen Proteinkorona und der zellulären Aufnahme genauer analysiert. Dabei sollten Interaktionspartner, d.h. Andockungsstellen, für Koronaproteine an die Zellmembran und damit mögliche zelluläre Aufnahmewege identifiziert werden. Die von mir verwendeten Nanopartikel wurden betreffend ihrer physiko chemischen Eigenschaften umfangreich charakterisiert. Im Anschluss erfolgte die Bestimmung der Proteinkorona in humanem Serum mittels verschiedener Methoden wie z.B. 2D Gelektrophorese in Kombination mit MALDI TOF/TOF Massenspektrometrie. Die zelluläre Aufnahme der Nanopartikel in humane THP-1 Monozyten untersuchte ich mittels Durchflusszytometrie und Fluoreszenzmikroskopie. Zudem wurden zelluläre Interaktionspartner über Pull Down Versuche identifiziert. Innerhalb dieser Arbeit konnte gezeigt werden, dass alle untersuchten physiko chemischen Eigenschaften einen Einfluss auf die Bildung und Zusammensetzung der Proteinkorona haben. Den am stärksten ausgeprägten Einfluss hatten die Oberflächenladung sowie die Hydrophobizität. Deren Zunahme resultierte in einer erhöhten Bindung von Proteinen. Ebenso bewirkte eine negative Ladung eine höhere Anzahl an gebundenen Proteinen im Vergleich zu einer positiv geladenen Nanopartikeloberfläche. Für die ausgewählten Partikelsysteme erzeugten Veränderungen in der Zusammensetzung der Proteinkorona Unterschiede in der zellulären Aufnahme in vitro. So korrelierte die in humane THP-1 Monozyten aufgenommene Menge an Nanopartikeln mit Veränderungen der Zusammensetzung der Proteinkorona. Zudem gelang es für die dendritischen Polyglycerole potentielle initiale Andockstellen auf humanen THP-1 Monozyten zu identifizieren und Hinweise auf einen möglichen Aufnahmemechanismus zu erhalten. So weist die Identifizierung von Serotransferin beispielsweise auf eine Clathrin-vermittelte Endozytose hin. Die erzielten Ergebnisse bestätigen den relevanten Einfluss der Proteikorona auf die zelluläre Aufnahme. Bestimmte Proteine (Opsonine) können die Aufnahme der Nanopartikel durch das mononukleär-phagozytäre System vermitteln und so die biologische Halbwertszeit sowie die systemische Zirkulationszeit verkürzen. Diese Resultate liefern einen wichtigen Beitrag zum optimierten Design der zukünftigen Nanocarrier

Biosensors for Diagnosis and Monitoring

Biosensor technologies have received a great amount of interest in recent decades, and this has especially been the case in recent years due to the health alert caused by the COVID-19 pandemic. The sensor platform market has grown in recent decades, and the COVID-19 outbreak has led to an increase in the demand for home diagnostics and point-of-care systems. With the evolution of biosensor technology towards portable platforms with a lower cost on-site analysis and a rapid selective and sensitive response, a larger market has opened up for this technology. The evolution of biosensor systems has the opportunity to change classic analysis towards real-time and in situ detection systems, with platforms such as point-of-care and wearables as well as implantable sensors to decentralize chemical and biological analysis, thus reducing industrial and medical costs. This book is dedicated to all the research related to biosensor technologies. Reviews, perspective articles, and research articles in different biosensing areas such as wearable sensors, point-of-care platforms, and pathogen detection for biomedical applications as well as environmental monitoring will introduce the reader to these relevant topics. This book is aimed at scientists and professionals working in the field of biosensors and also provides essential knowledge for students who want to enter the field

Fundamentals of SARS-CoV-2 Biosensors

COVID-19 diagnostic strategies based on advanced techniques are currently essential topics of interest, with crucial roles in scientific research. This book integrates fundamental concepts and critical analyses that explore the progress of modern methods for the detection of SARS-CoV-2

Self-assembling Peptide for HIV-1 Vaccine Design

Human immunodeficiency virus-1 (HIV-1) is a worldwide epidemic, which cannot be eliminated by any current therapeutics, even with highly active antiretroviral therapy, which only can control virus replication. A safe and effective vaccine against HIV-1 that can elicit both potent humoral and cellular responses has been considered a best solution to prevent the infection or to reduce the viral load. However, despite the fact that over 250 clinical trials have been conducted based on different concepts, no vaccine has been successfully developed. The extraordinary diversity of HIV-1, the capability of the virus to escape from the adaptive immunity, the difficulty in inducing broadly neutralizing antibodies, and the lack of clear immune correlates of protection represent the major challenges obstructing the development of HIV-1 vaccines. Designing a peptide-based vaccine that stimulates cytotoxic T lymphocytes (CTLs) specifically against the highly conserved epitopes in HIV-1 has been considered a promising strategy. This can provide two theoretical advantages: maximizing the immunological coverage and minimizing the viral escape from recognition of T cells. However, owning to the short sequence (normally 8-10 amino acids for CTL epitopes), these conserved epitopes are weakly immunogenic, requiring potent adjuvants to boost the efficiency. Some novel strategies have been reported to achieve an efficient adjuvant without causing any side effect. Among them, nanoparticle based delivery systems that can provide targeted delivery to immune cells and/or self-adjuvant effect, are emerging as a promising approach. In this thesis, we present a self-assembling peptide based delivery platform efficiently integrating antigenic peptides and immune potentiators in the formulation of a nanoparticle vaccine, and evaluate the immunogenicity in vitro and in vivo. Three parts are involved in this work: (1) feasibility study of the delivery of HIV-1 CTL epitope with the self-assembling peptide EAK16-II (sequence: AEAEAKAKAEAEAKAK) and the cross-presentation efficiency by dendritic cells (DCs); (2) co-delivery of an antigenic peptide and a toll like receptor (TLR) agonist within one nanoparticle to target and maturate DCs, leading to enhanced CTL response; (3) formulating a prophylactic peptide vaccine against HIV-1 by the combination of CD4 epitope-conjugated EAK16-II, CD8 epitope-conjugated EAK16-II, and a TLR agonist R848, which was subsequently assessed the immunogenicity in the transgenic mice. The peptide EAK16-II could self-assemble into nanofibers, which were stable in the acidic environment and in the presence of proteases. We hypothesized that by directly conjugating HIV-1 CD8 epitope with EAK16-II, the fibrillar structures of the conjugate would enhance the stability of epitope and thus improve the immunogenicity. To verify this, the CD8 epitope SL9 was conjugated with EAK16-II to obtain the epitope-loading peptide SL9-EAK16-II. Physicochemical characterizations revealed SL9-EAK16-II spontaneously assembled to short nanofibers in PBS, which were more stable in serum or oligopeptidase than unstructured SL9. Ex-vivo generated DCs that were pulsed with SL9-EAK16-II and activated by maturation cytokines, stimulated more poly-functional CD8+ T cells. This augment was explained by the evidence that SL9-EAK16-II was degraded more slowly than SL9 within DCs, therefore prolonging the stimulation to CD8 T cells. Moreover, the results from confocal microscopy suggested the cytosolic pathway for the cross-presentation of SL9-EAK16-II. However, SL9-EAK16-II itself failed to maturate DCs after internalization, which might cause antigen tolerance. To avoid the induction of tolerance and further enhance the antigenicity of epitope SL9, TLR agonist R837 or R848 was incorporated into the nanofiber formulation. The data from fluorescence spectra and calorimetric titration suggested the co-assembly between SL9-loaded nanofibers and TLR agonist was mainly driven by hydrogen bonding and hydrophobic interactions. The SL9-EAK16-II/R848 co-assemblies strongly facilitated the activation of DCs, and stimulated significantly more epitope specific CTLs when assessed in the form of DC based vaccine. The in vitro studies implied the potential of the self-assembling peptide EAK16-II as a nanocarrier in the formulation of vaccine. We further determined the applicability of this formulation in vivo. Since the activation of CD4+T cells plays a critical role in the generation of functional memory CTLs, we incorporated an additional CD4 epitope TL13 into the vaccine formulation, via conjugating with EAK16-II. The new formulation of antigen was characterized as nanofibers with average size of approximately 220 nm. The transgenic mice that were subcutaneously injected with these nanofibers produced as much as 1 fold increase in frequencies of SL9 specific CTLs, when compared with the mice vaccinated with either the mixture of epitopes and R848, or R848 alone. Moreover, almost 90% of the SL9 specific CTLs primed by the nanofibers were central memory CD8+ T cells (CD44+, CD62L+), which was the hallmark of the acquired immune response. The in-vivo study suggested not only enhanced magnitude, but also higher quality of T cell response was induced by the nanoparticle-based vaccine. Our findings demonstrated the self-assembling peptide had considerable promise as a delivery platform to integrate the principal components for cellular response-focusing vaccines