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The recent COVID19 pandemic has remarkably boosted the research on in vitro diagnosis assays to detect biomarkers in biological fluids. Specificity and sensitivity are mandatory for diagnostic kits aiming to reach clinical stages. Whilst the modulation of sensitivity can significantly improve the detection of biomarkers in liquids, this has been scarcely explored. Here, we report on the proof of concept and parametrization of a novel biosensing methodology based on the changes of AC magnetic hysteresis areas observed for magnetic nanoparticles following biomolecular recognition in liquids. Several parameters are shown to significantly modulate the transducing capacity of magnetic nanoparticles to detect analytes dispersed in saline buffer at concentrations of clinical relevance. Magnetic nanoparticles were bio-conjugated with an engineered recognition peptide as a receptor. Analytes are engineered tetratricopeptide binding domains fused to the fluorescent protein whose dimerization state allows mono- or divalent variants. Our results unveil that the number of receptors per particle, analyte valency and concentration, nanoparticle composition and concentration, and field conditions play a key role in the formation of assemblies driven by biomolecular recognition. Consequently, all these parameters modulate the nanoparticle transduction capacity. Our study provides essential insights into the potential of AC magnetometry for customizing biomarker detection in liquids.
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Técnicas Biosensibles , Nanopartículas , Magnetismo , Nanopartículas/química , Biomarcadores , Fenómenos Magnéticos , Técnicas Biosensibles/métodosRESUMEN
Iron oxide nanoparticles (IONPs) have become one of the most promising nanomaterials for biomedical applications because of their biocompatibility and physicochemical properties. This study demonstrates the use of protein engineering as a novel approach to design scaffolds for the tunable synthesis of ultrasmall IONPs. Rationally designed proteins, containing different number of metal-coordination sites, were evaluated to control the size and the physicochemical and magnetic properties of a set of protein-stabilized IONPs (Prot-IONPs). Prot-IONPs, synthesized through an optimized coprecipitation approach, presented good T1 and T2 relaxivity values, stability, and biocompatibility, showing potential for magnetic resonance imaging (MRI) applications.
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This work reports on the use of protein engineering as a versatile tool to rationally design metal-binding proteins for the synthesis of highly photoluminescent protein-stabilized gold nanoclusters (Prot-AuNCs). The use of a single repeat protein scaffold allowed the incorporation of a set of designed metal-binding sites to understand the effect of the metal-coordinating residues and the protein environment on the photoluminescent (PL) properties of gold nanoclusters (AuNCs). The resulting Prot-AuNCs, synthesized by two sustainable procedures, showed size-tunable color emission and outstanding PL properties. In a second stage, tryptophan (Trp) residues were introduced at specific positions to provide an electron-rich protein environment and favor energy transfer from Trps to AuNCs. This modification resulted in improved PL properties relevant for future applications in sensing, biological labeling, catalysis, and optics.
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Oro , Nanopartículas del Metal , Catálisis , Transferencia de Energía , Oro/química , Nanopartículas del Metal/química , Ingeniería de ProteínasRESUMEN
ConspectusThe last decades have witnessed unprecedented scientific breakthroughs in all the fields of knowledge, from basic sciences to translational research, resulting in the drastic improvement of the lifespan and overall quality of life. However, despite these great advances, the treatment and diagnosis of some diseases remain a challenge. Inspired by nature, scientists have been exploring biomolecules and their derivatives as novel therapeutic/diagnostic agents. Among biomolecules, proteins raise much interest due to their high versatility, biocompatibility, and biodegradability.Protein binders (binders) are proteins that bind other proteins, in certain cases, inhibiting or modulating their action. Given their therapeutic potential, binders are emerging as the next generation of biopharmaceuticals. The most well-known example of binders are antibodies, and inspired by them researchers have developed alternative binders using protein design approaches. Protein design can be based on naturally occurring proteins in which, by means of rational design or combinatorial approaches, new binding interfaces can be engineered to obtain specific functions or based on de novo proteins emerging from state-of-the-art computational methodologies.Among the novel designed proteins, a class of engineered repeat proteins, the consensus tetratricopeptide repeat (CTPR) proteins, stand out due to their stability and robustness. The CTPR unit is a helix-turn-helix motif constituted of 34 amino acids, of which only 8 are essential to ensure correct folding of the structure. The small number of conserved residues of CTPR proteins leaves plenty of freedom for functional mutations, making them a base scaffold that can be easily and reproducibly tailored to endow desired functions to the protein. For example, the introduction of metal-binding residues (e.g., histidines, cysteines) drives the coordination of metal ions and the subsequent formation of nanomaterials. Additionally, the CTPR unit can be conjugated with other peptides/proteins or repeated in tandem to encode larger CTPR proteins with superhelical structures. These properties allow for the design of both binder and nanomaterial-coordination modules as well as their combination within the same molecule, making the CTPR proteins, as we have demonstrated in several recent examples, the ideal platform to develop protein-nanomaterial hybrids. Generally, the fusion of two distinct materials exploits the best properties of each; however, in protein-nanomaterial hybrids, the fusion takes on a new dimension as new properties arise.These hybrids have ushered the use of protein-based nanomaterials as biopharmaceuticals beyond their original therapeutic scope and paved the way for their use as theranostic agents. Despite several reports of protein-stabilized nanomaterials found in the literature, these systems offer limited control in the synthesis and properties of the grown nanomaterials, as the protein acts just as a stabilizing agent with no significant functional contribution. Therefore, the rational design of protein-based nanomaterials as true theranostic agents is still incipient. In this context, CTPR proteins have emerged as promising scaffolds to hold simultaneously therapeutic and diagnostic functions through protein engineering, as it has been recently demonstrated in pioneering in vitro and in vivo examples.
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Nanotechnology-based approaches hold substantial potential to avoid chemoresistance and minimize side effects. In this work, we have used biocompatible iron oxide magnetic nanoparticles (MNPs) called MF66 and functionalized with the antineoplastic drug doxorubicin (DOX) against MDA-MB-231 cells. Electrostatically functionalized MNPs showed effective uptake and DOX linked to MNPs was more efficiently retained inside the cells than free DOX, leading to cell inactivation by mitotic catastrophe, senescence and apoptosis. Both effects, uptake and cytotoxicity, were demonstrated by different assays and videomicroscopy techniques. Likewise, covalently functionalized MNPs using three different linkers-disulfide (DOX-S-S-Pyr, called MF66-S-S-DOX), imine (DOX-I-Mal, called MF66-I-DOX) or both (DOX-I-S-S-Pyr, called MF66-S-S-I-DOX)-were also analysed. The highest cell death was detected using a linker sensitive to both pH and reducing environment (DOX-I-S-S-Pyr). The greatest success of this study was to detect also their activity against breast cancer stem-like cells (CSC) from MDA-MB-231 and primary breast cancer cells derived from a patient with a similar genetic profile (triple-negative breast cancer). In summary, these nanoformulations are promising tools as therapeutic agent vehicles, due to their ability to produce efficient internalization, drug delivery, and cancer cell inactivation, even in cancer stem-like cells (CSCs) from patients.
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There is still a need for improving the treatment of breast cancer with doxorubicin (DOX). In this paper, we functionalized magnetic nanoparticles (MNPs) with DOX and studied the DOX-induced antitumor effects in breast cancer cells (BT474) in the presence of magnetic hyperthermia (43 °C, 1 h). We show that i) intratumoral application of DOX-functionalized MNPs (at least at a concentration of 9.6 nmol DOX/100 mm3 tumor volume) combined with magnetic hyperthermia favors tumor regression in vivo, and there is evidence for an increased effect compared to magnetic hyperthermia alone or to the intratumoral application of free DOX and ii) the presence of the pseudopeptide NucAnt (N6L) on the MNP surface might well be beneficial in its function as carrier for MNP internalization into breast cancer cells in vitro, which could further augment the possibility of the induction of intracellular heating spots and cell death in the future.
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This work presents a simple in situ synthesis and stabilization of fluorescent gold nanoclusters (AuNCs) with different sizes using engineered protein scaffolds in water. The protein-AuNC hybrids show a dual emission (450 and 700 nm) with a record photoluminescence quantum yield of 20%. These features impelled us to apply them to biohybrid light-emitting diodes as color down-converting filters or biophosphors. Efficient white emission (x/y CIE color coordinates of 0.31/0.29) and stabilities of more than 800 h were achieved. This represents a 2 orders of magnitude enhancement compared to the prior art. Besides the outstanding performance, the protein scaffold also infers a unique anisotropic emission character that is considered as a proof-of-concept of high interest for single-point lighting and display.
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Oro/química , Sustancias Luminiscentes/química , Nanopartículas del Metal/química , Proteínas/química , Luz , Iluminación , Luminiscencia , Modelos Moleculares , Nanotecnología , Repeticiones de TetratricopéptidosRESUMEN
BACKGROUND: Identifying the precise location of cells and their migration dynamics is of utmost importance for achieving the therapeutic potential of cells after implantation into a host. Magnetic resonance imaging is a suitable, non-invasive technique for cell monitoring when used in combination with contrast agents. RESULTS: This work shows that nanowires with an iron core and an iron oxide shell are excellent materials for this application, due to their customizable magnetic properties and biocompatibility. The longitudinal and transverse magnetic relaxivities of the core-shell nanowires were evaluated at 1.5 T, revealing a high performance as T2 contrast agents. Different levels of oxidation and various surface coatings were tested at 7 T. Their effects on the T2 contrast were reflected in the tailored transverse relaxivities. Finally, the detection of nanowire-labeled breast cancer cells was demonstrated in T2-weighted images of cells implanted in both, in vitro in tissue-mimicking phantoms and in vivo in mouse brain. Labeling the cells with a nanowire concentration of 0.8 µg of Fe/mL allowed the detection of 25 cells/µL in vitro, diminishing the possibility of side effects. This performance enabled an efficient labelling for high-resolution cell detection after in vivo implantation (~ 10 nanowire-labeled cells) over a minimum of 40 days. CONCLUSIONS: Iron-iron oxide core-shell nanowires enabled the efficient and longitudinal cellular detection through magnetic resonance imaging acting as T2 contrast agents. Combined with the possibility of magnetic guidance as well as triggering of cellular responses, for instance by the recently discovered strong photothermal response, opens the door to new horizons in cell therapy and make iron-iron oxide core-shell nanowires a promising theranostic platform.
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Rastreo Celular/métodos , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita , Nanocables , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Línea Celular , Compuestos Férricos , Hierro , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Fantasmas de Imagen , Nanomedicina TeranósticaRESUMEN
Immunotherapy has become a promising cancer therapy, improving the prognosis of patients with many different types of cancer and offering the possibility for long-term cancer remission. Nevertheless, some patients do not respond to these treatments and immunotherapy has shown some limitations, such as immune system resistance or limited bioavailability of the drug. Therefore, new strategies that include the use of nanoparticles (NPs) are emerging to enhance the efficacy of immunotherapies. NPs present very different pharmacokinetic and pharmacodynamic properties compared with free drugs and enable the use of lower doses of immune-stimulating molecules, minimizing their side effects. However, NPs face issues concerning stability in physiological conditions, protein corona (PC) formation, and accumulation in the target tissue. PC formation changes the physicochemical and biological properties of the NPs and in consequence their therapeutic effect. This review summarizes the recent advances in the study of the effects of PC formation in NP-based immunotherapy. PC formation has complex effects on immunotherapy since it can diminish ("immune blinding") or enhance the immune response in an uncontrolled manner ("immune reactivity"). Here, future perspectives of the field including the latest advances towards the use of personalized protein corona in cancer immunotherapy are also discussed.
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Inmunoterapia , Nanopartículas , Corona de Proteínas/química , Corona de Proteínas/metabolismo , Nanomedicina Teranóstica , Animales , Ensayos Clínicos como Asunto , Composición de Medicamentos , Sistemas de Liberación de Medicamentos , Humanos , Inmunoterapia/efectos adversos , Inmunoterapia/métodos , Inyecciones , Nanopartículas/administración & dosificación , Nanopartículas/química , Neoplasias/inmunología , Neoplasias/terapia , Nanomedicina Teranóstica/métodosRESUMEN
Designed repeat proteins catalyze the 1,3-dipolar reaction between an imine and a π-deficient dipolarophile in THF solution to form unnatural nitroproline esters, a reaction that no enzyme can catalyze. NMR studies and mutation experiments show that both acidic and basic residues can catalyze the reaction. The diastereocontrol of the reaction depends on the flexibility of the protein and on the number and location of the active lysine and glutamate residues, which can participate independently or forming dyads that promote the formation of unusual diastereomeric cycloadducts. QM/MM calculations permit one to rationalize the origins of this Huisgenase activity and of its diastereocontrol.
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Reacción de Cicloadición , Enzimas/metabolismo , BiocatálisisRESUMEN
Protein-based hybrid nanomaterials have recently emerged as promising platforms to fabricate tailored multifunctional biologics for biotechnological and biomedical applications. This work shows a simple, modular, and versatile strategy to design custom protein hybrid nanomaterials. This approach combines for the first time the engineering of a therapeutic protein module with the engineering of a nanomaterial-stabilizing module within the same molecule, resulting in a multifunctional hybrid nanocomposite unachievable through conventional material synthesis methodologies. As the first proof of concept, a multifunctional system was designed ad hoc for the therapeutic intervention and monitoring of myocardial fibrosis. This hybrid nanomaterial combines a designed Hsp90 inhibitory domain and a metal nanocluster stabilizing module resulting in a biologic drug labelled with a metal nanocluster. The engineered nanomaterial actively reduced myocardial fibrosis and heart hypertrophy in an animal model of cardiac remodeling. In addition to the therapeutic effect, the metal nanocluster allowed for in vitro, ex vivo, and in vivo detection and imaging of the fibrotic disease under study. This study evidences the potential of combining protein engineering and protein-directed nanomaterial engineering approaches to design custom nanomaterials as theranostic tools, opening up unexplored routes to date for the next generation of advanced nanomaterials in medicine.
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Combining different therapies into a single nanomaterial platform is a promising approach for achieving more efficient, less invasive, and personalized treatments. Here, we report on the development of such a platform by utilizing nanowires with an iron core and iron oxide shell as drug carriers and exploiting their optical and magnetic properties. The iron core has a large magnetization, which provides the foundation for low-power magnetic manipulation and magnetomechanical treatment. The iron oxide shell enables functionalization with doxorubicin through a pH-sensitive linker, providing selective intracellular drug delivery. Combined, the core-shell nanostructure features an enhanced light-matter interaction in the near-infrared region, resulting in a high photothermal conversion efficiency of >80% for effective photothermal treatment. Applied to cancer cells, the collective effect of the three modalities results in an extremely efficient treatment with nearly complete cell death (â¼90%). In combination with the possibility of guidance and detection, this platform provides powerful tools for the development of advanced treatments.
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Sistemas de Liberación de Medicamentos/métodos , Compuestos Férricos/química , Nanocables/química , Neoplasias/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Doxorrubicina/química , Doxorrubicina/farmacología , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/instrumentación , Humanos , Hipertermia Inducida/instrumentación , Hierro/química , Luz , Fototerapia/instrumentaciónRESUMEN
In this paper we show that conjugation of magnetic nanoparticles (MNPs) with Gemcitabine and/or NucAnt (N6L) fostered their internalization into pancreatic tumor cells and that the coupling procedure did not alter the cytotoxic potential of the drugs. By treating tumor cells (BxPC3 and PANC-1) with the conjugated MNPs and magnetic hyperthermia (43⯰C, 60â¯min), cell death was observed. The two pancreatic tumor cell lines showed different reactions against the combined therapy according to their intrinsic sensitivity against Gemcitabine (cell death, ROS production, ability to activate ERK 1/2 and JNK). Finally, tumors (e.g. 3â¯mL) could be effectively treated by using almost 4.2â¯×â¯105 times lower Gemcitabine doses compared to conventional therapies. Our data show that this combinatorial therapy might well play an important role in certain cell phenotypes with low readiness of ROS production. This would be of great significance in distinctly optimizing local pancreatic tumor treatments.
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Hipertermia Inducida , Nanopartículas de Magnetita/química , Neoplasias Pancreáticas/patología , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Antígeno Ki-67/metabolismo , Nanopartículas de Magnetita/ultraestructura , Ratones Desnudos , Péptidos/farmacología , Fenotipo , Fase S/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Metal nanoclusters (NCs) are considered ideal nanomaterials for biological applications owing to their strong photoluminescence (PL), excellent photostability, and good biocompatibility. This study presents a simple and versatile strategy to design proteins, via incorporation of a di-histidine cluster coordination site, for the sustainable synthesis and stabilization of metal NCs with different metal composition. The resulting protein-stabilized metal NCs (Prot-NCs) of gold, silver, and copper are highly photoluminescent and photostable, have a long shelf life, and are stable under physiological conditions. The biocompatibility of the clusters was demonstrated in cell cultures in which Prot-NCs showed efficient cell internalization without affecting cell viability or losing luminescence. Moreover, the approach is translatable to other proteins to obtain Prot-NCs for various biomedical applications such as cell imaging or labeling.
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Among all new nanomaterials, metal nanoclusters (NCs) have attracted special attention due to their interesting optical properties, among others. Metal NCs have been recently studied and used as sensors for different analytes. However, there is a need to explore the potential of these new sensors in a systematic manner and to develop new systems to broaden the possibilities that sensing offers to the industry. In this work, we show the potential use of repeat protein scaffolds as versatile templates for the synthesis and stabilization of various metal NCs, specifically Au, Ag, and CuNCs. The resulting protein-metal NCs hybrids are evaluated as sensors for different stimuli such as temperature, ions, or reactive oxygen species (ROS). Among the three protein-metal NCs, all performed nicely as temperature sensors, AuNCs responded to metal ions, and AgNCs were able to detect ROS.
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Técnicas Biosensibles/instrumentación , Nanopartículas del Metal/química , Nanoestructuras/química , Proteínas/químicaRESUMEN
Nanomedicine nowadays offers novel solutions in cancer therapy by introducing multimodal treatments in one single formulation. In addition, nanoparticles act as nanocarriers changing the solubility, biodistribution and efficiency of the therapeutic molecules, thus generating more efficient treatments and reducing their side effects. To apply these novel therapeutic approaches, efforts are focused on the multi-functionalization of the nanoparticles and will open up new avenues to advanced combinational therapies. Pancreatic ductal adenocarcinoma (PDAC) is a cancer with unmet medical needs. Abundant expression of the anti-phagocytosis signal CD47 has also been observed on pancreatic cancer cells, in particular a subset of cancer stem cells (CSCs) responsible for resistance to standard therapy and metastatic potential. CD47 receptor is found on pancreatic cancer and highly expressed on CSCs, but not on normal pancreas. Inhibiting CD47 using monoclonal antibodies has been shown as an effective strategy to treat PDAC in vivo. However, CD47 inhibition effectively slowed tumor growth only in combination with Gemcitabine or Abraxane. In this work, we present the generation of multifunctionalized iron oxide magnetic nanoparticles (MNPs) that include the anti-CD47 antibody and the chemotherapeutic drug Gemcitabine in a single formulation. We demonstrate the in vitro efficacy of the formulation against CD47-positive pancreatic cancer cells. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Antígeno CD47/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Portadores de Fármacos , Magnetismo/métodos , Nanopartículas de Magnetita , Nanomedicina/métodos , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Antígeno CD47/inmunología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/química , Desoxicitidina/metabolismo , Desoxicitidina/farmacología , Composición de Medicamentos , Humanos , Nanopartículas de Magnetita/química , Células Madre Neoplásicas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Propiedades de Superficie , Células Tumorales Cultivadas , GemcitabinaRESUMEN
Exploiting and combining different properties of nanomaterials is considered a potential route for next generation cancer therapies. Magnetic nanowires (NWs) have shown good biocompatibility and a high level of cellular internalization. We induced cancer cell death by combining the chemotherapeutic effect of doxorubicin (DOX)-functionalized iron NWs with the mechanical disturbance under a low frequency alternating magnetic field. (3-aminopropyl)triethoxysilane (APTES) and bovine serum albumin (BSA) were separately used for coating NWs allowing further functionalization with DOX. Internalization was assessed for both formulations by confocal reflection microscopy and inductively coupled plasma-mass spectrometry. From confocal analysis, BSA formulations demonstrated higher internalization and less agglomeration. The functionalized NWs generated a comparable cytotoxic effect in breast cancer cells in a DOX concentration-dependent manner, (~60% at the highest concentration tested) that was significantly different from the effect produced by free DOX and non-functionalized NWs formulations. A synergistic cytotoxic effect is obtained when a magnetic field (1 mT, 10 Hz) is applied to cells treated with DOX-functionalized BSA or APTES-coated NWs, (~70% at the highest concentration). In summary, a bimodal method for cancer cell destruction was developed by the conjugation of the magneto-mechanical properties of iron NWs with the effect of DOX producing better results than the individual effects.
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Muerte Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Nanocables/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Magnetismo/métodos , Propilaminas/administración & dosificación , Albúmina Sérica Bovina/administración & dosificación , Silanos/administración & dosificaciónRESUMEN
This chapter will focus on the description of protein-based nanostructures. How proteins can be used as molecular units in order to generate complex materials and structures? What are the key aspects to achieve defined final properties, including shape, stability, function, and order at different length scales by modifying the protein sequence at the modular level?As described in other chapters of the book, we will review the basic concepts and the latest achievements in protein engineering toward nanotechnological applications. Particularly in this chapter the main focus will be on a particular type of proteins, repeat proteins. Because of their modular nature, these proteins are better suited to be used as building blocks than other protein scaffolds. First, we describe general concepts of the protein-based assemblies. Then we introduce repeat proteins and describe the properties that will impact their use in nanotechnology. In particular, we focus on a system based on a synthetic protein, the consensus tetratricopeptide repeat (CTPR). We review recent works from other groups and our group in which the potential of these repeat protein scaffolds is exploited for the fabrication of different protein assemblies, and as biomolecular templates to arrange different molecules and nanoscale objects.
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Nanoestructuras/química , Ingeniería de Proteínas/métodos , Proteínas/química , Secuencias Repetitivas de Aminoácido , Proteínas/genéticaRESUMEN
Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.
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Compuestos Férricos/química , Receptores de Hialuranos/metabolismo , Nanopartículas/química , Línea Celular Tumoral , Química Farmacéutica/métodos , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Compuestos Férricos/administración & dosificación , Humanos , Magnetismo/métodos , Nanomedicina/métodos , Nanopartículas/administración & dosificación , Células Neoplásicas Circulantes/metabolismo , Células Madre Neoplásicas/metabolismo , Distribución Tisular/fisiología , GemcitabinaRESUMEN
Proteins have been used as templates to stabilize fluorescent metal nanoclusters thus obtaining stable fluorescent structures, and their fluorescent properties being modulated by the type of protein employed. Designed consensus tetratricopeptide repeat (CTPR) proteins are suited candidates as templates for the stabilization of metal nanoclusters due to their modular structural and functional properties. Here, we have studied the ability of CTPR proteins to stabilize fluorescent gold nanoclusters giving rise to designed functional hybrid nanostructures. First, we have investigated the influence of the number of CTPR units, as well as the presence of cysteine residues in the CTPR protein, on the fluorescent properties of the protein-stabilized gold nanoclusters. Synthetic protocols to retain the protein structure and function have been developed, since the structural and functional integrity of the protein template is critical for further applications. Finally, as a proof-of-concept, a CTPR module with specific binding capabilities has been used to stabilize gold nanoclusters with positive results. Remarkably, the protein-stabilized gold nanocluster obtained combines both the fluorescence properties of the nanoclusters and the functional properties of the protein. The fluorescence changes in nanoclusters fluorescence have been successfully used as a sensor to detect when the specific ligand was recognized by the CTPR module.