AB toxins as high-affinity ligands for cell targeting in cancer therapy

Conventional targeted therapies for the treatment of cancer have limitations, including the development of acquired resistance. However, novel alternatives have emerged in the form of targeted therapies based on AB toxins. These biotoxins are a diverse group of highly poisonous molecules that show a nanomolar affinity for their target cell receptors, making them an invaluable source of ligands for biomedical applications. Bacterial AB toxins, in particular, are modular proteins that can be genetically engineered to develop high-affinity therapeutic compounds. These toxins consist of two distinct domains: a catalytically active domain and an innocuous domain that acts as a ligand, directing the catalytic domain to the target cells. Interestingly, many tumor cells show receptors on the surface that are recognized by AB toxins, making these high-affinity proteins promising tools for developing new methods for targeting anticancer therapies. Here we describe the structure and mechanisms of action of Diphtheria (Dtx), Anthrax (Atx), Shiga (Stx), and Cholera (Ctx) toxins, and review the potential uses of AB toxins in cancer therapy. We also discuss the main advances in this field, some successful results, and, finally, the possible development of innovative and precise applications in oncology based on engineered recombinant AB toxins

Microtubule cytoskeleton-disrupting activity of MWCNTs: applications in cancer treatment

Microtubules and carbon nanotubes (CNTs), and more particularly multi-walled CNTs (MWCNTs), share many mechanical and morphological similarities that prompt their association into biosynthetic tubulin filaments both, in vitro and in vivo. Unlike CNTs, microtubules are highly dynamic protein polymers that, upon interaction with these nanomaterials, display enhanced stability that has critical consequences at the cellular level. Among others, CNTs prompt ectopic (acentrosomal) microtubule nucleation and the disassembly of the centrosome, causing a dramatic cytoskeletal reorganization. These changes in the microtubule pattern trigger the generation of ineffective biomechanical forces that result in migration defects, and ultimately in spindle-assembly checkpoint (SAC) blockage and apoptosis. In this review, we describe the molecular mechanism involved in the intrinsic interference of CNTs with the microtubule dynamics and illustrate the consequences of this effect on cell biomechanics. We also discuss the potential application of these synthetic microtubule-stabilizing agents as synergetic agents to boost the effect of classical chemotherapy that includes spindle poisons (i.e. paclitaxel) or DNA interfering agents (5-fluorouracil)-, and list some of the advantages of the use of MWCNTs as adjuvant agents in preventing cell resistance to chemotherapy.This research was funded by ISCIII Projects ref. PI19/00349, DTS19/00033, cofunded by European Union FEDER Funds (European Regional Development Fund-ERDF) and IDIVAL for INNVAL19/18, INNVAL 20/13

Drug Nanoparticle Stability Assessment Using Isothermal and Nonisothermal Approaches

Many drugs are administered in the form of liquid-dispersed nanoparticles. Frequently, one of the overlooked aspects in the development of this drug delivery system is the loss of efficacy and the degradation of the carried drugs. Estimating the shelf life of drug products implies the storage of samples under controlled conditions of temperature and humidity for different periods, ranging from months to years, delaying decisions during development, manufacturing, and commercialization. Adapting well-known isothermal and nonisothermal methods to nanoparticles would allow correlating kinetic parameters obtained in a single mathematical model and predicting the shelf life faster than traditional methods. Unlike the traditional approaches, the isoconversional method (i) considers drug products as heterogeneous systems, without a unique kinetic order, (ii) establishes a maximum percentage of degradation, (iii) assumes the same kinetics for all processes regardless of the conditions, and (iv) includes the influence of humidity by a modification of Arrhenius equation. This method serves in calculating the kinetic parameters and shelf life derived from them, in a few weeks. In the same way, nonisothermal treatments allow obtaining these parameters by differential scanning calorimetry. Samples are subjected to different heating rates to establish the temperature at which the thermal decomposition event occurs and, thus, to calculate in a few days the activation energy and the preexponential factor using the Kissinger method. But this approach has limitations: the isoconversional method does not consider crystalline state of the sample, while nonisothermal method ignores the effect of the storage conditions. Processing nanoparticles for isothermal and nonisothermal treatments would allow accurate and fast prediction of the drug-loaded nanoparticle shelf life correlating parameters obtained using a single mathematical model. The accuracy of the prediction would be assessed by comparison of estimated shelf life versus data coming from traditional stability studies

Controlled drug delivery systems for cancer based on mesoporous silica nanoparticles

Abstract: The implementation of nanotechnology in medicine has opened new research horizons particularly in the field of therapeutic delivery. Mesoporous silica particles have emerged as biocompatible drug delivery systems with an enormous potential in the treatment of cancer among many other pathologies. In this review, we focus on the unique properties of these particles as chemotherapy delivery carriers. Here, we summarize the general characteristics of these nanomaterials ? including their physicochemical properties and customizable surfaces ? different stimuli that can be used to trigger targeted drug release, biocompatibility and finally, the drawbacks of these types of nanomaterials, highlighting some of the most important features of mesoporous silica nanoparticles in drug delivery.This work has been supported by the Spanish MINECO and European Union FEDER Funds under Project Ref. PI16/00496 (AES 2016), CTM 2017-84050-R, MAT 2015-69508-P, NanoBioApp Network (MINECO-17-MAT2016-81955-REDT), Xunta de Galicia (Centro Singular de Investigación de Galicia - Accreditation 2016-2019 and EM2014/035), European Union (European Regional Development Fund-ERDF) and IDIVAL INNVAL15/15 and INNVAL17/11

Nanoparticle biocoating to create ATP-powered swimmers capable of repairing proteins on the fly

In this study, we combine nanotechnology and biotechnology to design a biocompatible propulsion system based on the molecular chaperone Hsp90, a heat-shock protein (Hsp) that, in the presence of adenosine 5'-triphosphate (ATP), undergoes nanoscale conformational changes while trapping and renaturing other proteins. We show how, subjected to ATP availability in the medium, Hsp90-functionalized particles significantly enhance their diffusion motion, being able to achieve ballistic motion, while keeping the ability to restore the activity of surrounding heat-inactivated proteins. This biomechanics-based propulsion mechanism represents a promising strategy for the design of self-propelled nanodevices capable of performing sophisticated tasks in live biological contexts that include sensing the environment, recognizing and capturing, folding, and restoring defective proteins on the fly. In the short term, Hsp90-driven nanodevices could be applied to improve industrial processes that require enzymatic catalysis and high temperatures. But in the medium to long term, this bioactive coating could be used in the design of nanomachines that, like mini-robots, navigate the complex body cavities of biological tissues, deliver therapies and/or remove misfolded proteins in disorders such as Alzheimer's or Parkinson's disease.Acknowledgments: The authors acknowledge the financial support from the Spanish Instituto de Salud Carlos iii, and the European Union FEDER funds under Projects ref. PI22/00030, PI19/00349, from the Spanish Ministerio de Ciencia e Innovacion under project PID2020-119242RB-I00 and the European Union H2020-MSCA-RISE-2019 PEPSA-MATE project. ARR and MARD acknowledge financial support from IDIVAL (PREVAL19/04) and the Xunta de Galicia (2017- ED481A/322) respectively. We also acknowledge IDIVAL projects INNVAL19/12 and INNVAL21/1

Development of an accurate method for dispersion and quantification of carbon nanotubes in biological media

Understanding the biological effects triggered by nanomaterials is crucial, not only in nanomedicine but also in toxicology. The dose-response relation is relevant in biological tests due to its use for determining appropriate dosages for drugs and toxicity limits. Carbon nanotubes can trigger numerous unusual biological effects, many of which could have unique applications in biotechnology and medicine. However, their resuspension in saline solutions and the accurate determination of their concentration after dispersion in biological media are major handicaps to identify the magnitude of the response of organisms as a function of this exposure. This difficulty has led to inconsistent results and misinterpretations of their in vivo behavior, limiting their potential use in nanomedicine. The lack of a suitable protocol that allows comparing different studies of the content of carbon nanotubes and their adequate resuspension in culture cell media gives rise to this study. Here, we describe a methodology to functionalize, resuspend and determine the carbon nanotube concentration in biocompatible media based on UV-Vis spectroscopy. This method allows us to accurately estimate the concentration of these resuspended carbon nanotubes, after removing bundles and micrometric aggregates, which can be used as a calibration standard, for dosage-dependent studies in biological systems. This method can also be extended to any other nanomaterial to properly quantify the actual concentration.This work has been funded by the Instituto de Salud Carlos III (ISCiii) (ref. PI16/00496, PI19/00349, DTS19/00033); co-funded by ERDF/ESF, “Investing in your future”; the Spanish MINECO (project ref. PGC2018-101464-B-I00) and MICINN NanoBioApp Network (MINECO-17-MAT2016-81955-REDT). Authors also thank the networks Raman4Clinics (BM1401). CRL thanks the MINECO for the Juan de la Cierva Formación grant (ref. FJCI-2015-25306) and LGL the ISCiii for the Sara Borrell grant (ref. CD17/00105). The authors want to also thank the IDIVAL for financial support (refs. NVAL18/07, INNVAL18/28) and technical support

Engineering Sub-Cellular Targeting Strategies to Enhance Safe Cytosolic Silica Particle Dissolution in Cells

Mesoporous silica particles (MSP) are major candidates for drug delivery systems due to their versatile, safe, and controllable nature. Understanding their intracellular route and biodegradation process is a challenge, especially when considering their use in neuronal repair. Here, we characterize the spatiotemporal intracellular destination and degradation pathways of MSP upon endocytosis by HeLa cells and NSC-34 motor neurons using confocal and electron microscopy imaging together with inductively-coupled plasma optical emission spectroscopy analysis. We demonstrate how MSP are captured by receptor-mediated endocytosis and are temporarily stored in endo-lysosomes before being finally exocytosed. We also illustrate how particles are often re-endocytosed after undergoing surface erosion extracellularly. On the other hand, silica particles engineered to target the cytosol with a carbon nanotube coating, are safely dissolved intracellularly in a time scale of hours. These studies provide fundamental clues for programming the sub-cellular fate of MSP and reveal critical aspects to improve delivery strategies and to favor MSP safe elimination. We also demonstrate how the cytosol is significantly more corrosive than lysosomes for MSP and show how their biodegradation is fully biocompatible, thus, validating their use as nanocarriers for nervous system cells, including motor neurons.This research was funded by ISCIII Projects ref. PI16/00496, PI19/00349, DTS19/00033, co-funded by ERDF/ESF, "Investing in Your Future"; and MICINN Projects ref. CTM2017-84050-R, NanoBioApp Network (MINECO-17-MAT2016-81955-REDT), COST action Nano2Clinic CA17140, Xunta de Galicia (Centro Singular de Investigación de Galicia-Accreditation 2016-2019 and EM2014/035), European Union FEDER Funds (European Regional Development Fund-ERDF) and IDIVAL for INNVAL 17/11, INNVAL18/28, INNVAL19/18 and the technical support

Solid Lipid Particles for Lung Metastasis Treatment

Solid lipid particles (SLPs) can sustainably encapsulate and release therapeutic agents over long periods, modifying their biodistribution, toxicity, and side effects. To date, no studies have been reported using SLPs loaded with doxorubicin chemotherapy for the treatment of metastatic cancer. This study characterizes the effect of doxorubicin-loaded carnauba wax particles in the treatment of lung metastatic malignant melanoma in vivo. Compared with the free drug, intravenously administrated doxorubicin-loaded SLPs significantly reduce the number of pulmonary metastatic foci in mice. In vitro kinetic studies show two distinctive drug release profiles. A first chemotherapy burst-release wave occurs during the first 5 h, which accounts for approximately 30% of the entrapped drug rapidly providing therapeutic concentrations. The second wave occurs after the arrival of the particles to the final destination in the lung. This release is sustained for long periods (>40 days), providing constant levels of chemotherapy in situ that trigger the inhibition of metastatic growth. Our findings suggest that the use of chemotherapy with loaded SLPs could substantially improve the effectiveness of the drug locally, reducing side effects while improving overall survival.This research was funded by the European Regional Development Fund (ERDF) and the Spanish MINECO Refs. PI16/00496 (AES 2016), PI19/00349 (AES 2019), and DTS19/00033; IDIVAL Refs. INNVAL17/11 and INNVAL19/12. J.G. and M.B.-L. also acknowledge financial support from the Fundação para a Ciência e a Tecnologia and the ERDF through NORTE2020 (2014–2020 North Portugal Regional Operational Program) through the projects UTAP-EXPL/NTec/0038/2017 (NANOTHER) and NORTE-01-0145-FEDER-031142 (MAGTARGETON). Nano2clinics COST Action CA17140

TBCD Links Centriologenesis, Spindle Microtubule Dynamics, and Midbody Abscission in Human Cells

Microtubule-organizing centers recruit α- and β-tubulin polypeptides for microtubule nucleation. Tubulin synthesis is complex, requiring five specific cofactors, designated tubulin cofactors (TBCs) A–E, which contribute to various aspects of microtubule dynamics in vivo. Here, we show that tubulin cofactor D (TBCD) is concentrated at the centrosome and midbody, where it participates in centriologenesis, spindle organization, and cell abscission. TBCD exhibits a cell-cycle-specific pattern, localizing on the daughter centriole at G1 and on procentrioles by S, and disappearing from older centrioles at telophase as the protein is recruited to the midbody. Our data show that TBCD overexpression results in microtubule release from the centrosome and G1 arrest, whereas its depletion produces mitotic aberrations and incomplete microtubule retraction at the midbody during cytokinesis. TBCD is recruited to the centriole replication site at the onset of the centrosome duplication cycle. A role in centriologenesis is further supported in differentiating ciliated cells, where TBCD is organized into “centriolar rosettes”. These data suggest that TBCD participates in both canonical and de novo centriolar assembly pathways

Composición de nanofilamentos para el tratamiento de tumores

Composición de nanofilamentos para el tratamiento de tumores. La presente invención se refiere a composiciones que comprenden nanofilamentos y al menos un excipiente para su empleo en el tratamiento antitumoral, donde los nanofilamentos, que actúan como agentes neoplásicos, están funcionalizados con suero procedente de sangre humana. Asimismo, se contempla el procedimiento de obtención de dichas composiciones.Solicitud: 201400254 (27.03.2014)Nº Pub. de Solicitud: ES2478793A1 (22.07.2014)Nº de Patente: ES2478793B2 (23.01.2015