WO2021022387A1 - Stable fluorescent nanodiamonds for the detection of biomarkers for alzheimer's disease - Google Patents

Stable fluorescent nanodiamonds for the detection of biomarkers for alzheimer's disease Download PDF

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WO2021022387A1
WO2021022387A1 PCT/CL2019/050070 CL2019050070W WO2021022387A1 WO 2021022387 A1 WO2021022387 A1 WO 2021022387A1 CL 2019050070 W CL2019050070 W CL 2019050070W WO 2021022387 A1 WO2021022387 A1 WO 2021022387A1
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peptide
marker according
nds
fnds
fluorescence
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PCT/CL2019/050070
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Spanish (es)
French (fr)
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Jerónimo MAZE RÍOS
Alejandra Beatriz ÁLVAREZ ROJAS
América Valeska CHANDÍA CRISTI
Nathalie Valeska CASANOVA MORALES
Raúl Manuel GONZÁLEZ BROUWER
Marcelo Javier KOGAN
Francisco Andrés MORALES ZAVALA
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Pontificia Universidad Católica De Chile
Universidad De Chile
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Priority to PCT/CL2019/050070 priority Critical patent/WO2021022387A1/en
Publication of WO2021022387A1 publication Critical patent/WO2021022387A1/en

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    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/25—Diamond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
    • C30B29/02—Elements
    • C30B29/04—Diamond
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention is for more stable nanodiamond-based fluorescent markers harboring color centers for detecting beta-amyloid, a precursor to Alzheimer's disease.
  • the invention has useful applications in molecular diagnostics, biomedical, and nanotechnology fields.
  • fluorescent markers Since the discovery of Green Fluorescent Protein (GFP) in 1962, fluorescent markers have revolutionized the field of bioimaging. These markers have endowed different biomolecules and cells with the ability to fluoresce and are therefore detectable by conventional light microscopes. Fluorescent markers have made possible the localization of otherwise invisible organelles, the tracking of biomolecules within the cell, the study of chemical reactions of various biological processes, and the analysis of molecular interactions using fluorescent resonance energy transfer ( FRET), among other examples. Although all these new applications and techniques have greatly impacted the fields of biology and chemistry, the use and development of fluorescent markers still faces great challenges. Several fluorescent markers based on molecules and proteins exhibit photo-whitening and intermittency, reducing the reliability of the studies in which they are used.
  • Fluorescent markers should be linked or conjugated in order to label a specific molecule, organelle or study a specific process.
  • Nps nanoparticles
  • Passive targeting relies on the inherent properties of nanoparticles or abnormalities in tissue that allow them accumulate in specific locations as in the case of the Increased Permeability and Retention (EPR) effect present in some tumors.
  • Active targeting is based on the functionalization of the surface of nanoparticles with signaling molecules.
  • nanoparticles functionalized with different ligands such as small molecules, polysaccharides, peptides, proteins or even antibodies have been developed for diagnostic and therapeutic applications. Nanoparticles have been used, for example, in preclinical studies to attack tumors, improve drug delivery, and remove amyloid aggregates associated with Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • AD the most common form of dementia in the elderly, is a progressive neurodegenerative disorder characterized by cognitive and memory deficits.
  • AD brains mainly show the presence of senile plaques composed of aggregates of the Ab peptide
  • several types of nanoparticles have been proposed for the detection of this peptide as a highly specific biomarker for AD.
  • fluorescent markers available on the market, among them we find: Alexa Fluor 555, Alexa 488 and FITC. All of them show unstable fluorescence, especially when subjected to high-power excitation.
  • many color markers have a short life span compared to the time scale required in biological studies to draw safe conclusions. Therefore, stable and non-toxic fluorescent markers are necessary as tools to enable long and reliable biological studies. Said markers must not only exhibit a long lifetime under various test conditions and not exhibit photobleaching or intermittency, but also allow their conjugation or functionalization, which is a crucial step to be used in applications such as cell tracking, detection biomarkers and drug delivery.
  • the present invention would overcome the problems present in the state of the art and consists of stable and non-cytotoxic fluorescent markers based on nanodiamonds (Nds) functionalized with a bifunctional peptide, which is formed by a cell-penetrating peptide and a peptide of 6 amino acids of long beta leaf disruptor that is capable of recognizing aggregates of beta amiliode (Ab), a biomarker for Alzheimer's disease.
  • Nds nanodiamonds
  • Ab beta amiliode
  • patents US2017316487A1, US9616022B1, US2016138077A1 and US2014314850A1 were considered the most related to the present invention.
  • Patent US2017316487A1 describes a multitude of options for different applications. It is a document that in its description is extremely broad. In this document, nanodiamonds that can be functionalized are mentioned. In this case, functionalization ensures that the nanoparticles can enter different types of cells. However, this functionalization is not performed with peptides. The functionalization and the targeting molecule do not correspond to what is described in the present invention.
  • US9616022B1 describes nanodiamonds used for drug delivery. The content indicates that in the tests of modified nanodiamonds with the drug EFV, it was possible to cross the blood-brain barrier, and it also showed low toxicity. In addition, it is mentioned that nanodiamonds can be functionalized, and molecules with biological activity can be included, where, among the alternatives, they mention amino acids and proteins, among others.
  • US2016138077A1 describes nanodiamonds conjugated to fluorescent molecules. Alzheimer's is mentioned in the list of cited references. The content of the document indicates that the nanodiamond can be functionalized with a molecule that can be detected in the equipment of magnetic resonance imaging (MRI, for its acronym in English). Finally, document US2014314850A1 describes nanodiamonds for the release of nucleic acids for therapeutic purposes. The functionalization is indicated to be carried out with amino acids. In the content of the document, AD is mentioned as one of the diseases that can be treated with functionalized nanodiamonds.
  • the present invention consists of functionalized NDs (fNDs) - which harbor stable fluorescent color centers-, with a bifunctional peptide that is capable of recognizing extracellular amyloid beta aggregates (Ab) and their accumulation, which is believed to be the true reason for the Neuronal damage and cognitive decline in AD.
  • fNDs functionalized NDs
  • Ab extracellular amyloid beta aggregates
  • FNDs are non-cytotoxic and allow ultrasensitive detection (using picomolar concentrations of Nds) of amyloid fibrils and amyloid aggregates. Furthermore, the fluorescence of fNDs is more stable than the common color markers used to stain Ab such as Thioflavin T.
  • the present invention is a stable and non-cytotoxic fluorescent marker consisting of nanodiamonds with fluorescent color centers (fND) and a bifunctional peptide, which is constructed by a cell-penetrating peptide and a beta-sheet-breaking peptide 6 amino acids long that recognizes aggregates of beta amiliode that functions as a biomarker for AD.
  • fND fluorescent color centers
  • bifunctional peptide which is constructed by a cell-penetrating peptide and a beta-sheet-breaking peptide 6 amino acids long that recognizes aggregates of beta amiliode that functions as a biomarker for AD.
  • the NDs used in the biomarker described in the present invention have unconditionally stable fluorescence - even after many months under continuous wave excitation - they are biologically and chemically inert, and could serve as sub-wavelength resolution sensors.
  • diamonds based on fluorescent markers use centers of color defects as their active emitting part.
  • a common color center is the nitrogen-vacant (NV) center whose atomic structure is shown in Figure 1a. They can be approximated as a two-level system that after laser excitation at 532 nm show a wide emission around 700 nm ( Figure 1b).
  • the NDs used in the present invention contain nitrogen-vacant color centers that exist in two different charge states; neutrally charged (NVO) and negatively charged (NV-) ( Figure 1c).
  • the surface of NDs is functionalized with a bifunctional R7-CLPFFD peptide composed of the CLPFFD peptide and a RRRRRRR (R7) peptide.
  • the CLPFFD peptide is a beta sheet disruptor that recognizes toxic extracellular aggregates of amyloid Ab peptide present in the brain of AD patients.
  • the R7 section is a cell penetrating peptide (CPP) that enhances cellular uptake of its cargo.
  • CPP cell penetrating peptide
  • oligoarginines have been used to enhance drug delivery, such as insulin when administered intranasally. These CPPs are useful for the treatment of diseases that require the crossing of different types of cellular barriers, such as the blood-brain barrier (blood-brain barrier) in AD.
  • it is a fluorescent marker.
  • the fluorescent markers are based on NDs.
  • the NDs have a diameter of between 20 to 50 nm.
  • the fluorescent marker of the present invention uses 35 nm diameter NDs.
  • the NDs contain nitrogen-vacant color centers.
  • each ND contains an average of 15 nitrogen-vacant color centers.
  • the nitrogen-vacancy (NV) centers of the nanodiamonds exist in two different charge states: neutrally charged (NVO) and negatively charged (NV-) with zero phonon lines at 575 and 637 nm respectively, under a laser excitation of 532 nm ( Figure 1c).
  • the emission of NDs lies in the biological window of the tissues.
  • the NDs used in the present invention have unconditionally stable fluorescence, even after many months under continuous wave excitation.
  • the NDs used in the present invention are biologically and chemically inert, and could serve as sensors with sub-wavelength resolution.
  • the fluorescence of the ND does not show intermittency or photobleaching.
  • the fluorescence of the NDs is more stable than the common color markers used, such as Thioflavin T and FITC, among others.
  • the stable fluorescence of the NDs allows their detection at concentrations as low as picomolar using confocal microscopy.
  • the NDs are functionalized with a peptide.
  • the NDs are functionalized with a bifunctional peptide.
  • the bifunctional peptide confers different characteristics and functionalities to the NDs.
  • the bifunctional peptide is composed of two segments.
  • sequence of the bifunctional peptide is R7-CLPFFD.
  • the R7-CLPFFD peptide is composed of two segments: the CLPFFD segment and the R7 segment.
  • the CLPFFD segment has the ability to recognize Ab aggregates.
  • the CLPFFD segment includes hydrophobic residues Leu (L), Phe (F), and Phe (F).
  • the Asp (D) residue of the CLPFFD segment confers antipathy and a net charge of -1 to the molecule ( Figure 2a), increasing its solubility.
  • the R7 segment highly positive due to the presence of arginine (secondary amines), favors cell penetration.
  • the R7 peptide allows the crossing of cell membranes.
  • segment R7 has a net charge of +7.
  • the net positive charge of the R7 segment allows electrostatic bonding between the peptide and the negatively charged surface of the NDs ( Figure 2) containing carboxylate groups.
  • the surface of nanodiamonds containing fluorescent color centers can be functionalized to perform non-trivial and multiple tasks without damaging the stability of their fluorescence.
  • the fNDs are not cytotoxic.
  • the fNDs are capable of penetrating cells without affecting cell viability.
  • the fNDs could go across the blood-brain barrier.
  • the fNDs serve as a fluorescent marker for the detection of AD biomarkers.
  • the functionalized NDs bind fibrils of the Ab peptide and can be used for the indirect detection of extracellular Ab aggregates, associated with AD.
  • this functionalization of the peptide of NDs could be used in reliable and long-term experiments to detect Ab aggregates and follow their formation in AD.
  • Figure 1 Emission properties of NV color centers in diamond
  • FIG. 1 Functionalized nanodiamonds.
  • FIG. 1 Structure of nanodiamonds. HR-TEM. Electron micrographs showing (a) NDs and (b) fNDs.
  • FIG. 4 Cell internalization of fNDs in fibroblast cell line
  • APD avalanche photon detector
  • FIG. 1 Cell internalization of fNDs in the bEnd.3 cell line. Image of bEnd.3 cells incubated with NDs (upper panel) and fNDs (lower panel) at two concentrations (2 and 20 pM) for 6 hours.
  • Figure 6 Characterization of photo stability of the 555 Alexa Fluor conjugate and diamond-based color marker
  • Figure 7 The functionalization of NDs does not affect cell viability. Measurements of cell viability evaluated through the MTS reduction assay for cell lines (a) HT22 and (c) C3 10T1 / 2 incubated under different concentrations of fNDs (black bars) for 24 hours and non-functionalized NDs (gray bars) . Additional tests under high concentrations of non-functionalized nanodiamonds were performed for the cell lines (b) HT22 and (d) C3 10T1 / 2. The values correspond to the average percentage of viable cells relative to control cells (open bars). The error bars indicate the standard deviation estimated from three experiments each carried out in triplicate.
  • Example 1 Evaluation of the binding between the CLPFFD R7 peptide, peptide and the surface of NDs.
  • the RRR RRRRCLPFFD peptide was dissolved in ultra-pure Milli-Q water at a final concentration of 0.05 mg / ml.
  • the nanodiamonds were added to the solution of this peptide, remaining in a concentration of 0.8 nM in the final solution, and then they were incubated with vigorous shaking for 2 h.
  • the adsorption of the peptide on the surface of the nanocrystal was evaluated by the change in zeta potential (pZ) and hydrodynamic diameter (Dh) as an indicator of the electrostatic coating for this bifunctional peptide.
  • the colloidal suspension was centrifuged and washed three times.
  • HR-TEM transmission electron microscopy
  • Example 2 Evaluation of interactions of fNDs with cells and their properties as color markers.
  • fibroblasts 30,000 cells / ml were incubated with 8 pM fNDs. After 6 hours the samples were washed and fixed. In order to visualize the cells, microtubules (components of the cytoskeleton) were immunostained using anti beta tubulin antibodies (1: 1000) and Alexa Fluor 488 conjugated secondary antibodies (1: 1000). The samples were analyzed in a home confocal microscope setup equipped with an avalanche photon detector (APD) and an optical spectrometer.
  • APD avalanche photon detector
  • BEnd.3 cells are cerebral vascular endothelial cells and are commonly used in different in vitro models of drug transport across the blood-brain barrier.
  • BEnd.3 cells (ATCC CRL-2299) were incubated with nanodiamonds in a concentration range of 2 to 20 pM using both functionalized and non-functionalized nanodiamonds as a control.
  • Figure 4a shows a representative image of fibroblast cells incubated with fNDs.
  • this configuration allowed to use concentrations of NDs in the pM range and could even detect NDs containing unique emitters.
  • the analyzes of different regions of interest of the sample clearly show two distinguishable spectra: one corresponding to Alexa Fluor 488, and the other corresponding to fND ( Figure 4b and c, respectively).
  • the fNDs and Alexa 488 were observed in the same focal plane.
  • Figure 4a shows fNDs within the perimeter of the cell where no fNDs were observed outside the cell (washed samples) suggesting that the fNDs are within the cell. Furthermore, no fNDs were observed in the cell nucleus.
  • Example 3 Evaluation of the fluorescence stability of fNDs.
  • Example 4 Effects of fNDs on cell viability.
  • HT22 hippocampal neurons and C3 10T1 / 2 fibroblast cells were incubated with different concentrations of fNDs for 24 hours. Then, cell viability was measured using the [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS) assay.
  • MTS test is based on the estimation of the reduction of tetrazolium salts by cellular respiration of viable cells that generates as a purple formazan product that can be quantified at 492 nm. The purple product was measured at 492 nm using a reader (Autobio Phomo). The percentage reduction in MTT was compared to control cells not exposed to the material, which represented 100% of the reduction in MTT.
  • Example 5 Binding of fNDs to Ab fibers.

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Abstract

The present invention consists of functionalised NDs (fNDs) -housing stable fluorescent colour centres- with a bifunctional peptide capable of recognising extracellular amyloid-beta aggregates (Aß) and the accumulation thereof, which is considered the true cause of neuronal damage and cognitive decline in AD. fNDs are not cytotoxic and enable ultrasensitive detection (using picomolar Nd concentrations) of amyloid fibrils and amyloid aggregates. Moreover, the fluorescence of fNDs is more stable than the common colour markers used to dye Aß, such as Thioflavin T.

Description

NANODIAMANTES FLUORESCENTES ESTABLES PARA LA DETECCIÓN DE BIOMARCADORES DE LA ENFERMEDAD DE ALZHEIMER STABLE FLUORESCENT NANODIAMANTS FOR THE DETECTION OF BIOMARKERS OF ALZHEIMER'S DISEASE
Campo de la Invención Field of Invention
La presente invención es de marcadores fluorescentes más estables con base en nanodiamantes, que albergan centros de color, para detectar el beta-amiloide, un precursor de la enfermedad de Alzheimer. La invención tiene aplicaciones útiles en el diagnóstico molecular, biomédico y en campos nanotecnológicos. The present invention is for more stable nanodiamond-based fluorescent markers harboring color centers for detecting beta-amyloid, a precursor to Alzheimer's disease. The invention has useful applications in molecular diagnostics, biomedical, and nanotechnology fields.
Antecedentes Background
Desde el descubrimiento de la Proteína Fluorescent Verde (GFP, por sus siglas en inglés) en 1962, marcadores fluorescentes han revolucionado el campo de la bioimagen. Estos marcadores han dotado a diferentes biomoléculas y células de la capacidad de fluorescencia y por lo tanto son detectables por los microscopios ópticos convencionales. Marcadores fluorescentes han hecho posible la localización de orgánulos de otra forma invisibles, el rastreo de biomoléculas en el interior de la célula, el estudio de reacciones químicas de varios procesos biológicos, y el análisis de interacciones moleculares utilizando la transferencia de energía de resonancia fluorescente (FRET, por sus siglas en inglés), entre otros ejemplos. Aunque todas estas nuevas aplicaciones y técnicas han impactado enormemente en los campos de biología y química, el uso y desarrollo de marcadores fluorescentes todavía se enfrenta a grandes desafíos. Varios marcadores fluorescentes basados en moléculas y proteínas presentan foto blanqueamiento e intermitencia, disminuyendo la fiabilidad de los estudios en que se utilizan. Aunque el desarrollo de marcadores fluorescentes más estables, como los puntos cuánticos (QD, por sus siglas en inglés) han mostrado grandes progresos durante los últimos 5 años, muchos marcadores de color basados en semiconductores todavía son tóxicos para la célula. Asimismo, varios marcadores de color tienen un tiempo de vida corto en comparación con la escala de tiempo requerida para estudios biológicos con el fin de llegar a conclusiones fiables. Por lo tanto, marcadores fluorescentes estables son esenciales para experimentos largos. Por otro lado, la capacidad de un marcador para fluorescer no es suficiente. Marcadores fluorescentes deberían estar unidos o conjugados con el fin de etiquetar una molécula específica, orgánulo o estudiar un proceso específico. Por ejemplo, varias nanopartículas (Nps) has sido diseñadas para ser utilizadas en aplicaciones biomédicas y nanotecnologicas específicas dirigiéndolos al lugar correcto dentro del cuerpo, ya sea mediante direccionamiento pasivo o activo. El direccionamiento pasivo se basa en las propiedades inherentes de nanopartículas o anormalidades en el tejido que les permiten acumular en ubicaciones específicas como en el caso del efecto de Permeabilidad y Retención Aumentada (EPR, por sus siglas en inglés) presentes en algunos tumores. El direccionamiento activo se basa en la funcionalización de la superficie de nanopartículas con moléculas de señalización. En los últimos 30 años, varias nanopartículas dirigidas funcionalizadas con diferentes ligandos como moléculas pequeñas, polisacáridos, péptidos, proteínas o incluso anticuerpos han sido desarrolladas para aplicaciones diagnósticas y terapéuticas. Las nanopartículas se han utilizado, por ejemplo, en estudios preclínicos para atacar tumores, mejorar la entrega de medicamentos, y eliminar agregados amiloides relacionados con la enfermedad de Alzheimer (AD, por sus siglas en inglés). Since the discovery of Green Fluorescent Protein (GFP) in 1962, fluorescent markers have revolutionized the field of bioimaging. These markers have endowed different biomolecules and cells with the ability to fluoresce and are therefore detectable by conventional light microscopes. Fluorescent markers have made possible the localization of otherwise invisible organelles, the tracking of biomolecules within the cell, the study of chemical reactions of various biological processes, and the analysis of molecular interactions using fluorescent resonance energy transfer ( FRET), among other examples. Although all these new applications and techniques have greatly impacted the fields of biology and chemistry, the use and development of fluorescent markers still faces great challenges. Several fluorescent markers based on molecules and proteins exhibit photo-whitening and intermittency, reducing the reliability of the studies in which they are used. Although the development of more stable fluorescent markers such as quantum dots (QDs) have shown great progress over the past 5 years, many semiconductor-based color markers are still toxic to the cell. Also, several color markers have a short life span compared to the time scale required for biological studies in order to reach reliable conclusions. Therefore, stable fluorescent markers are essential for long experiments. On the other hand, the ability of a marker to fluoresce is not sufficient. Fluorescent markers should be linked or conjugated in order to label a specific molecule, organelle or study a specific process. For example, several nanoparticles (Nps) have been designed to be used in specific biomedical and nanotechnological applications by directing them to the correct place within the body, either through passive or active targeting. Passive targeting relies on the inherent properties of nanoparticles or abnormalities in tissue that allow them accumulate in specific locations as in the case of the Increased Permeability and Retention (EPR) effect present in some tumors. Active targeting is based on the functionalization of the surface of nanoparticles with signaling molecules. In the last 30 years, several targeted nanoparticles functionalized with different ligands such as small molecules, polysaccharides, peptides, proteins or even antibodies have been developed for diagnostic and therapeutic applications. Nanoparticles have been used, for example, in preclinical studies to attack tumors, improve drug delivery, and remove amyloid aggregates associated with Alzheimer's disease (AD).
AD, la forma de demencia más común en personas de edad avanzada, es un trastorno neurodegenerativo progresivo caracterizado por deficiencias cognitivas y de memoria. Como los cerebros con AD muestran principalmente la presencia de placas seniles compuestas de agregados del péptido Ab, varios tipos de nanopartículas han sido propuestos para la detección de este péptido como un biomarcador altamente específico para AD. Actualmente, encontramos varios marcadores fluorescentes disponibles en el mercado, entre ellos encontramos: Alexa Fluor 555, Alexa 488 y FITC. Todos ellos muestran fluorescencia inestable, especialmente cuando están sometidos a una excitación de gran potencia. Como se mencionó antes, muchos marcadores de color presentan un tiempo de vida corto en comparación con la escala de tiempo requerida en estudios biológicos para sacar conclusiones seguras. Por lo tanto, marcadores fluorescentes estables y no tóxicos son necesarios como herramientas para permitir estudios biológicos largos y confiables. Dichos marcadores no sólo deben exhibir un tiempo de vida largo bajo varias condiciones de ensayo y no presentar foto blanqueamiento o intermitencia, pero también, permitir su conjugación o funcionalización, lo cual es una etapa crucial para ser utilizados en aplicaciones tal como rastreo celular, detección de biomarcadores y la entrega de los fármacos. AD, the most common form of dementia in the elderly, is a progressive neurodegenerative disorder characterized by cognitive and memory deficits. As AD brains mainly show the presence of senile plaques composed of aggregates of the Ab peptide, several types of nanoparticles have been proposed for the detection of this peptide as a highly specific biomarker for AD. Currently, we find several fluorescent markers available on the market, among them we find: Alexa Fluor 555, Alexa 488 and FITC. All of them show unstable fluorescence, especially when subjected to high-power excitation. As mentioned before, many color markers have a short life span compared to the time scale required in biological studies to draw safe conclusions. Therefore, stable and non-toxic fluorescent markers are necessary as tools to enable long and reliable biological studies. Said markers must not only exhibit a long lifetime under various test conditions and not exhibit photobleaching or intermittency, but also allow their conjugation or functionalization, which is a crucial step to be used in applications such as cell tracking, detection biomarkers and drug delivery.
La presente invención superaría los problemas presentes en el estado del arte y consiste en marcadores fluorescentes estables y no citotóxicos basados en nanodiamantes (Nds) funcionalizadas con un péptido bifuncional, el cual está formado por un péptido de penetración celular y un péptido de 6 aminoácidos de largo disrruptor de hojas beta que es capaz de reonocer agregados de beta amiliode (Ab), un biomarcador para la enfermedad de Alzheimer. Para evaluar los méritos de la invención descritos en este documento, se presenta a continuación un breve resumen de los documentos más relevantes presentes en el arte para la presente invención. The present invention would overcome the problems present in the state of the art and consists of stable and non-cytotoxic fluorescent markers based on nanodiamonds (Nds) functionalized with a bifunctional peptide, which is formed by a cell-penetrating peptide and a peptide of 6 amino acids of long beta leaf disruptor that is capable of recognizing aggregates of beta amiliode (Ab), a biomarker for Alzheimer's disease. In order to evaluate the merits of the invention described in this document, a brief summary of the most relevant documents present in the art for the present invention is presented below.
La búsqueda se centró en documentos relacionados con los campos de nanotecnología; usos específicos o aplicaciones de nanoestructuras; medición o análisis de nanoestructuras; fabricación o tratamiento de nanoestructuras; nanobiotecnología o nanomedicina; nanotecnología para interactuar, detectar y actuar; tecnología de nanodiamantes y en AD. The search focused on documents related to the fields of nanotechnology; specific uses or applications of nanostructures; measurement or analysis of nanostructures; fabrication or treatment of nanostructures; nanobiotechnology or nanomedicine; nanotechnology to interact, detect and act; nanodiamond technology and in AD.
En general, existen documentos que divulgan el uso de nanodiamantes para diferentes propósitos. Sin embargo, el análisis realizado sugiere que no hay ningún sistema en el arte que posee todos los componentes que conforman la presente invención. In general, there are documents that disclose the use of nanodiamonds for different purposes. However, the analysis carried out suggests that there is no system in the art that has all the components that make up the present invention.
Entre los documentos encontrados, las patentes US2017316487A1 , US9616022B1 , US2016138077A1 y US2014314850A1 fueron considerados los más relacionados con la presente invención. Among the documents found, patents US2017316487A1, US9616022B1, US2016138077A1 and US2014314850A1 were considered the most related to the present invention.
La patente US2017316487A1 describe una multitud de opciones para diferentes aplicaciones. Es un documento que en su descripción es extremadamente amplio. En este documento, se mencionan los nanodiamantes que pueden ser funcionalizados. En este caso, la funcionalización procura que las nanopartículas puedan entrar en diferentes tipos de células. Sin embargo, esta funcionalización no es realizada con péptidos. La funcionalización y la molécula de direccionamiento no corresponden a lo descrito en la presente invención. Patent US2017316487A1 describes a multitude of options for different applications. It is a document that in its description is extremely broad. In this document, nanodiamonds that can be functionalized are mentioned. In this case, functionalization ensures that the nanoparticles can enter different types of cells. However, this functionalization is not performed with peptides. The functionalization and the targeting molecule do not correspond to what is described in the present invention.
El documento US9616022B1 describe nanodiamantes usados para la liberación de fármacos. En el contenido se indica que en las pruebas de nanodiamantes modificados con el fármaco EFV, fue posible cruzar la barrera sangre-cerebro, y además mostró una baja toxicidad. Además, se menciona que los nanodiamantes pueden ser funcionalizados, y se pueden incluir moléculas con actividad biológica, en donde, entre las alternativas, mencionan aminoácidos y proteínas, entre otros. US9616022B1 describes nanodiamonds used for drug delivery. The content indicates that in the tests of modified nanodiamonds with the drug EFV, it was possible to cross the blood-brain barrier, and it also showed low toxicity. In addition, it is mentioned that nanodiamonds can be functionalized, and molecules with biological activity can be included, where, among the alternatives, they mention amino acids and proteins, among others.
El documento US2016138077A1 describe nanodiamantes conjugados con moléculas fluorescentes. Se menciona el Alzheimer en la lista de las referencias citadas. El contenido del documento indica que el nanodiamante puede ser funcionalizado con una molécula que puede ser detectado en el equipo de imagen de resonancia magnética (MRI, por sus siglas en inglés). Finalmente, el documento US2014314850A1 describe nanodiamantes para la liberación de ácidos nucleicos con fines terapéuticos. Se indica que la funcionalización se lleva a cabo con aminoácidos. En el contenido del documento, AD es mencionada como una de las enfermedades que pueden ser tratadas con nanodiamantes funcionalizadas. US2016138077A1 describes nanodiamonds conjugated to fluorescent molecules. Alzheimer's is mentioned in the list of cited references. The content of the document indicates that the nanodiamond can be functionalized with a molecule that can be detected in the equipment of magnetic resonance imaging (MRI, for its acronym in English). Finally, document US2014314850A1 describes nanodiamonds for the release of nucleic acids for therapeutic purposes. The functionalization is indicated to be carried out with amino acids. In the content of the document, AD is mentioned as one of the diseases that can be treated with functionalized nanodiamonds.
En resumen, se encontró que ninguna patente previa describe un marcador fluorescente con las mismas características que el marcador de la presente invención. Sin embargo, en la búsqueda de publicaciones científicas se encontró la publicación hecha por los inventores, publicado el 10 de agosto de 2018 (Morales-Zavala y cois., 2018). J Nanobiotechnology. Vol. 16(1):60. doi: 10.1186/s12951 -018-0385-7). In summary, it was found that no previous patent describes a fluorescent marker with the same characteristics as the marker of the present invention. However, in the search for scientific publications, the publication made by the inventors was found, published on August 10, 2018 (Morales-Zavala et al., 2018). J Nanobiotechnology. Vol. 16 (1): 60. doi: 10.1186 / s12951 -018-0385-7).
Resumen de la invención Summary of the invention
La presente invención consiste en NDs funcionalizados (fNDs) - que albergan centros de color fluorescente estable-, con un péptido bifuncional que es capaz de reconocer agregados beta amiloide extracelular (Ab) y su acumulación, lo cual se cree que es la verdadera razón del daño neuronal y la declinación cognitiva en AD. The present invention consists of functionalized NDs (fNDs) - which harbor stable fluorescent color centers-, with a bifunctional peptide that is capable of recognizing extracellular amyloid beta aggregates (Ab) and their accumulation, which is believed to be the true reason for the Neuronal damage and cognitive decline in AD.
Los fNDs no son citotóxicos y permiten la detección ultrasensible (mediante el uso de concentraciones picomolar de Nds) de fibrillas amiloides y agregados de amiloide. Más aún, la fluorescencia de fNDs es más estable que los marcadores de color común utilizados para teñir Ab tal como la Tioflavina T. FNDs are non-cytotoxic and allow ultrasensitive detection (using picomolar concentrations of Nds) of amyloid fibrils and amyloid aggregates. Furthermore, the fluorescence of fNDs is more stable than the common color markers used to stain Ab such as Thioflavin T.
Descripción detallada de la invención Detailed description of the invention
La presente invención es un marcador fluorescente estable y no citotóxico que consiste en nanodiamantes con centros de color fluorescente (fND) y un péptido bifuncional, el cual es construido por un péptido de penetración celular y un péptido de 6aminoácidos de largo que rompe hojas beta que reconoce agregados de beta amiliode que funciona como un biomarcador de AD. The present invention is a stable and non-cytotoxic fluorescent marker consisting of nanodiamonds with fluorescent color centers (fND) and a bifunctional peptide, which is constructed by a cell-penetrating peptide and a beta-sheet-breaking peptide 6 amino acids long that recognizes aggregates of beta amiliode that functions as a biomarker for AD.
Los NDs utilizados en el biomarcador descrito en la presente invención, tienen una fluorescencia incondicionalmente estable - incluso después de muchos meses bajo una excitación de onda continua - son biológica y químicamente inertes, y podrían servir como sensores con resolución de sub-longitud de onda. En general, los diamantes basados en marcadores fluorescentes utilizan centros de defectos de color como su parte emisora activa. Un centro de color común es el centro nitrógeno-vacante (NV) cuya estructura atómica se muestra en la Figura 1 a. Se pueden aproximar como sistema de dos niveles que después de la excitación láser a 532 nm muestran una amplia emisión en alrededor de 700 nm (Figura 1b). Los NDs utilizados en la presente invención contienen centros de color nitrógeno-vacante que existen en dos estados de cargas diferentes; cargados neutralmente (NVO) y cargados negativamente (NV-) (Figura 1c). The NDs used in the biomarker described in the present invention have unconditionally stable fluorescence - even after many months under continuous wave excitation - they are biologically and chemically inert, and could serve as sub-wavelength resolution sensors. In general, diamonds based on fluorescent markers use centers of color defects as their active emitting part. A common color center is the nitrogen-vacant (NV) center whose atomic structure is shown in Figure 1a. They can be approximated as a two-level system that after laser excitation at 532 nm show a wide emission around 700 nm (Figure 1b). The NDs used in the present invention contain nitrogen-vacant color centers that exist in two different charge states; neutrally charged (NVO) and negatively charged (NV-) (Figure 1c).
La superficie de NDs está funcionalizada con un péptido bifuncional R7-CLPFFD compuesto por el péptido CLPFFD y un péptido RRRRRRR (R7). El péptido CLPFFD es un disrruptor de hojas beta que reconoce agregados extracelulares tóxicos del péptido Ab amiloide presentes en el cerebro de los pacientes de AD. La sección R7 es un péptido penetración celular (CPP, por sus siglas en inglés) que mejora la captación celular de su carga. Por ejemplo, se han utilizado oligoargininas para mejorar la entrega de fármacos, tal como insulina cuando es administrada intranasalmente. Estos CPP son útiles para el tratamiento de enfermedades que requieren del cruce de diferentes tipos de barreras celulares, tal como la barrera sangre-cerebro (barrera hematoencefálica, por sus siglas en inglés) en AD. The surface of NDs is functionalized with a bifunctional R7-CLPFFD peptide composed of the CLPFFD peptide and a RRRRRRR (R7) peptide. The CLPFFD peptide is a beta sheet disruptor that recognizes toxic extracellular aggregates of amyloid Ab peptide present in the brain of AD patients. The R7 section is a cell penetrating peptide (CPP) that enhances cellular uptake of its cargo. For example, oligoarginines have been used to enhance drug delivery, such as insulin when administered intranasally. These CPPs are useful for the treatment of diseases that require the crossing of different types of cellular barriers, such as the blood-brain barrier (blood-brain barrier) in AD.
En una realización preferida de la presente invención es un marcador fluorescente. In a preferred embodiment of the present invention it is a fluorescent marker.
En una realización preferida los marcadores fluorescentes se basan en NDs. In a preferred embodiment the fluorescent markers are based on NDs.
En una realización preferida los NDs tienen un diámetro de entre 20 a 50 nm. In a preferred embodiment the NDs have a diameter of between 20 to 50 nm.
En una realización específica el marcador fluorescente de la presente invención utiliza NDs de 35 nm de diámetro. In a specific embodiment the fluorescent marker of the present invention uses 35 nm diameter NDs.
En una realización específica los NDs contienen centros de color nitrógeno-vacante.In a specific embodiment the NDs contain nitrogen-vacant color centers.
En una realización específica cada ND contiene un promedio de 15 centros de color nitrógeno-vacante. In a specific embodiment each ND contains an average of 15 nitrogen-vacant color centers.
En una realización específica los centros nitrógeno-vacante (NV) de los nanodiamantes existen en dos estados de cargas diferentes: cargados neutralmente (NVO) y cargados negativamente (NV-) con líneas de fonones cero a 575 y 637 nm respectivamente, bajo un láser de excitación de 532 nm (Figura 1c). In a specific embodiment the nitrogen-vacancy (NV) centers of the nanodiamonds exist in two different charge states: neutrally charged (NVO) and negatively charged (NV-) with zero phonon lines at 575 and 637 nm respectively, under a laser excitation of 532 nm (Figure 1c).
En una realización preferida la emisión de NDs radica en la ventana biológica de los tejidos.In a preferred embodiment the emission of NDs lies in the biological window of the tissues.
En una realización preferida los NDs utilizados en la presente invención, tienen una fluorescencia incondicionalmente estable, incluso después de muchos meses bajo una excitación de onda continua. En una realización preferida los NDs utilizados en la presente invención son biológica y químicamente inerte, y podrían servir como sensores con resolución de sub-longitud de onda. In a preferred embodiment the NDs used in the present invention have unconditionally stable fluorescence, even after many months under continuous wave excitation. In a preferred embodiment the NDs used in the present invention are biologically and chemically inert, and could serve as sensors with sub-wavelength resolution.
En una otra realización preferida la fluorescencia del ND no presenta intermitencia ni foto blanqueamiento. In another preferred embodiment, the fluorescence of the ND does not show intermittency or photobleaching.
En una realización preferida la fluorescencia de los NDs es más estable que los marcadores de color común utilizados, tal como Thioflavin T y FITC, entre otros. In a preferred embodiment the fluorescence of the NDs is more stable than the common color markers used, such as Thioflavin T and FITC, among others.
En una realización preferida la fluorescencia estable de los NDs permite su detección a concentraciones tan bajas como picomolar utilizando microscopía confocal. In a preferred embodiment the stable fluorescence of the NDs allows their detection at concentrations as low as picomolar using confocal microscopy.
En una realización preferida los NDs son funcionalizados con un péptido. In a preferred embodiment the NDs are functionalized with a peptide.
En una realización específica los NDs son funcionalizados con un péptido bifuncional.In a specific embodiment the NDs are functionalized with a bifunctional peptide.
En una realización preferida el péptido bifuncional confiere diferentes características y funcionalidades a los NDs. In a preferred embodiment the bifunctional peptide confers different characteristics and functionalities to the NDs.
En una realización específica el péptido bifuncional está compuesto de dos segmentos.In a specific embodiment the bifunctional peptide is composed of two segments.
En una realización específica la secuencia del péptido bifuncional es R7-CLPFFD. In a specific embodiment the sequence of the bifunctional peptide is R7-CLPFFD.
En una realización preferida el péptido R7-CLPFFD está compuesto de dos segmentos: el segmento CLPFFD y el segmento R7. In a preferred embodiment the R7-CLPFFD peptide is composed of two segments: the CLPFFD segment and the R7 segment.
En una realización preferida el segmento CLPFFD tiene la capacidad de reconocer agregados Ab. In a preferred embodiment the CLPFFD segment has the ability to recognize Ab aggregates.
En una realización específica el segmento CLPFFD incluye residuos hidrófobos Leu (L), Phe (F), y Phe (F). In a specific embodiment the CLPFFD segment includes hydrophobic residues Leu (L), Phe (F), and Phe (F).
En una realización específica el residuo Asp (D) del segmento CLPFFD confiere antipatía y carga neta de -1 a la molécula (Figura 2a), aumentando su solubilidad. In a specific embodiment, the Asp (D) residue of the CLPFFD segment confers antipathy and a net charge of -1 to the molecule (Figure 2a), increasing its solubility.
En una realización preferida el segmento R7, altamente positivo debido a la presencia de arginina (aminas secundarias), favorecer la penetración celular. In a preferred embodiment, the R7 segment, highly positive due to the presence of arginine (secondary amines), favors cell penetration.
En una realización específica el péptido R7 permite el cruce de las membranas celulares. En una realización preferida específica el segmento R7 tiene una carga neta de + 7. En una realización preferida la carga neta positiva del segmento R7 permite la unión electrostática entre el péptido y la superficie de carga negativa de los NDs (Figura 2) que contienen grupos de carboxilato. En otra realización preferida, la superficie de los nanodiamantes que contienen centros de color fluorescente pueden ser funcionalizados para realizar tareas no triviales y múltiples sin dañar la estabilidad de sus fluorescencias.In a specific embodiment the R7 peptide allows the crossing of cell membranes. In a specific preferred embodiment segment R7 has a net charge of +7. In a preferred embodiment the net positive charge of the R7 segment allows electrostatic bonding between the peptide and the negatively charged surface of the NDs (Figure 2) containing carboxylate groups. In another preferred embodiment, the surface of nanodiamonds containing fluorescent color centers can be functionalized to perform non-trivial and multiple tasks without damaging the stability of their fluorescence.
En una realización preferida los fNDs no son citotóxicos. In a preferred embodiment the fNDs are not cytotoxic.
En una realización preferida los fNDs son capaces de penetrar en las células sin afectar la viabilidad celular. In a preferred embodiment the fNDs are capable of penetrating cells without affecting cell viability.
En una realización específica los fNDs podrían ir a través de la barrera hematoencefálica.In a specific embodiment the fNDs could go across the blood-brain barrier.
En una realización preferida los fNDs sirven como un marcador fluorescente para la detección de biomarcadores de AD. In a preferred embodiment the fNDs serve as a fluorescent marker for the detection of AD biomarkers.
En una realización preferida los NDs funcionalizados unen fibrillas del péptido Ab y pueden ser utilizados para la detección indirecta de agregtados Ab extracelulares, asociados con AD. In a preferred embodiment the functionalized NDs bind fibrils of the Ab peptide and can be used for the indirect detection of extracellular Ab aggregates, associated with AD.
En una realización preferida particular, esta funcionalización del péptido de NDs podrían ser utilizados en experimentos fiables y a largo plazo para detectar agregados Ab y siguen su formación en AD. In a particular preferred embodiment, this functionalization of the peptide of NDs could be used in reliable and long-term experiments to detect Ab aggregates and follow their formation in AD.
Breve descripción de los dibujos Brief description of the drawings
Figura 1. Propiedades de emisión de centros de color de NV en diamante (a) configuración atómica de centros de color de NV en diamante. Un nitrógeno (azul) y tres carbonos (verde) son adyacentes al lugar vacante. El centro de NV puede existir en dos configuraciones de carga, centro cargado neutralmente NVO y el centro cargado negativamente NV-. (b) Modelo de dos niveles de las transiciones electrónicas del centro de color de NV-. (c) Espectro de emisión de los nanodiamantes. El espectro muestra una línea de fonones de cero a 575 nm para el centro de NVO y a 637 nm para el centro de NV-. Ambos centros muestran una amplia banda lateral de fonones. Figure 1. Emission properties of NV color centers in diamond (a) Atomic configuration of NV color centers in diamond. One nitrogen (blue) and three carbons (green) are adjacent to the vacant spot. The center of NV can exist in two charge configurations, the neutrally charged center NVO and the negatively charged center NV-. (b) Two-level model of the electronic transitions of the color center of NV-. (c) Emission spectrum of nanodiamonds. The spectrum shows a phonon line from zero at 575 nm for the center of NVO and at 637 nm for the center of NV-. Both centers show a wide lateral band of phonons.
Figura 2. Nanodiamantes funcionalizados. (a) Péptido bifuncional compuesto de un péptido R7 de penetración celular (área adjunta circular a la izquierda) que permite a sus cargas mejorar su captación celular y un péptido CLPFFD que rompe hojas beta (área adjunta circular a la derecha) que reconoce agregados Ab tóxicos presentes en AD. (b) Potencial Zeta (Zp, por sus siglas en inglés), diámetro hidrodinámico (HD, por sus siglas en inglés) e índice de polidispersidad (PDI, por sus siglas en inglés) de NDs desnudos (c) Ilustración y propiedades (ZP, HD y PDI) de fNDs. Figure 2. Functionalized nanodiamonds. (a) Bifunctional peptide composed of a cell-penetrating R7 peptide (circular attached area on the left) that allows its cargoes to improve their cellular uptake and a peptide CLPFFD that breaks beta sheets (circular attached area on the right) that recognizes Ab aggregates toxins present in AD. (b) Zeta potential (Zp), hydrodynamic diameter (HD) and polydispersity index (PDI) of naked NDs (c) Illustration and properties (ZP, HD and PDI) of fNDs.
Figura 3. Estructura de nanodiamantes. HR-TEM. Micrografías de electrones mostrando (a) NDs y (b) fNDs. Figure 3. Structure of nanodiamonds. HR-TEM. Electron micrographs showing (a) NDs and (b) fNDs.
Figura 4. Internalización celular de fNDs en línea celular de los fibroblastos (a) Imagen compuesta de células de fibroblastos con tubulina marcada con Alexa 488 excitada bajo iluminación láser (verde) de 488 nm y fNDs excitados bajo iluminación (rojo) de 532 nm. En ambos casos la emisión fue registrada utilizando un detector de fotones de avalancha (APD, por sus siglas en inglés) (b) Espectro fluorescente de nanodiamantes mostrando la característica de líneas de fonones cero a 637 y 575 nm. (c) Espectro de fluorescencia de Alexa 488. Figure 4. Cell internalization of fNDs in fibroblast cell line (a) Composite image of fibroblast cells with Alexa 488-labeled tubulin excited under 488 nm laser illumination (green) and excited fNDs under 532 nm illumination (red). In both cases the emission was recorded using an avalanche photon detector (APD) (b) Fluorescent spectrum of nanodiamonds showing the characteristic of zero phonon lines at 637 and 575 nm. (c) Alexa 488 fluorescence spectrum.
Figura 5. Internalización celular de fNDs en la línea celular bEnd.3. Imagen de células bEnd.3 incubadas con NDs (panel superior) y fNDs (panel inferior) a dos concentraciones (2 y 20 pM) por 6 horas. Figure 5. Cell internalization of fNDs in the bEnd.3 cell line. Image of bEnd.3 cells incubated with NDs (upper panel) and fNDs (lower panel) at two concentrations (2 and 20 pM) for 6 hours.
Figura 6. Caracterización de la estabilidad de la foto del marcador de color basado en diamante y conjugado 555 Alexa Fluor (a) Trazas de fluorescencia bajo iluminación láser de longitud de onda de 532 nm continua, de conjugado 555 Alexa Fluor (marcadores negros) y fNDs que contienen centros de color nitrógeno-vacante (marcadores grises) para varias potencias de láser (b) Tasa de descomposición de conjugado 555 Alexa Fluor versus la potencia de excitación. La fluorescencia disminuye su intensidad a una tasa de 0,8 Hz/mW mientras que la de las fNDs permaneció estable (c) Intensidad de fluorescencia vs. tiempo de iluminación de Alexa Fluor 488 y (d) para FITC. Figure 6. Characterization of photo stability of the 555 Alexa Fluor conjugate and diamond-based color marker (a) Traces of fluorescence under continuous 532 nm wavelength laser illumination, of 555 Alexa Fluor conjugate (black markers) and fNDs containing nitrogen-vacant color centers (gray markers) for various laser powers (b) 555 Alexa Fluor conjugate decomposition rate versus excitation power. Fluorescence decreases its intensity at a rate of 0.8 Hz / mW while that of the fNDs remained stable (c) Fluorescence intensity vs. lighting time for Alexa Fluor 488 and (d) for FITC.
Figura 7. La funcionalización de NDs no afecta a la viabilidad celular. Las mediciones de la viabilidad celular evaluada a través del ensayo de reducción MTS para líneas celulares (a) HT22 y (c) C3 10T1/2 incubadas bajo diferentes concentraciones de fNDs (barras negras) por 24 horas y NDs no funcionalizados (barras grises). Pruebas adicionales bajo altas concentraciones de nanodiamantes no funcionalizados fueron realizados para las líneas celulares (b) HT22 y (d) C3 10T1/2. Los valores corresponden al porcentaje medio de células viables con respecto a las células de control (barras blancas). Las barras de error indican la desviación estándar estimada a partir de tres experimentos llevados a cabo cada uno en triplicado. Figure 7. The functionalization of NDs does not affect cell viability. Measurements of cell viability evaluated through the MTS reduction assay for cell lines (a) HT22 and (c) C3 10T1 / 2 incubated under different concentrations of fNDs (black bars) for 24 hours and non-functionalized NDs (gray bars) . Additional tests under high concentrations of non-functionalized nanodiamonds were performed for the cell lines (b) HT22 and (d) C3 10T1 / 2. The values correspond to the average percentage of viable cells relative to control cells (open bars). The error bars indicate the standard deviation estimated from three experiments each carried out in triplicate.
Figura 8. Asociación de fNDs con fibras y placas Ab. (a) Imagen TEM que muestra las fibras de Ab y fNDs juntos (las flechas muestran dos fNDs específicos, como ejemplos). Regiones sin fibras que muestran casi ningún NDs funcionalizado. (b) Imagen que muestra el hipocampo del tejido de cerebro de ratón con AD marcado para detectar placas Ab con un anticuerpo 4G8 anti Ab y anticuerpo secundario Alexa 488 (puntos verdes); e imágenes ampliadas con fNDs iluminados con láser de 532 nm. El primer recuadro muestra una ampliación de imagen confocal de 50x50 pm2 en las cercanías de la placa Ab. El segundo recuadro muestra una ampliación de imagen confocal de 5x5 pm2. Finalmente, se muestra un espectro de emisión típico de un fND detectado bajo una excitación de 532 nm en las cercanías de la placa Ab. Figure 8. Association of fNDs with Ab fibers and plates. (a) TEM image showing Ab fibers and fNDs together (arrows show two specific fNDs, as examples). Fiber-free regions showing almost no functionalized NDs. (b) Image showing the hippocampus of AD-labeled mouse brain tissue for Ab plaques with an anti-Ab 4G8 antibody and Alexa 488 secondary antibody (green dots); and magnified images with 532 nm laser illuminated fNDs. The first inset shows a 50x50 pm 2 confocal image magnification in the vicinity of plate Ab. The second inset shows a 5x5 pm 2 confocal image magnification. Finally, a typical emission spectrum of an fND detected under an excitation of 532 nm in the vicinity of plate Ab is shown.
Los siguientes son ejemplos de realizaciones para la presente invención de acuerdo a lo descrito anteriormente: The following are examples of embodiments for the present invention as described above:
Ejemplo 1 : Evaluación de la unión entre el péptido CLPFFD R7, péptido y la superficie de NDs. Example 1: Evaluation of the binding between the CLPFFD R7 peptide, peptide and the surface of NDs.
Objetivo del experimento: Para evaluar la adsorción del péptido R7-CLPFFD a la superficie de NDs. Objective of the experiment: To evaluate the adsorption of the R7-CLPFFD peptide to the surface of NDs.
Descripción del experimento: Primero, para la funcionalización de Nds, el péptido RRR RRRRCLPFFD fue disuelto en agua Milli-Q ultra pura en una concentración final de 0,05 mg/ml. Los nanodiamantes fueron añadidos a la solución de este péptido quedando en una concentración de 0,8 nM en la solución final, y luego fueron incubados con una agitación vigorosa durante 2 h. La adsorción del péptido en la superficie del nanocristal fue evaluada mediante el cambio en el potencial zeta (pZ) y diámetro hidrodinámico (Dh) como un indicador del revestimiento electrostático para este péptido bifuncional. La suspensión coloidal se centrifugó y se lavó tres veces. Los fNDs lavados fueron reevaluados mediante pZ y Dh para asegurar que la funcionalización permaneció. Finalmente, la funcionalización del nanodiamante fue analizado mediante microscopía de transmisión de electrones de alta resolución (HR-TEM, por sus siglas en inglés) tiñiendo las muestras con ácido fosfotungstico (1%) con el fin de evaluar la presencia del péptido que rodea al nanodiamante. Resultados: Primero, el valor de pZ después de la adsorción del péptido aumentó de -29,7 ± 1 ,6 a +29,1 ± 4,0 mV indicando que el nanocristal fue funcionalizado positivamente. Más aún, el aumento en Dh desde 74,3 ± 0,5 nm to 163,3 ± 2,0 nm, alrededor de dos veces el diámetro del nanocristal desnudo, confirmada la funcionalización. Los parámetros pZ y Dh de los fNDs permanecieron estables después de tres lavados (Figura 2). Description of the experiment: First, for the functionalization of Nds, the RRR RRRRCLPFFD peptide was dissolved in ultra-pure Milli-Q water at a final concentration of 0.05 mg / ml. The nanodiamonds were added to the solution of this peptide, remaining in a concentration of 0.8 nM in the final solution, and then they were incubated with vigorous shaking for 2 h. The adsorption of the peptide on the surface of the nanocrystal was evaluated by the change in zeta potential (pZ) and hydrodynamic diameter (Dh) as an indicator of the electrostatic coating for this bifunctional peptide. The colloidal suspension was centrifuged and washed three times. The washed fNDs were re-evaluated by pZ and Dh to ensure that functionalization remained. Finally, the functionalization of the nanodiamond was analyzed using high resolution transmission electron microscopy (HR-TEM) staining the samples with phosphotungstic acid (1%) in order to evaluate the presence of the peptide surrounding the nanodiamond. Results: First, the pZ value after peptide adsorption increased from -29.7 ± 1.6 to +29.1 ± 4.0 mV indicating that the nanocrystal was positively functionalized. Furthermore, the increase in Dh from 74.3 ± 0.5 nm to 163.3 ± 2.0 nm, about twice the diameter of the naked nanocrystal, confirmed the functionalization. The pZ and Dh parameters of the fNDs remained stable after three washes (Figure 2).
Más aún, las imágenes de microscopía de transmisión de electrones de alta resolución (HR-TEM) de los fNDs comparados con nanocristales desnudos también indican la funcionalización con el péptido. La gradiente de densidad electrónica es más densa en el centro que en la periferia de las partículas indicando que los NDs están rodeados de péptidos. El diámetro de partícula promedio es 199 ± 56 nm, más grande que el diámetro del ND desnudo. Más aún, utilizando HR-TEM fue posible observar que los NDs están rodeados por una gruesa capa de péptido, posiblemente formando una multicapa de péptidos (Figura 3). Por otro lado, el espectro de fluorescencia de los NDs no cambió después de la funcionalización y tres lavados consecutivos. Furthermore, high resolution transmission electron microscopy (HR-TEM) images of fNDs compared to naked nanocrystals also indicate functionalization with the peptide. The electron density gradient is denser at the center than at the periphery of the particles, indicating that the NDs are surrounded by peptides. The average particle diameter is 199 ± 56 nm, larger than the diameter of the bare ND. Furthermore, using HR-TEM it was possible to observe that the NDs are surrounded by a thick layer of peptide, possibly forming a multilayer of peptides (Figure 3). On the other hand, the fluorescence spectrum of the NDs did not change after functionalization and three consecutive washes.
Conclusiones: En conjunto, estos resultados sustentan la adsorción exitosa del péptido R7-CLPFFD sobre la superficie de NDs. Conclusions: Taken together, these results support the successful adsorption of the R7-CLPFFD peptide on the surface of NDs.
Ejemplo 2: Evaluación de interacciones de fNDs con células y sus propiedades como marcadores de color. Example 2: Evaluation of interactions of fNDs with cells and their properties as color markers.
Objetivo del experimento: Evaluar la interacción con las células, internalización celular y detección ultrasensible de fNDs. Objective of the experiment: To evaluate the interaction with cells, cellular internalization and ultrasensitive detection of fNDs.
Descripción del experimento: Para evaluar la internalización de fNDs, se incubaron fibroblastos (30.000 cells/ml) con fNDs de 8 pM. Después de 6 horas las muestras fueron lavadas y fijadas. Con el fin de visualizar las células, se inmunotiñeron microtúbulos (componentes del citoesqueleto) utilizando anticuerpos anti beta tubulina (1 :1000) y anticuerpos secundarios conjugados con Alexa Fluor 488 (1 :1000). Las muestras fueron analizadas en una configuración de microscopio confocal casero equipado con un detector de fotones de avalancha (APD) y un espectrómetro óptico. Description of the experiment: To evaluate the internalization of fNDs, fibroblasts (30,000 cells / ml) were incubated with 8 pM fNDs. After 6 hours the samples were washed and fixed. In order to visualize the cells, microtubules (components of the cytoskeleton) were immunostained using anti beta tubulin antibodies (1: 1000) and Alexa Fluor 488 conjugated secondary antibodies (1: 1000). The samples were analyzed in a home confocal microscope setup equipped with an avalanche photon detector (APD) and an optical spectrometer.
Después de evaluar la internalización celular de fNDs mediante el análisis de su espectro de emisión, la capacidad de penetración celular de fNDs fue además evaluada en un modelo celular más estrechamente relacionado con el contexto biológico de la barrera hematoencefálica. Las células bEnd.3 son células endoteliales vasculares cerebrales y son comúnmente utilizadas en diferentes modelos in vitro de transportación de fármacos a través de la barrera hematoencefálica. Las células bEnd.3 (ATCC CRL-2299) fueron incubadas con nanodiamantes en un rango de concentración de 2 a 20 pM utilizando tanto nanodiamentes funcionalizados como no funcionalizados como un control. After evaluating the cellular internalization of fNDs by analyzing their emission spectrum, the cellular penetration capacity of fNDs was further evaluated in a cellular model more closely related to the biological context of the blood-brain barrier. BEnd.3 cells are cerebral vascular endothelial cells and are commonly used in different in vitro models of drug transport across the blood-brain barrier. BEnd.3 cells (ATCC CRL-2299) were incubated with nanodiamonds in a concentration range of 2 to 20 pM using both functionalized and non-functionalized nanodiamonds as a control.
Resultados: Los resultados de los análisis de internalización de fNDs en células de fibroblastos se muestran en la figura 4. La figura 4a muestra una imagen representativa de células de fibroblastos incubados con fNDs. Primero, debido a la alta sensibilidad de APD, esta configuración permitió utilizar concentraciones de NDs en el rango pM y podría incluso detectar NDs que contengan emisores únicos. Segundo, los análisis de diferentes regiones de interés de la muestra muestran claramente dos espectros distinguibles: uno correspondiente a la Alexa Fluor 488, y la otra correspondiente al fND (Figura 4b y c, respectivamente). Los fNDs y Alexa 488 fueron observados en el mismo plano focal. Además, la Figura 4a muestra fNDs dentro del perímetro de la célula en donde no se observaron fNDs fuera de la célula (muestas lavadas) sugiriendo que los fNDs están dentro de la célula. Además, no se observaron fNDs en el núcleo celular. Results: The results of the analysis of internalization of fNDs in fibroblast cells are shown in Figure 4. Figure 4a shows a representative image of fibroblast cells incubated with fNDs. First, due to the high sensitivity of APD, this configuration allowed to use concentrations of NDs in the pM range and could even detect NDs containing unique emitters. Second, the analyzes of different regions of interest of the sample clearly show two distinguishable spectra: one corresponding to Alexa Fluor 488, and the other corresponding to fND (Figure 4b and c, respectively). The fNDs and Alexa 488 were observed in the same focal plane. Furthermore, Figure 4a shows fNDs within the perimeter of the cell where no fNDs were observed outside the cell (washed samples) suggesting that the fNDs are within the cell. Furthermore, no fNDs were observed in the cell nucleus.
Los resultados de la segunda parte del experimento utilizando células bEnd.3 mostraron que los fNDs podrían ser internalizados en estas células y que la penetración de nanopartículas funcionalizadas en células bEnd.3 es aumentada en fNDs en comparación a las nanopartículas no funcionalizadas. The results of the second part of the experiment using bEnd.3 cells showed that fNDs could be internalized in these cells and that the penetration of functionalized nanoparticles in bEnd.3 cells is increased in fNDs compared to non-functionalized nanoparticles.
Conclusiones: Los resultados sugieren que la internalización celular de los fNDs es mayor en comparación a los ND desnudos, en células de fibroblastos y en células bEnd.3, una línea celular endotelial vascular cerebral comúnmente utilizado en modelos in vitro para probar el transporte a través de barrera hematoencefálica. Conclusions: The results suggest that the cellular internalization of fNDs is greater compared to naked NDs, in fibroblast cells and in bEnd.3 cells, a cerebral vascular endothelial cell line commonly used in in vitro models to test transport through blood-brain barrier.
Ejemplo 3: Evaluación de la estabilidad de la fluorescencia de fNDs. Example 3: Evaluation of the fluorescence stability of fNDs.
Objetivo del experimento: Evaluar la estabilidad de fluorescencia de fNDs. Objective of the experiment: To evaluate the fluorescence stability of fNDs.
Descripción del experimento: Con el fin de evaluar la estabilidad de fluorescencia de fNDs, su intensidad de fluorescencia fue comparada a la de Alexa Fluor 555 a través de la vigilancia de las muestras durante 5 minutos bajo varias longitudes de onda de excitación. Description of the experiment: In order to evaluate the fluorescence stability of fNDs, their fluorescence intensity was compared to that of Alexa Fluor 555 by monitoring the samples for 5 minutes under various excitation wavelengths.
Resultados: Se observó que la fluorescencia de Alexa Fluor 555 a diferentes potencias de láser disminuyó a lo largo del tiempo a una tasa de 0,8 Flz/mW (Figura 6a y b). La Figura 6c y d también muestran la estabilidad de fluorescencia de Alexa 488 y FITC respectivamente, bajo varias potencias excitación láser. En contraste, la fluorescencia de los fNDs permaneció constante. Por lo tanto, los diamantes basados en marcadores fluorescentes son más estables que otros marcadores fluorescentes. La fluorescencia no muestra ningún deterioro perceptible después de varios días bajo excitación láser, y después de meses, o incluso años sin excitación láser continua, permitiendo la experimentación a largo plazo. Results: It was observed that the fluorescence of Alexa Fluor 555 at different laser powers decreased over time at a rate of 0.8 Flz / mW (Figure 6a and b). Figure 6c and d also show the fluorescence stability of Alexa 488 and FITC respectively, under various laser excitation powers. In contrast, the fluorescence of the fNDs remained constant. Therefore, fluorescent marker-based diamonds are more stable than other fluorescent markers. Fluorescence does not shows no perceptible deterioration after several days under laser excitation, and after months, or even years without continuous laser excitation, allowing long-term experimentation.
Conclusiones: Los resultados mostraron que la estabilidad de la fluorescencia de fNDs es superior a la de los marcadores de color comúnmente utilizados. Por lo tanto, estos resultados podrían permitir estudios más largos y confiables de agregados de Ab. Conclusions: The results showed that the fluorescence stability of fNDs is superior to that of commonly used color markers. Therefore, these results could allow longer and more reliable studies of Ab aggregates.
Ejemplo 4: Efectos de los fNDs sobre la viabilidad celular. Example 4: Effects of fNDs on cell viability.
Objetivo del experimento: Probar los efectos de los fNDs sobre la viabilidad celular.Objective of the experiment: To test the effects of fNDs on cell viability.
Descripción del experimento: Para el ensayo de viabilidad se incubaron neuronas hipocampales HT22 y células de fibroblastos C3 10T1 /2 con diferentes concentraciones de fNDs durante 24 horas. Luego, la viabilidad celular se midió utilizando el ensayo de [3- (4,5- dimetiltiazol-2-il) -5- (3-carboximetoxifenil) -2- (4-sulfofenil) -2H-tetrazolio (MTS). La prueba MTS se basa en la estimación de la reducción de las sales de tetrazolio mediante respiración celular de células viables que genera como producto formazan de color púrpura que puede cuantificarse a 492 nm. El producto púrpura se midió a 492 nm utilizando un lector (Autobio Phomo). El porcentaje de reducción de MTT se comparó con las células de control no expuestas al material, el cual representaba el 100% de la reducción de MTT. Description of the experiment: For the viability test, HT22 hippocampal neurons and C3 10T1 / 2 fibroblast cells were incubated with different concentrations of fNDs for 24 hours. Then, cell viability was measured using the [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS) assay. The MTS test is based on the estimation of the reduction of tetrazolium salts by cellular respiration of viable cells that generates as a purple formazan product that can be quantified at 492 nm. The purple product was measured at 492 nm using a reader (Autobio Phomo). The percentage reduction in MTT was compared to control cells not exposed to the material, which represented 100% of the reduction in MTT.
Resultados: Las células tratadas (con fNDs) no mostraron diferencias significativas en la viabilidad celular en comparación con los grupos de control (Figura 7). Además, no se observaron diferencias significantes en la viabilidad celular utilizando una concentración más alta de NDs no funcionalizados en cualquier línea celular. Results: The treated cells (with fNDs) did not show significant differences in cell viability compared to the control groups (Figure 7). Furthermore, no significant differences in cell viability were observed using a higher concentration of non-functionalized NDs in any cell line.
Conclusiones: Las propiedades físicas y químicas de los NDs y fNDs no afectan a la viabilidad celular. Esta sería una de las principales ventajas de nuestro nanosistema de marcador celular comparado con puntos cuánticos, los cuales son altamente tóxicos bajo ciertas condiciones. Conclusions: The physical and chemical properties of NDs and fNDs do not affect cell viability. This would be one of the main advantages of our cell marker nanosystem compared to quantum dots, which are highly toxic under certain conditions.
Ejemplo 5: Unión de fNDs a fibras Ab. Example 5: Binding of fNDs to Ab fibers.
Objetivo del experimento: Evaluar la capacidad de fNDs para unir agregados de fibrilar Ab. Objective of the experiment: To evaluate the ability of fNDs to bind aggregates of fibrillar Ab.
Descripción del experimento: Las fibras Ab fueron cultivadas in vitro y posteriormente se incubaron con fNDs bajo agitación constante durante 30 min. Como control los fNDs se incubaron con agregados fibrilares de albúmina. Se evaluó la unión de fNDs a las fibras Ab mediante microscopía electrónica se barrido con módulo de transmisión (STEM, por sus siglas en inglés). Description of the experiment: Ab fibers were cultured in vitro and subsequently incubated with fNDs under constant shaking for 30 min. As a control, the fNDs were incubated with fibrillar albumin aggregates. The binding of fNDs to Ab fibers was evaluated Scanning electron microscopy with transmission module (STEM).
Por otro lado, con el fin de visualizar la asociación entre fNDs y placas Ab se incubaron cortes de tejidos cerebrales de ratones transgénicos con AD que sobreexpresan Ab, con fNDs. Los cortes fueron además co-incubados con el anticuerpo 4G8 (anticuerpo contra Ab) y luego con un anticuerpo secundario conjugado con Alexa 488 para la visualización de placas Ab. On the other hand, in order to visualize the association between fNDs and Ab plaques, sections of brain tissues of transgenic mice with AD that overexpress Ab were incubated with fNDs. The sections were further co-incubated with the 4G8 antibody (antibody against Ab) and then with a secondary antibody conjugated to Alexa 488 for the visualization of Ab plaques.
Resultados: Se observó que los fNDs co-localizan con fibras Ab, decorando los agregados fibrilares (figura 8a). No se observó casi ningún fNDs en regiones sin fibras. En el ensayo de control con albúmina fibrilar, no se observó ninguna interacción entre estos agregados y fNDs. Results: It was observed that the fNDs co-localize with Ab fibers, decorating the fibrillar aggregates (figure 8a). Almost no fNDs were observed in regions without fibers. In the fibrillar albumin control assay, no interaction was observed between these aggregates and fNDs.
La visualización de fNDs y placas Ab (utilizando el anticuerpo 4G8 y luego con un anticuerpo secundario con Alexa 488) en cortes de tejidos cerebrales de ratones transgénicos con AD, mostraron asociación entre ambas señales fluorescentes (fNDs y las señales 488) en el halo de placas Ab (Figura 8b), indicando la detección del péptido Ab por los dos marcadores. En regiones donde no se encuentras presentes las placas Ab, no se encontraron fNDs. Visualization of fNDs and Ab plaques (using the 4G8 antibody and then with a secondary antibody with Alexa 488) in sections of brain tissues of transgenic mice with AD, showed an association between both fluorescent signals (fNDs and 488 signals) in the halo of Ab plates (Figure 8b), indicating the detection of the Ab peptide by the two markers. In regions where Ab plaques are not present, no fNDs were found.
Conclusiones: Los resultados sugieren una interacción específica entre los fNDs y fibras Ab, probablemente debido a la región CLPFFD del péptido bifuncional R7-CLPFFD presente sobre la superficie de los fNDs. Por lo tanto, los fNDs pueden ser utilizados como sondas fluorescentes para detectar regiones de agregados de Ab. Conclusions: The results suggest a specific interaction between the fNDs and Ab fibers, probably due to the CLPFFD region of the bifunctional peptide R7-CLPFFD present on the surface of the fNDs. Therefore, fNDs can be used as fluorescent probes to detect aggregate regions of Ab.
Estos resultados sugieren que los fNDs se pueden convertir en un método poderoso y ultrasensible para detectar la formación de agregados de Ab durante el desarrollo de AD. These results suggest that fNDs can become a powerful and ultrasensitive method to detect the formation of Ab aggregates during AD development.

Claims

Reivindicaciones Claims
1. Marcador fluorescente estable y no citotóxico CARACTERIZADO porque consiste en nanodiamantes (ND) con centros de color fluorescente (fND) y un péptido bifuncional, el cual es construido por un péptido de penetración celular y un péptido de 6 aminoácidos de largo que funciona como disrruptor de hojas beta y que reconoce agregados de beta amiliode, donde los agregadeos de beta amiloide son un biomarcador de la enfermedad de Alzheimer. 1. Stable and non-cytotoxic fluorescent marker CHARACTERIZED because it consists of nanodiamonds (ND) with fluorescent color centers (fND) and a bifunctional peptide, which is constructed by a cell-penetrating peptide and a 6 amino acid long peptide that functions as beta sheet disrupter and recognizes beta amyloid aggregates, where beta amyloid aggregates are a biomarker of Alzheimer's disease.
2. Marcador de acuerdo a la reivindicación 1 , CARACTERIZADO porque los NDs utilizados en el biomarcador tienen una fluorescencia incondicionalmente estable - incluso después de muchos meses bajo una excitación de onda continua - son biológica y químicamente inertes. 2. Marker according to claim 1, CHARACTERIZED in that the NDs used in the biomarker have an unconditionally stable fluorescence - even after many months under continuous wave excitation - they are biologically and chemically inert.
3. Marcador de acuerdo a la reivindicación 1 , CARACTERIZADO porque funcionan como sensores con resolución de sub-longitud de onda. 3. Marker according to claim 1, CHARACTERIZED in that they function as sensors with sub-wavelength resolution.
4. Marcador de acuerdo a la reivindicación 1 , CARACTERIZADO porque los ND basados en marcadores fluorescentes utilizan centros de defectos de color como su parte emisora activa, donde un centro de color común es el centro nitrógeno-vacante (NV). 4. Marker according to claim 1, CHARACTERIZED in that ND based on fluorescent markers use centers of color defects as their active emitting part, where a common color center is the nitrogen-vacant center (NV).
5. Marcador de acuerdo a la reivindicación 4, CARACTERIZADO porque los ND contienen centros de color nitrógeno-vacante que existen en dos estados de cargas diferentes; cargados neutralmente (NVO) y cargados negativamente (NV-). 5. Marker according to claim 4, CHARACTERIZED in that the DNs contain nitrogen-vacant color centers that exist in two different charge states; neutrally charged (NVO) and negatively charged (NV-).
6. Marcador de acuerdo a la reivindicación 1, CARACTERIZADO porque el péptido bifuncional es R7-CLPFFD, compuesto por el péptido CLPFFD y un péptido RRRRRRR (R7). 6. Marker according to claim 1, CHARACTERIZED in that the bifunctional peptide is R7-CLPFFD, composed of the CLPFFD peptide and a RRRRRRR (R7) peptide.
7. Marcador de acuerdo a la reivindicación 6, CARACTERIZADO porque el péptido CLPFFD es un disrruptor de hojas beta que reconoce agregados extracelulares tóxicos del péptido Ab amiloide presentes en el cerebro de los pacientes de AD. 7. Marker according to claim 6, CHARACTERIZED in that the CLPFFD peptide is a beta sheet disruptor that recognizes toxic extracellular aggregates of amyloid Ab peptide present in the brain of AD patients.
8. Marcador de acuerdo a la reivindicación 6, CARACTERIZADO porque la sección R7 es un péptido penetración celular que mejora la captación celular de su carga. 8. Marker according to claim 6, CHARACTERIZED in that the R7 section is a cell penetration peptide that improves cell uptake of its cargo.
9. Marcador de acuerdo a la reivindicación 8, CARACTERIZADO porque el péptido de penetración celular permite el cruce de diferentes tipos de barreras celulares, específicamente la barrera hematoencefálica. 9. Marker according to claim 8, CHARACTERIZED in that the cell penetration peptide allows the crossing of different types of cell barriers, specifically the blood-brain barrier.
"lO.Marcador de acuerdo a la reivindicación 1 , CARACTERIZADO porque los NDs tienen un diámetro de entre 20 a 50 nm. " 10. Marker according to claim 1, CHARACTERIZED in that the NDs have a diameter of between 20 to 50 nm.
11. Marcador de acuerdo a la reivindicación 1, CARACTERIZADO porque el marcador fluorescente utiliza NDs de 35 nm de diámetro. 11. Marker according to claim 1, CHARACTERIZED in that the fluorescent marker uses 35 nm diameter NDs.
12. Marcador de acuerdo a la reivindicación 1 , CARACTERIZADO porque los NDs contienen centros de color nitrógeno-vacante. 12. Marker according to claim 1, CHARACTERIZED in that the NDs contain nitrogen-vacant color centers.
13. Marcador de acuerdo a la reivindicación 1 , CARACTERIZADO porque cada ND contiene un promedio de 15 centros de color nitrógeno-vacante. 13. Marker according to claim 1, CHARACTERIZED in that each ND contains an average of 15 nitrogen-vacant color centers.
14. Marcador de acuerdo a la reivindicación 4, CARACTERIZADO porque los centros nitrógeno-vacante (NV) de los nanodiamantes existen en dos estados de cargas diferentes: cargados neutralmente (NVO) y cargados negativamente (NV-) con líneas de fonones cero a 575 y 637 nm respectivamente, bajo un láser de excitación de 532 nm. 14. Marker according to claim 4, CHARACTERIZED because the nitrogen-vacancy centers (NV) of the nanodiamonds exist in two different charge states: neutrally charged (NVO) and negatively charged (NV-) with phonon lines zero to 575 and 637 nm respectively, under a 532 nm excitation laser.
15. Marcador de acuerdo a la reivindicación 1, CARACTERIZADO porque la emisión de NDs radica en la ventana biológica de los tejidos. 15. Marker according to claim 1, CHARACTERIZED in that the emission of NDs lies in the biological window of the tissues.
16. Marcador de acuerdo a la reivindicación 1, CARACTERIZADO porque la fluorescencia del ND no presenta intermitencia ni foto blanqueamiento.16. Marker according to claim 1, CHARACTERIZED in that the fluorescence of the ND does not show intermittence or photo bleaching.
17. Marcador de acuerdo a la reivindicación 1, CARACTERIZADO porque la fluorescencia de los NDs es más estable que los marcadores de color común utilizados, tal como Thioflavin T y FITC, entre otros. 17. Marker according to claim 1, CHARACTERIZED in that the fluorescence of the NDs is more stable than the common color markers used, such as Thioflavin T and FITC, among others.
18. Marcador de acuerdo a la reivindicación 1, CARACTERIZADO porque la fluorescencia estable de los NDs permite su detección a concentraciones en órdenes de magnitud picomolar utilizando microscopía confocal. 18. Marker according to claim 1, CHARACTERIZED in that the stable fluorescence of the NDs allows their detection at concentrations in picomolar orders of magnitude using confocal microscopy.
19. Marcador de acuerdo a la reivindicación 1, CARACTERIZADO porque los NDs funcionalizados unen fibrillas del peptido Ab y pueden ser utilizados para la detección indirecta de agregados Ab extracelulares, asociados con AD. 19. Marker according to claim 1, CHARACTERIZED in that the functionalized NDs bind fibrils of the Ab peptide and can be used for the indirect detection of extracellular Ab aggregates associated with AD.
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