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    • 专利摘要:
    Use of a composition comprising a combination of fluorescent nanoparticles, wherein said composition comprises nanoparticles that emit radiation at a wavelength corresponding to blue, nanoparticles that emit radiation at a wavelength corresponding to green, nanoparticles that emit radiation at a wavelength corresponding to red and nanoparticles that emit radiation at a wavelength corresponding to red/purple to determine depths of porous materials, to determine the thickness of porous material sheets, to determine the root structure of a plant or to elaborate a diagnostic agent for the diagnosis of skin cancer in a human subject. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2684057A1
    申请号:ES201730451
    申请日:2017-03-28
    公开日:2018-10-01
    发明作者:Edelweiss MOYANO RODRÍGUEZ；Antonio José CAAMAÑO FERNÁNDEZ；Julio RAMIRO BARGUEÑO；Jose Luis ROJO ÁLVAREZ；Francisco Javier Ramos López；Víctor DE LA PEÑA O'SHEA；Daniel JAQUE GARCÍA
    申请人:Imdea (instituto Madrileno De Estudios Avanzados) Energia；Imdea Inst Madrileno De Estudios Avanzados Energia；Universidad Autonoma de Madrid；Universidad Rey Juan Carlos；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    The present invention relates to a composition comprising a combination of fluorescent nanoparticles that can be applied in depth detection.
    10 applied in different scientific fields such as: Medicine, Chemistry, surface science and Botany. Specifically, the present invention relates to the use of the composition to determine sheet thickness of porous materials, to determine the root structure of a plant or to develop a diagnostic agent for the diagnosis of skin cancer in a human subject.
    BACKGROUND OF THE INVENTION
    Fluorescent tracers have the ability to fluoresce at different wavelengths of the visible range of the electromagnetic spectrum, depending on their chemical composition,
    20 texture, structure and morphology, when exposed to coherent radiation, typically in the near infrared range. In recent years techniques that use "quantum dats" have been proposed; but these compounds show problems: they need ultraviolet radiation (which causes potential tissue damage) and a reduction in fluorescence efficiency occurs.
    25 Compounds based on inorganic matrices doped with rare earth elements, known as "upconversion" have been described.
    The upconversion process is an intrinsic process of the lanthanides, by means of which
    30 produces a chain excitation of the electrons after radiation of the compound with near infrared, to subsequently lead to a relaxation of the electrons and therefore an emission of the energy accumulated in said excitation.
    The relaxation of these electrons, from the excited to the fundamental state, results in the emission of electromagnetic radiation with short wavelengths. In order for this process to be carried out, three elements that will be part of the crystalline network of the compound are necessary, each acting for a specific purpose in the process:
    -Stabilizer: They are the materials that form the crystalline matrix of the compound. Being this5 the base of the crystalline structure thereof.
    - Sensitizer: Rare earth that acts by donating energy to activators, with the trivalent lanthanide elements showing the highest upconversion efficiency.
    10 -Activator: This element accumulates sensitizer energy and emits radiation with shorter wavelengths than the energy received.
    This process is observed in all transition metals. However, those with greater efficiency are lanthanides, due to their electromagnetic configuration.
    15 The lanthanides that show the highest upconversion efficiency are trivalent ions. Therefore, the usual configuration of upconversion fluorescent nanoparticles is formed with the trivalent ions of lanthanides bound to harmless compounds that act as stabilizers
    In the state of the art there are few methods described for measuring the thickness of the passivation layers of metallic materials. The most commonly used technique is the so-called electrochemical impedance spectroscopy, which involves a negative action on the material studied that can damage the measured layer.
    DESCRIPTION OF THE INVENTION
    The present invention provides the use of a composition comprising a combination of fluorescent nanoparticles, wherein said composition comprises 30 nanoparticles that emit radiation at a wavelength corresponding to blue, nanoparticles that emit radiation at a wavelength corresponding to green, nanoparticles that emit radiation at a wavelength that corresponds to red and nanoparticles that emit radiation at a wavelength that corresponds to red / purple to determine depths of porous materials, to determine sheet thickness of 35 porous materials, to determine the structure root of a plant or to make a

    diagnostic agent for the diagnosis of skin cancer in a human subject, where said use comprises:
    (to) applying said composition on said porous material or applying said composition on the growth medium of said plant or administering said composition to said human subject,
    (b) irradiating the porous material or the growth medium of said plant or the skin of said human subject with near-infrared wavelength radiation in the 8001000 nm range or with visible wavelength radiation in the 500-700 range nm,
    (and) detect the radiation emitted by said nanoparticles and
    (d) determine depths of porous materials, thickness of sheets of materials, root structure of a plant or depth of the skin to which the tumor is based on the determination of the depth at which the nanoparticles are.
    Another embodiment is the use of the invention, wherein the composition comprises:-nanoparticles comprising the NaYF4 stabilizer, the sensitize Yb and an activator,where said activator is Er or Ho (NaYF4: Yb, activator), which emit radiation at lengthswave corresponding to blue, green and red,-nanoparticles comprising the NaYF4 stabilizer, the Yb sensitizer and the activatorTm (NaYF4: Yb, Tm), which emit radiation at wavelengths corresponding to blue andred / purple,-nanoparticles comprising the LaF3 stabilizer, the Tm sensitizer and an activator,where said activator is Er or Ho (LaF3: Tm, activator), which emit radiation at lengths ofwave corresponding to red and-nanoparticles comprising the Y20 3 stabilizer, the sensitize Yb and an activator,where said activator is Er or Ho (Y20 3: Yb, activator), which emit radiation at lengths ofwave corresponding to red.
    Another embodiment is the use of the invention, wherein said nanoparticles comprisespecific molecules against skin tumors.
    Another embodiment is the use of the invention, wherein said specific molecules againstSkin tumors are antibodies.
    Another embodiment is the use of the invention, wherein said skin tumors are selected.from the group consisting of melanomas, carcinomas, lymphomas and sarcomas.

    The use of the composition of the invention to make a diagnostic agent for the diagnosis of skin cancer in a human subject allows an imaging diagnosis of epithelial cancer, allowing to determine the depth of the affected areas. This use does not require radioactive tracers or ionizing radiation, which is used in the state of the art.
    The use of the composition of the invention to make a diagnostic agent for the diagnosis of skin cancer in a human subject uses fluorescent markers that do not pose any toxicity to the organism. An advantage of this use of the invention is that it does not require equipment of great economic cost or of great technological complexity.
    In the use of the composition of the invention to make a diagnostic agent for the diagnosis of skin cancer in a human subject, different formulations can be used that allow its use in various diagnostic conditions.
    Another alternative embodiment is a composition comprising a combination of fluorescent nanoparticles, wherein said composition comprises nanoparticles that emit radiation at a wavelength that corresponds to blue, nanoparticles that emit radiation at a wavelength that corresponds to green, nanoparticles that emit radiation at a wavelength corresponding to red and nanoparticles that emit radiation at a wavelength corresponding to red / purple for use in a method of diagnosing skin cancer in a human subject, wherein said procedure comprises:
    (to) administering said composition to said human subject,
    (b) irradiating the skin of said human subject with near-infrared wavelength radiation in the 800-1000 nm range or with visible wavelength radiation in the 500-700 nm range,
    (C) detect the radiation emitted by said nanoparticles and
    (d) determine the depth of the skin at which the tumor is based on the determination of the depth at which the nanoparticles are found.
    Another alternative embodiment is a method of diagnosing skin cancer in a human subject comprising:
    (to) administering a composition comprising a combination of fluorescent nanoparticles, wherein said composition comprises nanoparticles that emit radiation at a wavelength corresponding to blue, nanoparticles that emit radiation at a
    wavelength that corresponds to green, nanoparticles that emit radiation at a wavelength that corresponds to red and nanoparticles that emit radiation at a wavelength that corresponds to red / purple to said human subject,
    (b) irradiating the skin of said human subject with infrared wavelength radiation
    5 close in the 800-1000 nm range or with wavelength radiation in the visible in the500-700 nm range,
    (C) detect the radiation emitted by said nanoparticles and
    (d) determine the depth of the skin at which the tumor is based on the determination of the depth at which the nanoparticles are found.
    The nanoparticles of the composition of the invention have a high efficiency of radiation emission in the visible range.
    The combination of nanoparticles of the invention shows a differentiated spectrum in the visible range at different depths of the biological tissue. This is due to a different response to different wavelengths of biological tissue.
    In the present invention, near-infrared radiation generates a chain excitation of the elements (rare earths) that make up said nanoparticles, which
    20 allows a fluorescence response in the visible region.
    This visible fluorescence allows to locate tumors. For this, the surface of the nanoparticles is modified with specific molecules against skin tumors so that the nanoparticles will bind to the tumor cells when they are introduced into the torrent
    25 blood. Such specific molecules against skin tumors may be antibodies.
    The different wavelengths of the visible region of the spectrum show different attenuation to the tissue layers. Knowing said attenuation determines the depth at which the nanoparticles are located and therefore the tumor.
    The present invention provides the use of the composition of the invention to determine sheet thickness of porous materials. This use allows to measure the thickness of the layers generated in passivation, because they are inert compounds that would keep the material intact after its measurement.

    To this end, a layer of upconversion fluorescent nanoparticles is placed on the material and subsequent passivation is obtained. Through the incision of infrared radiation on the passivation sheet, a light response is obtained which, depending on the material studied and the thickness of the layer, would correspond to a wavelength of the visible range, that is, a discriminative response in color would be obtained.
    The most common passivation is that of aluminum, which forms a layer of aluminum oxide known as alumina and protects the material from the action of external agents. Alumina is a material that begins to degrade with frequencies below 1 MHz, which determines that infrared and visible radiation would be inert to it.
    The discriminative color signal appears because the absorbance of the material decreases from blue to red in the visible spectrum (MS Aw et al., Transition metal pairs on ceriapromoted, ordered mesoporous alu mine as catalyst for methane-C02 reforming reaction. Catalysis Seienee & Teehnology., 2016.6, 3797-3805).
    In order to understand said output signal, following the premise that the passivation layers are porous, the exponential extinction law developed in the preferred embodiments (equations 7-11) is applied, taking into account the effective attenuation coefficient that It will be specific to each material and each attenuation signal of the same with respect to the wavelength.
    The present invention provides the use of a composition comprising a combination of fluorescent nanoparticles to determine the root structure of a plant.
    There are several studies in which various experiments based on plant growth have been developed in a medium that contains high amounts of upconversion fluorescent nanoparticles, resulting in growth and development equal to that of control plants (Peng, J. et. al., Upconversion Nanoparticles Dramatically Promote Plant Growth Without Toxicity. Nano Researchs. November 2012, Volume 5, Issue 11, pp 770-782). In addition to growing in said medium, the upconversion fluorescent nanoparticles become in the vascular system of the plant, eliminating the need to subsequently incorporate upconversion fluorescent nanoparticles to them to develop growth measures
    relevant. On the other hand, the advantage of upconversion fluorescent nanoparticles is that they are harmless to plants.
    It is well known that, due to photosynthesis, the elements that make up plants
    5 such as chlorophyll and carotenoids, have absorption bands in blue and red in thevisible region, these differences of absorption of visible light would allow to observe theplant root growth by assessing their thicknessaccording to the light response they generate.
    10 As with biological tissue, this light response must be studied to know the depth of the emission signal, which will be carried out knowing the exponential extinction suffered by the emission spectrum of the nanoparticles introduced into the plant.
    15 For this, it must be taken into account that, unlike the biological tissue, in the plant, the percentage of water is between 70% and 90%, and is mostly in the roots. As for the calculations of the depth of the signal, they will have to be elaborated with respect to the effective attenuation coefficient of the water that would mainly affect the infrared signal.
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1: Scanning electron microscopy images of different powdered compounds: NaYF4: Yb, Er (first image, starting from above), NaYF4: Yb, Tm (second image, starting from above), LaF3: Tm, Er ( third image, starting at the top) and YZ0 3: Yb, Er (fourth image, starting at the top).
    Figure 2: The dashed curve represents the emission or fluorescence of the compound and the curve continues the absorption of the compounds: NaYF4: Yb, Er (first graph, starting 30 above), NaYF4: Yb, Tm (second graph, starting above ), LaF3: Tm, Er (third graph, starting at the top) and Y203: Yb, Er (fourth graph, starting at the top).
    Figure 3: Different crystalline phases present in each compound, determined by X-ray diffraction: NaYF4: Yb, Er (first graph, starting at the top), NaYF4: Yb, 35 Tm (second graph, starting at the top), LaF3: Tm , Er (third graph, starting at the top) and Y20 3: Yb, Er (fourth graph, starting at the top).
    Figure 4: Schematic representation of the transfer of radiation to and from fluorescent nanoparticles in a biological tissue.
    5 Figure 5: Melanoma model at different depths. First row, epidermis (0-0.04mm); second row, dermis (0.04-4 mm); third row, subdermis (4-60 mm). First column,position of the mela noma with respect to the surface of the skin; second column, nanoparticlesfluorescents randomly attached to the surface of the noma mela, the nanoparticlesrepresented in light tone are those that are illuminated, those represented in tone
    Dark 10 indicates nanoparticles in a dark or unlit area. The third column represents the resulting visible light color pattern on the surface of the skin relative to the depth of the fluorescent nanoparticles. The color pattern to determine the depth is established by the wavelengths of visible light Al and A2.
    15 Figure 6: Root structure model of a plant. The colorimetric differentiation is established with the wavelengths of visible light Al, A2 and A3 so ordered due to the effective absorption coefficient of water, mostly in the roots.
    Figure 7: Model for determining the thickness of passivation sheets. Al and A2 represent
    20 the wavelength of visible light emitted by the nanoparticles, representing the wavelengths that allow the passivation layer to be traversed based on its thickness.
    PREFERRED MODES OF REALIZATION
    25 Example 1. Synthesis of upconversion fluorescent nanoparticles
    The most relevant parameters of nanoparticles that directly affect the physical process and can be modified in the synthesis are:
    30 -Size. - Crystal phase purity. -Morphology. -Monodispersity.
    35 The most studied methods for controlling these parameters are: coprecipitation method; thermal decomposition, sol-gel process and solvothermic method. All these processes have limitations, especially in terms of dispersion of nanoparticles and size distributions, except for the solvothermic method. This method allows the synthesis of nanocrystals with a size, morphology, optical and magnetic properties controlled through temperature and reaction time, the concentration of
    5 reagents, the pH value and the different reaction precursors. Therefore, thesolvothermic method for the synthesis of upconversion fluorescent nanoparticlespresented in this work.
    In order to synthesize these nanoparticles, the following steps have been carried out:
    10 -Preparation of the stock solution: this solution contains rare earths (Ianthanides) that act as: stabilizer, sensitizer (energy donor) and activator (energy emitter) to carry out the upconversion process. The solution used has 0.5 M Ln (N03) where the percentage of each lanthanide is:
    15-Percentage X of stabilizer. - 0.2X percentage of sensitizer. -0.002X percentage of activator.
    20 All these percentages are calculated relative to the number of moles. Finally, when the solution is prepared, it is stirred at a temperature between 40-50 ° C until the components have completely dissolved and the color changes from a white to transparent color.
    - Preparation of solution A: it is a mixture of oleic acid and ethanol, which
    25 act respectively as a controller of growth and solubility of the particles in the mixture. The amount (in moles) of these compounds is calculated as follows:
    - Oleic acid, relative to the amount of stabilizer:
    30 Ionic Mobid = 81.5 Mobilizer (1)
    - Ethanol, relative to the amount of oleic acid:
    MolEtOH = 8.2 Molacidoleolic (2)
    35 -Preparation of solution B: This mixture is formed by NaF and NaOH. The first of these components is used to form the crystal matrix. The second is used as a precipitation agent to give rise to solid particles. The amount thereof (in moles) is calculated as:
    -NaF: An excess of compound is necessary with respect to the stabilizer. In this case5 an amount three times greater will be used.
    MolNaF = 3 Annoying (3)
    - NaOH: calculated relative to the amount of NaF considering an excess over it, 10 excess was considered sufficient after several tests.
    MolNaoH = 2.5 MolNaF (4)
    The next step is the mixing of solutions A and 8 for the time required to ensure the action of each component.
    Once the final solution has been obtained, it is introduced into an autoclave (the volume of the solution introduced will depend on the volume of the autoclave used) which in turn will be introduced in a muffle oven to generate the ideal conditions for the formation of the
    20 nanoparticles In this case the conditions were 230 oC for 24 hours. When this process is finished, the resulting precipitate obtained inside the autoclave is washed with ethanol and water several times with the help of a centrifuge.
    To do this, ethanol and water will be introduced into a tube together with the precipitate and the mixture 25 will be taken to a centrifuge at 6000 rpm. for 5 min, after which the liquid part will be discarded leaving the precipitate clean.
    The final step in the synthesis is the introduction of the particles washed in an oven to eliminate possible residues that could not be eliminated with centrifugal washing. This process is called calcination and its conditions are 400 oC for 4 hours.
    The upconversion fluorescent nanoparticles that were synthesized are:
    NaYF4: Yb, Er 35 NaYF4: Yb, Tm LaF3: Tm, Er
    These nanoparticles were selected because they have different fluorescence spectra with which the entire range of the visible spectrum can be covered.
    Example 2. Characterization of upconversion fluorescent nanoparticles
    In order to carry out the combination of upconversion fluorescent nanoparticles suitable to achieve a differentiated response in depth, one must first characterize them individually. The characteristics that most affect the upconversion process are:
    - Structure of the nanoparticle: a high surface volume ratio favors the efficiency of the process.
    15-Absorption and emission spectrum: they are key elements to know the response in depth of the materials.
    In order to characterize the nanoparticles, a scanning electron microscope (Scanning Electron Microscope (SEM »(Hitachi TM-1000) was used. It generates an image from
    20 of an electron beam, which is detected after interacting with the observation sample, which allows defining the structure, texture and possible chemical composition of the sample.
    An X-ray diffraction (X-Ray Diffraction (XRD)) was performed. For this, the Philips 25 PW 3040100 X'Per! MPD / MRD.
    All this allowed to characterize the crystalline structure of the nanoparticles at the atomic and molecular level.
    30 To characterize the absorption spectrum, a Perkin Elmer Lambda 1050 UV device is used. For this, the compounds are irradiated with a 980 nm laser (1kW power) and their response is detected with a calibrated StellarNet Inc spectrometer that allows the display of emission peaks. With all these results, it can be seen that relationships directly affect the emission power of the compounds, and therefore
    35 the wavelength and intensity of it. This would make possible (or not) the visualization of the upconversion process of the compounds through the different porous materials.

    The most important relationship is determined by the images obtained from the SEM, where it is deduced that the emission intensity is directly dependent on the size of the nanoparticles. Simultaneously, thanks to the analysis of the XRD spectra, it is observed that there are different crystalline phases and compositions in the synthesized nanoparticles which determines that the different morphologies, observed in the images of the SEM, in the same nanoparticle do not correspond to the same crystalline phases and compositions
    It was observed that in those compounds with representative peaks of the hexagonal crystalline phase of the upconversion fluorescent nanoparticles, the emission intensity is greater than those in which phase mixtures appear. This reduction in the emission intensity may be due to the different emissions presented by different crystalline phases and to the presence of phases that do not carry out the upconversion process as is the case of NaF and that weaken the infrared absorption by the nanoparticles. fluorescent upconversion and therefore also its emission. Therefore, it would be necessary to search for new reaction conditions that would allow a total control of the resulting compounds, in this way, concrete compounds could be generated to measure the depth of concrete materials.
    Four different upconversion fluorescent nanoparticles have been generated and characterized. The crystalline structure of the upconversion fluorescent nanoparticles with NaYF4 base has been characterized as long hexagonal rods, but, due to the solvotermal synthesis process, we also find products (upconversion fluorescent nanoparticles and not upconversion fluorescent nanoparticles) with cubic structure. The crystalline structure of both the LaF3: Tm, Er upconversion fluorescent nanoparticles and the Yz03: Yb, Er upconversion fluorescent nanoparticles and the products (non-upconversion fluorescent nanoparticles with cubic structure) also appear in the resulting compound. These products seem to hinder the upconversion process so that the control of the synthesis process is necessary so that the purity of the upconversion fluorescent nanoparticles obtained is raised.
    Additionally, upconversion fluorescent nanoparticles with NaYF4 bases show greater efficiency to the upconversion process than those with cubic phase. As the upconversion process is a surface process, upconversion fluorescent nanoparticles with a greater surface volume ratio (Ex: long and thin hexagonal rods)
    they will be more efficient than those with low relationships (eg, cubic nanostructures).
    Although all upconversion fluorescent nanoparticles have a process
    5 upconversion, when illuminated with ambient white light (typical conditions ofwork), those with hexagonal structure are more efficient than those withcubic structures
    Example 3. Design upconversion fluorescent nanoparticles as a diagnostic agent
    UC materials are ideal for working in the first and second biological window (Smith, AM et al., Second window for in vivo imaging, Nature Nanotechnology, 4 (11): 710711 (2009). These are respectively located in the electromagnetic spectrum in 650-950 nm and 1000-1350nm, the second window can be used for the illumination of the UCNP while the first one is ideal for the transmission of the fluorescent response.In addition, the interest of upconversion fluorescent nanoparticles in the field of The diagnosis of cancer is that it is possible to establish a direct union of these compounds with cancer cells.Thanks to the mutation of the surface of these cells, changes in their antigenic production occur, therefore, on the surface of the tumor tissue 20 antigens that are not found with such abundance in healthy tissue, so the upconversion fluorescent nanoparticles are doped with antibodies related to In tumor tumor, the upconversion fluorescent nanoparticles and cancer cells will be allowed to bind due to the formation of the antigen-antibody complex. There are other ways of binding cancer cells, either through physical processes or by the
    25 binding of upconversion fluorescent nanoparticles with molecules that mimic the structure of biomolecules present in the human body.
    The use of these nanoparticles is well indicated in the field of diagnosis for the following reasons:
    - Possibility of using both visible and infrared radiation to generate images in biological tissue that do not cause harmful effects to cells or irradiated tissue.
    - In addition, the use of both radiations has an additional effect that allows the resulting signal-to-noise ratio to be increased 35 to one hundred times. This fact is due to the combination of the presence of both biological windows (low noise channel) and the absence of self-flowering by irradiated tissue (absence of interference).
    - Upconversion fluorescent nanoparticles are not toxic and are, in fact, biologically inert.
    Next, it will be explained how radiation transfer takes place in the two biological windows of the tissue. Additionally, the model for the treatment of images in the depth detection processes in the diagnosis of subcutaneous tumors is included.
    Radiation transfer in biological medium
    The spectrum of interest will be directly affected by two effects: i) absorption and ii) dispersion of photons in the biological tissue. Both can modify the number of photons that go from the Source (UCNP) to the surface of the tissue but will not modify its energy, that is, its wavelength.
    The probability of photon absorption by a medium per unit of path length (in cm-1) is given by the absorption coefficient o / Ja. The probability of dispersion of photons by a medium per unit of path length (cm- '), is given by the dispersion coefficient or 1Js. The probability of dispersion in the biological tissue is normally at least three orders of magnitude greater than that of absorption.
    However, the dispersion in a biological medium, which is highly anisotropic, presents notable differences to those found in isotropic media considered as standard in this type of applications. Thus, it must be considered that the type of dispersion that takes place is in diffusive regime, in which the probability of dispersion of a photon per unit of path length is reduced by at least two orders of magnitude with respect to the absorption coefficient, due to anisotropy. This effect is taken into account considering the reduced dispersion coefficient, which estimates the equivalent probability of the dispersion of a photon in isotropic medium in diffusive regime and is defined as:
    35 ,,; = ", (1 -g) (5)
    where g is the mean of the dispersion cosine of the polar angle by single dispersion.
    The diffusion is governed by Fick's law, which leads to the diffusion coefficient, which is linked to the influence of the gradient, and can be defined as (in units of cm · 1):
    D -1 (6)
    - 3 (flli + fI ~)
    In addition, the exponential extinction law that determines the intensity of the photon beam that passes through a length z in an anisotropic medium, defined as:
    Where I and I are the input and output intensities respectively, and Jieel effective attenuation coefficient, which quantifies the influence of the decay rate at distances far from the emission source (defined in cm · 1):
    1 ', = chi (8)
    The effective attenuation coefficient can also be interpreted as the depth at which the intensity of the light decreases to 1 / e of the initial intensity, that is, I l ':::: 36, 79% of
    the.
    The extinction of light in biological medium is experimentally measured as fJe. The effective attenuation coefficient has been measured in different biological media, mainly in oxygenated and deoxygenated hemoglobin, human skin and fat.
    Attenuation model for biological tissue
    A channel model or attenuation model was used. This model allowed to define the attenuation for the spectrum of the upconversion fluorescent nanoparticles at different depths in biological tissue, taking into account both the attenuation of the emitting source (NIR) and that of the fluorescence in the different types of biological media of the tissue (visible ).
    In previous sections it has been determined that Pe is the parameter responsible for the attenuation of light in an anisotropic medium. If in this case the light is NIR or visible, the first and second biological window of the tissue will be applied. Therefore, a source of near-infrared radiation in contact with the skin is defined, illuminating upconversion fluorescent nanoparticles located at a depth with lapump intensity
    z. Thanks to the ranges of the second biological window, it is known that the Ipump (Iopump is 980 nm) will be suitable for exciting upconversion fluorescent nanoparticles. The light emitted by the particles can be phenomenologically modeled in terms of
    10 laser excitation intensity as:
    Where A, B and K are positive constants. However, since the illumination takes place at a certain depth of the biological tissue, the near infrared laser attenuation follows the law of exponential attenuation that can be observed in Eq. 7:
    I ~ (l, z) = "(1," m,) ', (10)
    1 + Alpump +8 (Ip ump)
    - l 'e-Il ~ z
    20 AND 1pump -pump.
    If the exponential extinction law is also applied to the emission spectrum, the intensity of the emitted radiation (with AF wavelength) of the upconversion fluorescent nanoparticles located at a depth z within the biological tissue (with a coefficient of
    25 effective attenuation of Pe (A »illuminated with a laser (with Ap wavelength) can easily be calculated as:
    30 Where == I ~ ump The previous equation will be used to calculate the expected response of upconversion fluorescent nanoparticles at different depths with different components of biological tissue.
    5 Adaptation of upconversion fluorescent nanoparticles to form images oftumors
    Taking into account PeCA) and the measurement of the emission peaks of the upconversion fluorescent nanoparticles of the previous section, it can be determined that they are 10 emission lines found in the first biological window (between 650 and 950 nm).
    Considering the results obtained in the characterization processes, the control parameters and improvement of the properties of the upconversion fluorescent nanoparticles for the process of interest have been determined. The bases and sensitizers 15 can be used to obtain higher emission fluorescence intensity, while modifying the activator can be used to change the emission peaks that would allow the spectrum to be modified. It can be seen that it is easy to devise an optimization strategy by combining different upconversion fluorescent nanoparticles, which would result in identifiable combinations of resulting spectra. This
    20 optimization strategy can be developed through combinatorial chemical research or by simulation of the expected spectrum.
    The present application explored the use of a simple combination of upconversion fluorescent nanoparticles synthesized with different emission behaviors to predict their response spectra on the skin surface. The combination of the materials selected in each case gives rise to systems in which a combination of different emission lines appears, each of which has an associated attenuation relative to the components of the biological tissue at different depths, resulting in a pattern of colors which allows diagnosis at different depths
    30 epithelials
    Example 4. Results obtained with the attenuation model for biological tissue
    They have been considered at three levels of depth of diagnosis at the skin level (O -0.04 35 mm), at the level of the dermis (0.04 -4 mm) and at the level of the subdermis (4 mm -6 cm).

    The tumor model has been extracted from a real melanoma and for simplicity has been adjusted to a geometric model, ellipsoid type.
    Figure 5 shows the results obtained. The position and the tumor model observed are shown in the first and second columns. The exposed tumor surface is observed in the third column. Assuming that upconversion fluorescent nanoparticles randomly adhere to the tumor surface, only those exposed to near infrared illumination can undergo the upconversion process. Thus, the areas of light gray tones represent the upconversion fluorescent nanoparticles susceptible to upconversion and therefore those that will be fluorescent, while darker tones are those that are not irradiated and therefore will not emit light.
    Finally, the last column represents the image that will be observed on the surface of the skin according to the combination of upconversion fluorescent nanoparticles previously described and undergoing attenuation consistent with the different components of the biological tissue at each depth.
    In the first row, there is a noma mela located at the surface level, so the attenuation coefficient relevant to this level is only that of the skin. At this level the capillaries are extremely small and their contribution is irrelevant. In addition, there is no fat present in the epidermis, therefore skin attenuation is very weak and the spectrum can be observed on the surface without suffering attenuation.
    In the second row of the same figure, the situation is shown in which the mela just with the same dimensions as in the previous level is in the dermis. Here, the relevant attenuation coefficients are those of the skin and deoxygenated blood (deoxygenated is selected as having an attenuation coefficient higher than that of the oxygenated). It is considered a Jie resulting from the contribution of 2/3 / J (skin) and 1/3 / Je (HG oeox) due to the increased presence of capillaries in this region.
    In this case, different colors are observed depending on the depth. The intensity of the green wavelengths is greater in areas closer to the surface. However, red wavelengths begin to appear in deeper regions.
    Finally, in the third row, the case of a melanoma at the subdermal level is shown, where the attenuation coefficient considered in a contribution of skin, blood and fat in a proportion 1/3, eu (skin), 1/3, eu (HGoeOx) and 1/3, eu (fat), because fat appears as a component of the tissue in the subdermis. In the latter case, only the red color is observed and this is due to the passage band existing in the biological windows for these wavelengths.
    The results presented demonstrate the feasibility of using upconversion fluorescent nanoparticles to obtain differentiated color patterns depending on the depth of tissue at which a particular tumor is found.
    权利要求:
    Claims (7)
    [1]
    one. Method for determining the depth of a material characterized in that said method
    understands:
    S (a) applying on said material a composition comprising a combination of
    fluorescent nanoparticles, wherein said combination comprises:
    -at least one nanoparticle comprising NaYF4 as a stabilizer, Yb as
    sensitize te and an activator selected from Er or Ho, in which said nanoparticle
    It is capable of emitting radiation at wavelengths corresponding to blue, green and
    OR Red,
    -at least one nanoparticle comprising NaYF4 as a stabilizer, Yb as
    sensitizer and Tm as activator, in which said nanoparticle is capable of emitting
    radiation at wavelengths corresponding to blue and red / purple,
    -at least one nanoparticle comprising LaF3 as a stabilizer, Tm as
    fifteen sensitizer and an activator selected from Er or Ho, wherein said nanoparticle
    It is capable of emitting radiation at wavelengths corresponding to red and
    -at least one nanoparticle comprising Y20 3 as a stabilizer, Yb as
    sensitizer and an activator selected from Er or Ho, wherein said nanoparticle
    It is capable of emitting radiation at wavelengths corresponding to red,
    twenty (b) irradiate the material of step (a) with infrared wavelength radiation
    close in the 800-1000 nm range or with wavelength radiation in the visible in the
    500-700 nm range, to obtain radiation emitting nanoparticles,
    (c) detecting the radiation emitted by said nanoparticles from step (b),
    (d) determine from the radiation detected in step (c) the depth at which
    2S find the nanoparticles and
    (e) determine the depth of the material from the depth at which they are
    the nanoparticles of step (d).
    [2]
    2. Method for determining the depth of a material according to claim 1,
    30 characterized in that the material is selected from: a porous material, the root of a
    plant or skin of a human subject.
    [3]
    3. Method according to claim 2, wherein said porous material has laminar structure.
    [4]
    Four. Method according to claim 2, wherein said material is the skin of a human subject.
    [5]
    5. Method for determining the depth of a material according to claim 4,
    S characterized in that the composition of step (a) comprises at least one molecule capable of recognizing an antigen in cells of a skin tumor.
    [6]
    6. Method according to claim 5, characterized in that said molecule is an antibody.
    OR
    [7]
    7. Method according to any of claims 5 or 6, characterized in that said skin tumor is selected from the group consisting of a melanoma, a carcinoma, a lymphoma and a sarcoma.
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    同族专利:
    公开号 | 公开日
    ES2684057B1|2019-07-04|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2013112856A2|2012-01-26|2013-08-01|The Regents Of The University Of Colorado, A Body Corporate|Multifunctional nanomaterials for the treatment of cancer|
    WO2013181076A1|2012-05-30|2013-12-05|University Of Massachusetts Medical School|Coated up-conversion nanoparticles|
    WO2015102535A1|2014-01-06|2015-07-09|National University Of Singapore|Uniform core-shell tio2 coated upconversion nanoparticles and use thereof|
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