Thermometric composition comprising biodegradable periodic mesoporous organosilica and at least two dyes

Abstract

Provided herein is a thermometric composition, comprising a plurality of biodegradable periodic mesoporous organosilica, B-PMO, particles, wherein: - each B-PMO particle is loaded with a first dye and a second dye, - the first dye and second dye are both fluorescent, - a fluorescent light emission by the first dye causes a luminescent excitaon of the second dye, - the first dye and / or the second dye exhibit(s) a temperature-dependence fluorescence. Further is provide a method for preparing such thermometric composition, and a method for obtaining temperature data of a cell, a tissue or a subject, using such thermometric composition.

Description

[0001] THERMOMETRIC COMPOSITION COMPRISING BIODEGRADABLE PERIODIC MESOPOROUS ORGANOSILICA AND AT LEAST TWO DYES

[0002] Field of the invention

[0003] The present invention is in the field of thermal monitoring and thermal-mapping, and more particular in the field of thermometric composition which may be used therefore. These thermometric compositions are preferably suitable to be used as (nano-)thermometers, which can be employed in vivo and preferably subcutaneously.

[0004] Background to the invention

[0005] Temperature is a fundamental quantity related to many natural biological processes. As a result, thermal monitoring plays a paramount role in disease diagnosis. The conventional techniques for temperature readout (e.g. thermomcouples, thermistors, IR cameras), although reliable for many applications, are not performing satisfactorily for sophisticated in vivo and in vitro temperature mapping, either because they are invasive, or unable to detect temperature below the skin surface. Employing luminescence for in vivo temperature assessment has attracted substantial interest in the past decade. Luminescent (nano)thermometers can be developed because some materials possess luminescent properties strongly dependent on temperature. This allows engineering of sensors capable of readouts at the nanoscale that can work in a non-contact and minimally invasive manner. Such luminescent nanothermometers are a promising new tool for early-stage diagnosis of inflammation states or cancer (such cells have slightly elevated temperatures), as well as for treatment control (e.g. during photothermal ablation where the heating process needs to be carefully controlled to avoid damage of nearby healthy tissue).

[0006] However, despite their great promise and impact, to date, luminescent nanothermometers have not surpassed preclinical stages, as many developed nanomaterials, are potential toxicity and bioaccumulation in the human body considering the ultimate objective of clinical applications. Since most reported nanothermometers currently are not degradable materials and are mainly based on the incorporation of heavy metal ions, these aspects remain of genuine concerns in the fields of nanomedicine, nanobiotechnology, nanotoxicology, and nanopharmacology.

[0007] Hence, there is a demand for thermometric compositions, preferably suitable to be used as nanothermometers, with a low toxicity and preferably do not accumulate in the body. However, these thermometric compositions need to be sensitive, and express a high signal to noise ratio. Preferably these thermometric compositions are easy to produce, and allow a large variation in used wavelength, detection equipment and modes of excitement. There is a specific demand for thermometric compositions that can be used through the skin or through tissue. Summary of the invention

[0008] The thermometric compositions and methods disclosed herein may meet one or more demands as set out above. The inventors have found that loading two fluorescent dyes in a biodegradable PMO particle, wherein the emission of one dye, excited the other dye; may at least meet some of the herein formulated demands. Having the two dyes in a B-PMO particle ensures efficient energy transfer between the two dyes, making the overall system very sensitive.

[0009] More particularly the invention provides a thermometric composition, comprising a plurality of biodegradable periodic mesoporous organosilica, B-PMO, particles, wherein:

[0010] - each B-PMO particle is loaded with a first dye and a second dye,

[0011] - the first dye and second dye are both fluorescent,

[0012] - a fluorescent light emission by the first dye causes a luminescent excitation of the second dye,

[0013] - the first dye and / or the second dye exhibit(s) a temperature-dependence fluorescence.

[0014] In some embodiments, the first dye and second dye are both organic.

[0015] In some embodiments, each B-PMO particle comprises multiple polymeric chains of alkoxysilane of the formula I

[0016] - (RO)3Si-R' -,

[0017] (formula I) wherein R is CH3-, or CH2CH3-; and, wherein R' is a substrate for an enzyme.

[0018] In some embodiments, R' is [-(CH2)x-(S-S)n-(CH2)x-]; wherein x is equal to or greater than 1; and, wherein n equal to or greater than 1.

[0019] In some embodiments, the first dye has an emission peak in the wavelength range 500 to 1200 nm, and wherein the second dye has an absorption peak in the in the wavelength range 500 to 1200 nm.

[0020] In some embodiments, the absorption band, preferably the absorption peak, of the first dye is lower in wavelength compared to the emission band, preferably the emission peak, of the second dye. In some embodiments, the first dye is selected to have: an absorption peak between 500 and 750 nm; and, an emission peak of at least 600 nm: and, wherein the second dye is selected to have: an absorption spectrum overlapping with the emission spectrum of the first dye; an emission peak at least 20 nm removed from the emission peak of the first dye.

[0021] In some embodiments, the emission peak of the second dye has a wavelength between 600 and 950 nm.

[0022] In some embodiments, the first dye and the second dye are independently selected from the list comprising: a rhodamine derivative, a coumarin derivative, an acridine derivative, a carbopyronin derivative, an oxazine derivative, a cyanine dye, or a xanthene dye.

[0023] In some embodiments, the ratio between the first dye and the second dye, equals the ratio between the intensities of their respective emission band ± 20%, preferably ± 15%, preferably ± 10%, preferably ± 5% of that ratio.

[0024] The invention further provides in a method of preparing the thermometric composition according to an embodiment described herein, comprising the step of:

[0025] -soaking B-PMO particles unloaded, with a combination of the first dye and the second dye.

[0026] In some embodiments, the method involves applying a lipid bilayer on the surface of the loaded B- PMO particles.

[0027] The invention further provides in a method for obtaining temperature data of a cell, a tissue or a subject comprising: obtaining, using a light sensor, light-emission data of the subject, where the light sensor is disposed external to a cell, a tissue or a subject; determining from the light-emission data, the temperature data of a subject, wherein the cell, the tissue or the subject been provided with the thermometric composition of any one of the previous claims, and the light sensor is configured for detection of light emissions by thethermometric composition.

[0028] In some embodiments, the method involves irradiating the cell, the tissue or the subject with light with a wavelength within the absorption band of the first dye.

[0029] In some embodiments, the method is performed in vitro.

[0030] Figure Legends

[0031] Figure 1 provides a sketch of a possible layout of absorption bands and emission bands of the first and second dye used in a thermometric composition according to an embodiment of the invention.

[0032] Figure 2 depicts various temperature dependent fluorescence of the first and second dye.

[0033] Detailed description of invention

[0034] Before the present system and method of the invention are described, it is to be understood that this invention is not limited to particular systems and methods or combinations described, since such systems and methods and combinations may, of course, vary. It is also to be understood that the terminology used herein is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0035] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0036] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. It will be appreciated that the terms "comprising", "comprises" and "comprised of" as used herein comprise the terms "consisting of", "consists" and "consists of".

[0037] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. The term "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

[0038] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear perse, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0039] All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference.

[0040] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0041] In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0042] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination. In the present description of the invention, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration only of specific embodiments in which the invention may be practiced. Parenthesized or emboldened reference numerals affixed to respective elements merely exemplify the elements by way of example, with which it is not intended to limit the respective elements. Unless otherwise indicated, all figures and drawings in this document are not to scale and are chosen for the purpose of illustrating different embodiments of the invention. In particular the dimensions of the various components are depicted in illustrative terms only, and no relationship between the dimensions of the various components should be inferred from the drawings, unless so indicated.

[0043] It is to be understood that other embodiments may be utilised and structural or logical changes may be made without departing from the scope of the present invention. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims.

[0044] Provided herein is a thermometric composition, comprising a plurality of biodegradable periodic mesoporous organosilica, B-PMO, particles, wherein:

[0045] - each B-PMO particle (of the plurality) is loaded with a first dye and a second dye,

[0046] - the first dye and second dye are both fluorescent,

[0047] - preferably, the first dye and second dye are both organic, and preferably free of heavymetals, preferably free of metals,

[0048] - a fluorescent light emission by the first dye causes a luminescent excitation of the second dye, and,

[0049] - the first dye and / or the second dye exhibit(s) a temperature-dependence fluorescence.

[0050] Such thermometric compositions may have the advantage that the compositions are biodegradable and therefore not accumulated in a subject, making the thermometric compositions to be suitable for in vivo use. Furthermore, the thermometric composition can be free of metals and especially heavy metals, what makes the toxicity of the thermometric composition low or even non existing. As the two dyes are confined withing the B-PMO, the dyes are in close proximity of each other, what ensures an efficient energy transfer between the two dyes. This allows for ratiometric thermometers to be based on the thermometric compositions. Due to the two different dyes being used, the correlation between the temperature and the fluorescence is high, which may allow detection of very small temperature differences. Even more, because of two dyes being used working together, the signal to noise ratio is high, and the sensitivity is high. This may allow for using low amounts of thermometric composition, which is in its turn advantage as the addition of low amounts of the thermometric composition may not interfere with biological processes which are investigated. The thermometric composition allows further for a flexibility in (commercial) dyes being used, which would allow to tune the thermometric composition to the desired situation, e.g. envisaged temperature rage, envisaged wavelengths, available detection method, available irradiation source, .... The B-PMO particles as used in the invention may be easy to synthesis and / or easy up-scalable, especially compared to any metal-based nanoparticles. As also commercially available dyes may be used, the thermometric composition is easily accessible.

[0051] In some embodiments, the thermometric composition is a ratiometric thermometer. In some embodiments, the thermometric composition is used as a ratiometric thermometer, preferably in a biological system. As used herein the term "ratiometric thermometers" are thermometers in which measurements are based on the intensity change of two emission bands. These ratiometric thermometers, may be considered to be more reliable temperature probe compared to those based only on a single emission band. In ratiometric thermometers, the intensity ratio between two transitions may not be compromised by experimental disadvantages such as small material inhomogeneities, variations of the sensor concentration or optoelectronic drifts of the excitation source and detectors.

[0052] In some embodiments, one of the first dye or the second dye exhibit(s) a temperature-dependence fluorescence, whereas the other dye exhibits a temperature-independent fluorescence. In such embodiments, the fluorescence of the dye exhibits a temperature-independent fluorescence can be used as a reference emission signal, which may be used for calibrating.

[0053] As used herein the term "periodic mesoporous organosilica" or abbreviated to PMO refers to a type of silica comprising organic groups that give rise to a periodic mesoporous structure. PMO may be obtained by the sol-gel process from an organo-bridged alkoxysilane in the presence of a structuredirecting agent, e.g. a surfactant. The porous framework of the PMO may be based on organic functional group covalently linking siloxane domains. After synthesis the structure directing agent is removed from the pores of the PMO. In general, PMOs may have a well-ordered mesoporous structure, a high thermal and / or mechanical stability. Typically, PMOs may have a rather uniform distribution of organic functionalities in the pore walls.

[0054] In some embodiment, the PMO may be a material made from: a mixture of organo-bridged alkoxysilanes; or, a mixture of organo-bridged alkoxysilanes mixed with a silica source, such as tetraethyl orthosilicate (TEOS).

[0055] In some embodiments, the mixture may further comprise silsesquioxanes.

[0056] As used herein the term "silsesquioxane" may refer to an organosilicon compound with the chemical formula [RSiOa / zJn (R = H, alkyl, aryl, alkenyl or alkoxyl.). Silsesquioxane may adopt cage-like or polymeric structures with Si-O-Si linkages and tetrahedral Si vertices, wherein inorganic silicate (SiCh) cores are formed with an organic exterior or shell.

[0057] As used herein, the term "biodegradable" refers to the property of a compound or material to be degraded by biological processes, preferably to non-toxic compounds. Preferably, these biological processes occur in a body, preferably a human body.

[0058] In some embodiments, the biodegradable periodic mesoporous organosilica particles are metabolizable PMO particles, preferably metabolizable by an animal or human body.

[0059] In some embodiments, the biodegradable periodic mesoporous organosilica particles are eliminable from the animal or human body. In some embodiments, elimination half-life of the biodegradable periodic mesoporous organosilica particles from the animal or human body, is at most 72.0 hours, preferably at most 48.0 hours, preferably at most 24.0 hours, preferably at most 18.0 hours, preferably at most 12.0 hours, preferably at most 6.0 hours.

[0060] As used herein the term "fluorescent" refers to the property of a compound, such as a dye, to re-emit light after light excitation. The re-emited light can be any light, such as ultraviolet, visible light or infrared.

[0061] As used herein the term "dye" are compounds, preferably organic molecules, with extended conjugation, comprising a framework of o-bonds and an associated n-system, which interact only specific wavelengths of the light spectrum. Hence, due to this specific interaction, a dye may exhibit specific colours. Dyes may differ from organic pigments in the fact that dyes are soluble.

[0062] As used herein, the term "temperature-dependence fluorescence" may refer to changes in fluorescent behavior of a compound due to the influence of temperature, so can the wavelength of the emitted light change under the influence of temperature, can the emission intensity change under the influence of temperature and / or can the lifetime of the fluorescence change under the influence of temperature. Hence, compounds showing such a temperature-dependence fluorescence may be used for luminescence thermometry. Preferably, the temperature-dependence fluorescence manifest itself in the visual light or infrared section of the light spectrum.

[0063] In some embodiments, at least one of the first dye or the second dye is a thermochromic dye, preferably both the first dye and the second dye are thermochromic dyes.

[0064] As used herein the term "thermochromic" refers to the property of a substance to change colour due to the change in temperature.

[0065] As used herein, the term "B-PMO particle being loaded with a dye" refers to the dye being partially or fully impregnated in the B-PMO particle or the dye being non-covalently coupled to the B-PMO particle. In other words, the B-PMO particle may be doped with the dye. The dye may be (reversable) encapsulated in the B-POM pores, may be (reversable) (non-covalently) coupled to the walls of the B- PMO particle.

[0066] In some embodiments, the first dye and the second dye are non-covalently coupled to the B-PMO particles, or impregnated in the B-PMO particles.

[0067] In some embodiment, a lipid bilayer is provided on the surface of the loaded B-PMO particles. This lipid bilayer may prevent premature leakage of the dyes from the B-PMO particles.

[0068] In some embodiments, the thermometric composition is a dispersion, preferably a dispersion of the B-PMO particles in an aqueous phase, preferably in an isotonic solution.

[0069] In some embodiments, each B-PMO particle comprises multiple polymeric chains of alkoxysilane of the formula I

[0070] - (RO)3Si-R' -,

[0071] (formula I) wherein R is CH3-, or CH2CH3-; and, wherein R' is a substrate for an enzyme, preferably cellular enzyme, preferably an intra-cellular enzyme. As R' is a substrate for an enzyme, R' may provide the biodegradablele property of the B- PMO. Preferably, R' comprises at least one disulphide bridge. In some embodiments, R' is [-(CH2)x-(S-S)n-(CH2)x-]; wherein x is equal to or greater than 1, and preferably less than 15, preferably less than 10, preferably less than 5; and, wherein n equal to or greater than 1, preferably equal to or greater than 2, like n = 2, 4, or 6. Preferably n is at most 10, preferably at most 8, preferably at most 6.

[0072] In some embodiments, 90% of the B-PMO particles have a particle size (d90) of at least 20.0 nm to at most 650.0 nm, preferably at least 30.0 nm to at most 600.0 nm, preferably at least 50.0 nm to at most 500.0 nm, preferably at least 70.0 nm to at most 400.0 nm, preferably at least 100.0 nm to at most 300.0 nm, preferably at least 150.0 nm to at most 250.0 nm. Such particle sizes are advantageous as the particles are small enough to be used in the field of medicine, to reach a desired target, and still large enough to hold enough dye.

[0073] In some embodiments, the average wall thickness of the B-PMO is at least 5.0 to at most 100.0 nm, preferably at least 7.0 to at most 75.0 nm, preferably at least 10.0 to at most 50.0 nm, preferably at least 20.0 to at most 40.0 nm. Such wall thickness may be a compromise between rigidity providing mechanical stability and softness and flexibility, which is beneficial for delivery purposes and ability of entering cells.

[0074] In some embodiments, 90% of the pores of the B-PMO have a pore size of at least 0.5 nm, preferably at least 0.7 nm, preferably at least 1.0 nm, preferably at least 1.0 nm, preferably at least 2.0 nm. In some embodiments, 90% of the pores of the B-PMO have a pore size of at least 0.5 nm to at most 10.0 nm, preferably at least 0.7 nm to at most 7.0 nm, preferably at least 1.0 nm to at most 5.0 nm, preferably at least 1.0 nm to at most 4.0 nm, preferably at least 2.0 nm to at most 3.0 nm. Such pore sizes allow an easy uptake of the dyes.

[0075] In some embodiments, the BET surface area of the B-PMO particles before loading of the dyes is at least 100 m2 / g, preferably at least 200 m2 / g, preferably at least 400 m2 / g, preferably at least 600 m2 / g, preferably at least 800 m2 / g, preferably at least 1000 m2 / g, preferably at least 1200 m2 / g, preferably at least 1400 m2 / g, preferably at least 1500 m2 / g. After loading of the dyes, these BET surface areas may significantly be lower, for example in the 20 m2 / g or 50 m2 / g range.

[0076] In some embodiments, the B-PMO particles are non-aggregated. In some embodiments the B-PMO particles may be non-hollow particles. In some embodiments, the B-PMO particles may be hollow B-PMO particles. Hollow particles may have the advantage that they can deform more easily compared to non-hollow particles, and are therefore easier to be taken up by a cell.

[0077] In some embodiments, the B-PMO particles are synthesised from at least the following precursors, preferably in the presence of a structure-directing agent: tetra-alkyl orthosilicate to form a core particle, preferably selected from the list comprising tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate, preferably tetraethyl orthosilicate; and / or, a mixture comprising : o a di- or tetra- sulphide silica precursor, preferably wherein the di- or tetra- sulphide silica precursor is bis(3-trial koxysilylyalkyl ) disulphide and / or bis(3-trial koxysilylyalkyl ) tetrasulphide; o and optionally, a bis(trialkoxysilyl)hydrocarbon, preferably selected from the list comprising bis(trialkoxysilyl)ethylene, bis(trialkoxysilyl)methane, bis(trialkoxysilyl)ethane, bis(trialkoxysilyl)ethyn, bis(trialkoxysilyl)benzene; and, preferably, wherein the alkoxy groups are independently an methoxy-, ethoxy- or propoxygroup; and, preferably wherein the alkyl groups are independently a methyl-, ethyl-, propyl- or butylgroup; to form a B-PMO-shell around said core particles or to form a B-PMO particle if no core particle is synthesised.

[0078] The ratio between the bis(trialkoxysilyl)hydrocarbon and the di- or tetra- sulphide silica precursor in the mixture may be amended to achieve a certain speed of biodegradation. These precursors have the advantage that they do not degrade into toxic compounds, are not too bulky so porosity of the PMO gets lost, and / or do not show luminescence properties, which might interfere with the dyes.

[0079] In some embodiments, the volume ratio of bis(trialkoxysilyl)hydrocarbon over di- or tetra-sulphide silica precursor in the mixture is at least 0.0 to at most 10.0, preferably at least 0.1 to at most 7.5, preferably at least 0.3 to at most 5.0, preferably at least 0.4 to at most 3.0, preferably at least 0.5 to at most 2.5, preferably at least 0.6 to at most 2.0, preferably at least 0.7 to at most 1.5, preferably at least 0.8 to at most 1.3, preferably at least 0.9 to at most 1.1, like about 1.0. In some embodiment, the core particles are etched away after the synthesis of the B-PMO shell around said core particles, preferably with an etching agent such as NajCOs, NaOH, polyvinylpyrrolidone (PVP). This may lead to hollow B-PMO particles.

[0080] In some embodiments, the B-PMO particles are synthesised from at least the following precursors, preferably in the presence of a structure-directing agent: a mixture comprising : o a di- or tetra- sulphide silica precursor, preferably wherein the di- or tetra- sulphide silica precursor is bis(3-trial koxysilylyalkyl ) disulphide and / or bis(3-trial koxysilylyalkyl ) tetrasulphide; o and optionally, a bis(trialkoxysilyl)hydrocarbon, preferably selected from the list comprising bis(trialkoxysilyl)ethylene, bis(trialkoxysilyl)methane, bis(trialkoxysilyl)ethane, bis(trialkoxysilyl)ethyn, bis(trialkoxysilyl)benzene; and, preferably, wherein the alkoxy groups are independently an methoxy-, ethoxy- or propoxygroup; and, preferably wherein the alkyl groups are independently a methyl-, ethyl-, propyl- or butylgroup. Such B-PMO particles may be non-hollow particles.

[0081] A preferred embodiment of the bis(trialkoxysilyl)hydrocarbon is bis(triethoxysilyl)ethylene, and may be represented by formula [A]:

[0082] Bis(triethoxysilyl)ethylene has CAS number 87061-56-1.

[0083] A preferred embodiment of the di- or tetra- sulphide silica precursor is bis(3-triethoxy- silylypropyl)disul phide, and may be represented by formula [B]:

[0084] Bis(3-triethoxysilylypropyl)disulphide has CAS number 56706-10-6.

[0085] A preferred embodiment of the di- or tetra- sulphide silica precursor is bis(3-triethoxy- silylypropyl)tetrasulphide, and may be represented by formula [C]:

[0086] Bis(3-triethoxysilylypropyl)tetrasulphide has CAS number 40372-72-3.

[0087] In some embodiments, the B-PMO particles are synthesised from at least: tetraethyl orthosilicate (TEOS); bis(3-triethoxysilylypropyl) disulphide and / or bis(3-triethoxysilylypropyl) tetrasulphide; and, optionally, bis(triethoxysilyl)ethylene.

[0088] In some embodiments, the synthesis of the B-PMO may be aided by the use of a structure-directing agent, preferably a surfactant, preferably cetyltrimethylammonium bromide. The structure-directing agent is preferably removed from the particles after the synthesis of the B-PMO particles.

[0089] In some embodiments, other reagents may be used in the synthesis of the B-PMO particles such as ammonia, ethanol, distilled water, HCI, and / or NajCOs.

[0090] In some embodiments, the hollow B-PMO particles are prepared in a multi-step synthesis, wherein: in a first step, SiOj cores are prepared, preferably starting from a tetra-alkyl orthosilicate, in the presence of an aqueous base, such as aqueous ammonia; in a second step, a disulphide based organic shell is provided around the SiOj core, preferably by contacting the SiOj cores with the structure-directing agent and the mixture as described herein, preferably in an aqueous solvent; and, in a third step, the structure-directing agent is removed from the shell-core structure obtained in the second step, preferably by contacting the shell-core structures with an acidified alcohol, such as acidified ethanol; in a fourth step, etching away the SiOj cores with an etching agent.

[0091] Unless otherwise mentioned, the wavelengths mentioned herein (for the emission band, emission peak, absorption band and absorption peak) for the dyes are inside the B-PMO particles and wherein the B-PMO particles are suspended in an aqueous environment (preferably at a temperature in the middle of the intended temperature range). The skilled person appreciates that the emission band, emission peak, absorption band and absorption peak of dyes in an organic environment may differ from the ones in an aqueous environment. When selecting the dyes to be included in the B-PMO, a skilled person can make a selection based on reported values in an aqueous environment. One or experiments to confirm the wavelength of the emission band, emission peak, absorption band and absorption peak of the dyes in the B-PMO suspended in an aqueous environment may alternatively or additionally be performed to confirm the selected dyes to be included in an embodiment of the invention.

[0092] In some embodiments, the first dye has an emission peak in the wavelength range 500 to 1200 nm, and wherein the second dye has an absorption peak in the in the wavelength range 500 to 1200 nm.

[0093] In some embodiments, the first and second dyes both have an absorption peak and an emission peak both in the wavelength range 500 to 1200 nm.

[0094] In some embodiments, the first dye has an emission peak which partially overlaps with an absorption peak of the second dye.

[0095] In some embodiments, the absorption band, preferably the absorption peak, of the first dye is lower in wavelength compared to the emission band, preferably the emission peak, of the second dye.

[0096] In some embodiments, the emission band, preferably the emission peak, of the first dye is lower in wavelength compared to the emission band, preferably the emission peak, of the second dye. Preferably, the first and second dyes are different, preferably the first and second dyes have different chemical structures, Preferably, the first and second dyes have different absorption and / or emission spectra. Preferably, the first dye and thee second dye are photoluminescent dyes.

[0097] In some embodiments, the first and second dyes are: water stable, meaning that the absorption and emission spectrum of the dye does not change over time, when dissolved in water; pH stable, meaning that the absorption and emission spectrum of the dye does not change when exposed to different pH values; low-toxic, which may be determined by a cell viability assay; thermal stable, especially over the temperature range from at least 20°C to at most 60°C, preferably from at least 25°C to at most 50°C, meaning that the dye does not decompose in said temperature range; photo- stable, meaning that the absorption and emission spectrum of the dye does not change after irradiation with light; and / or, high quantum yielding.

[0098] In some embodiments, the first dye is selected to have (preferably inside the B-PMO, suspended in an aqueous solvent and at a temperature at the middle of the desired temperature range of the envisaged thermometric composition): an absorption peak between 500 and 750 nm; and, an emission peak of at least 600 nm, preferably in the far red to near infra-red spectrum, preferably between 600 and 800 nm, preferably between 650 and 800 nm. whereas the second dye is selected to have (preferably inside the B-PMO suspended in an aqueous solvent and at a temperature at the middle of the desired temperature range of the envisaged thermometric composition): an absorption spectrum overlapping with the emission spectrum of the first dye; an emission peak at least 20 nm, preferably at least 40 nm, preferably at least 60 nm, preferably at least 80 nm, preferably at least 90 nm removed from the emission peak of the first dye.

[0099] An example of such a first dye is Rh800®, which has in an aqueous environment: an absorption spectrum between 550 and 720 nm, with a maximum at 680 nm; an emission spectrum between 650-850 nm, with a peak at 705 nm; in combination with ICG® as a second dye, which has in an aqueous environment: an absorption spectrum between 600 and 750 nm; an emission peak at 815 nm. This system is preferably exited at a wavelength of 624 nm or

[0100] 660 nm.

[0101] Preferably, the thermometric composition gets excited by irradiation with light with a wavelength equal to the peak of the absorption spectrum of the first dye ± 50 nm, preferably ± 35 nm, preferably ± 25 nm, preferably ± 20 nm, preferably ± 15 nm, preferably ± 10 nm, preferably ± 5 nm, preferably ± 2 nm.

[0102] In some embodiments, the emission peak of the second dye has a wavelength between 500 to 2100 nm. This allows detection of the emitted light by existing technology. Preferably, the emission peak of the second dye has a wavelength between 500 to 900 nm. This allows detection of the emitted light by conventional CCD cameras.

[0103] In some embodiments, the emission peak of the second dye has a wavelength between 600 and 950 nm. This is the "first biological window", and may allow the light to penetrate and spread into tissue. Both emission in the infra-red spectrum or emission in the biological window might be challenging to achieve with lanthanide nanomaterials (= prio art).

[0104] In some embodiments, the emission peak of the second dye has a wavelength between 780 and 1400 nm, which may allow the light to travel through the skin.

[0105] In some embodiments, the emission peak of the second dye has a wavelength between 500 and 2100 nm, preferably between 500 and 1000 nm, preferably between 500 and 900 nm, preferably between 600 and 900 nm, preferably between 780 and 900 nm.

[0106] In some embodiments, both the first dye and the second dye are water-soluble. In some embodiments, the first dye and the second dye are dissolved in a solvent and encapsulated in the B-PMO, whereas the solvent is preferably an alcohol, preferably ethanol.

[0107] In some embodiments, the first and second dyes are not covalently linked together.

[0108] In some embodiments, the ratio between the first dye and the second dye depends on the intensity of their respective emission bands. The ratio between the first dye and the second dye, equals the ratio between the intensities of their respective emission band ± 20%, preferably ± 15%, preferably ± 10%, preferably ± 5% of that ratio. Such ratios have the advantage that the emission of one dye does not make the emission of the other dye undetectable. Hence, preferably both the emissions of the first dye and the second dye are distinguishable from the noise level.

[0109] In some embodiments, the molar ratio between the first dye and the second dye is between 1 / 99 and 99 / 1, preferably between 5 / 95 to 95 / 5, preferably between 10 / 90 and 90 / 10, preferably between 25 / 75 and 75 / 25, preferably around 50 / 50. Such ratios have the advantage that the emission of one dye does not make the emission of the other dye undetectable. Hence, preferably both the emissions of the first dye and the second dye distinguishable from the noise level.

[0110] In some embodiments, the absorption wavelength band peak of the first dye is different from the emission wavelength band peak of the second dye, preferably different in height, more preferably different in wavelength range.

[0111] In some embodiments, the first dye and / or second dyes are / is a fluorescent dye, preferably meaning that the emission of light by the dye ends after excitation of the dye stops.

[0112] Reference to the following dyes is made throughout this text:

[0113] Rh800®, a xanthene dye, CAS 137993-41-0, which may be represented by formula [X];

[0114] Atto610®, a carbopyronin derivative, CAS 303952-45-6, which may be represented by formula [XI];

[0115] IR806®, a cyanine dye, CAS 76783-59-0, which may be represented by formula [XII]; and, ICG®, or Indocyanine green, a cyanine dye, CAS 3599-32-4, which may be represented by formula [XIII],

[0116] [X] [XI]

[0117] [XII] [XIII]

[0118] Other dyes disclosed in here are: Table 1: overview of possible dyes

[0119] In some embodiments, the first dye and the second dye can independently be: a rhodamine derivative, a coumarin derivative, an acridine derivative, a carbopyronin derivative, an oxazine derivative, a cyanine dye, or a xanthene dye.

[0120] In this field, some dyes derived from rhodamine, coumarin, acridine, carbopyronin or oxazine are sold by Atto-Tec, and are therefore referred to as Atto dyes, which are particularly suitable to be used as first dye or second dye.

[0121] In some embodiments, the first dye is a xanthene dye or a derivative of rhodamine, coumarin, acridine, carbopyronin or oxazine, whereas the second dye is a cyanine dye.

[0122] In some embodiments, the first dye is a derivative of rhodamine, coumarin, acridine, carbopyronin or oxazine, whereas the second dye is a cyanine dye. In some embodiments, the first dye is a derivative of carbopyronin, whereas the second dye is a cyanine dye; e.g. Atto610® as first dye and IR806® as second dye.

[0123] In some embodiments, the first dye is a xanthene dye, whereas the second dye is a cyanine dye; e.g.

[0124] Rh800® as first dye and ICG® as second dye.

[0125] In some embodiments, the combination of the first dye and the second dye is selected from the following pairs:

[0126] Table 2: possible combinations of first dyes and second dyes It should be understood that based on the teachings herein, a skilled person is capable of selecting a combination of other dyes, which would be suitable as first dye and second dye as disclosed herein, and that therefore the invention is not limited to only the dyes disclosed herein.

[0127] In some embodiments, the thermometric composition comprises:

[0128] 1.0 mol% Atto610® as first dye and 99.0 mol% IR806® as second dye,

[0129] 1.5 mol% Atto610® as first dye and 98.5 mol% IR806® as second dye,

[0130] 50.0 mol% Rh800® as first dye and 50.0 mol% ICG® as second dye,

[0131] 25.0 mol% Rh800® as first dye and 75.0 mol% ICG® as second dye, or

[0132] 75.0 mol% Rh800® as first dye and 25.0 mol% ICG® as second dye; wherein the mol% are expressed compared to the total number of moles in the dye mixture in the thermometric composition.

[0133] Provided herein is a method of preparing the thermometric composition according to an embodiment disclosed herein, comprising:

[0134] - soaking B-PMO particles unloaded, with a combination of the first dye and the second dye, preferably as a solution, more preferably an aqueous solution.

[0135] In some embodiments, ultrasound is used during the soaking. This may ensure a good dispersion of the B-PMO particles in the solution.

[0136] In some embodiments, the B-PMO particles and the solution of the first dye and the second dye is agitated, preferably stirred, during the soaking.

[0137] In some embodiment, the soaking last from at least 1 hour to at most 72 hours, preferably at least 2 hours to at most 36 hours, preferably at least 6 hours to at most 48 hours, preferably at least 12 hours to at most 24 hours.

[0138] In some embodiments, the loaded B-PMO particles may be harvested from the solution by filtration and preferably centrifugation.

[0139] In some embodiment, the harvested loaded B-PMO particles are washed, preferably with water. In some embodiments, the harvested loaded B-PMO particles or the washed loaded B-PMO particles may be dried. In some embodiment, the method involves applying a lipid bilayer on the surface of the loaded B-PMO particles. This lipid bilayer may prevent leakage of the dyes from the B-PMO particles and may enhance the dispersibility of the particles in an aqueous solution. Preferably the lipid bilayer is a 1,1-dioleoyl- sn-glycero-3-phosphocholine (DOPC) bilayer. Preferably, the loaded B-PMO particles are contacted with a solution of lipids in an aqueous alcohol (preferably ethanol) solution. Ultrasound may be applied during this contacting step. The coated and loaded B-PMO particles may be harvested by filtration and preferably centrifugation, and optionally be dried.

[0140] Provided is a method for obtaining temperature data of a cell, a tissue or a subject comprising: obtaining, using a light sensor, light-emission data of the subject, where the light sensor is disposed external to a cell, a tissue or a subject; determining from the light-emission data, the temperature data of a subject, wherein the cell, the tissue or the subject been provided with the thermometric composition according to an embodiment disclosed herein, and the light sensor is configured for detection of light emissions by thethermometric composition.

[0141] In some embodiments, the light-emission is infra-red light. This may have the advantage that this data can penetrate the skin or tissue material and that temperature data can be obtained from within the tissue or subject.

[0142] In some embodiments, the method involves irradiating the cell, the tissue or the subject with light with a wavelength within the absorption band of the first dye; preferably with light with a wavelength equal to the peak of the absorption spectrum of the first dye ± 50 nm, preferably ± 35 nm, preferably ± 25 nm, preferably ± 20 nm, preferably ± 15 nm, preferably ± 10 nm, preferably ± 5 nm, preferably ± 2 nm. Preferably, the light with a wavelength within the absorption band of the first dye is Infra-red light. This may have the advantage that it can penetrate deep into tissue and skin.

[0143] In some embodiments, the method is performed in vitro, preferably for obtaining temperature data of the cell and tissue.

[0144] In some embodiments, the light sensor is a part of a spectrofluorometer, which may also comprise a light source for irradiating the cell, the tissue, or the subject. In some embodiments, the light sensor comprises an imaging sensor, such as a CCD camera.

[0145] In some embodiments, the the thermochromic composition is provided to the subject by venous injection.

[0146] In some embodiments, the temperature data is of a target below the skin of the subject.

[0147] In some embodiments, the target is a (benign or cancerous) tumour, or temperature abnormalities originating from other health conditions e.g. inflammation states or arthritis. Preferably the target is associated with melanoma cancer, breast cancer, head and / or neck cancer. These cancers can be particularly selected as target as these are not too deep under the shin, and well within the probing range of the thermometric compositions according to an embodiment disclosed herein.

[0148] In some embodiments, determining from the light-emission data, the temperature data of a subject is done via calibration cures, preferably a calibration curve specific for the thermometric composition used. In some embodiments, the calibration curve is a curve of the wavelength of the emission peak of the second dye in function of the temperature.

[0149] The invention will be more readily understood by reference to the following examples, which are included merely for purpose of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

[0150] Methods

[0151] Emission spectra and peaks of dyes are recorded using a spectrofluorometer. Fluorescence emission spectra show the change in fluorescence intensity as a function of the wavelength of the emission light. For the measurement the wavelength of excitation is set to a wavelength of known absorption by the sample (usually the strongest absorption point is selected), and the wavelength of the emission monochromator is scanned across the desired emission range and the intensity of the fluorescence is recorded on the detector as a function of emission wavelength.

[0152] For all measurements inhere, a FLS980 spectrofluorometer from Edinburgh Instruments was used.

[0153] Absorption spectra and peaks of dyes are recorded using a spectrophotometer. Absorption spectra show the change in absorbance of a sample as a function of the wavelength of incident light. Absorption spectra are measured by varying the wavelength of the incident light using a monochromator and recording the intensity of transmitted light on a detector. The intensity of light transmitted through the sample "Isampie" (such as an analyte dissolved in solvent) and the intensity of light through a blank "blank" (solvent only) are recorded, and the absorbance of the sample calculated using: A = IOgl0(lBlank / lsample).

[0154] Excitation of dyes are recorded using a spectrofluorometer. Fluorescence excitation spectra show the change in fluorescence intensity as a function of the wavelength of the excitation light. The wavelength of the emission monochromator is set to a wavelength of known fluorescence emission (usually the strongest emission intensity) by the sample, and the wavelength of the excitation monochromator is scanned across the desired excitation range and the intensity of fluorescence recorded on the detector as a function of excitation wavelength.

[0155] Particle size of the B-PMO in here is determined using Transmission Electron Microscopy

[0156] (TEM). Alternatively Scanning Electron Microscopy (SEM) may also be used.

[0157] Using an imaging processing software, such as ImageJ, or equivalent, the size of multiple particles is measured. Usually at least 50-100 particles are evaluated from different areas of the TEM grid. This is then usually plotted in the form of a histogram to show the average size and variations from the size.

[0158] Wall thickness of the B-PMO is evaluated employing Transmission Electron Microscopy (TEM).

[0159] Multiple images are taken and then using a visualisation software (ImageJ or equivalent) the wall thickness of multiple B-PMO particles (at least 50-100) are measured and averaged. Preparation of a hollow B-PMO particles

[0160] In this example, the hollow B-PMO particles were synthesized in a three-step procedure. First, the SiOj template particles were prepared. For this, 15 mL of aqueous ammonia (25%) was added to 135 mL of ethanol and 2 mL distilled water. After leaving the mixture to stir for 5 minutes, 6 mL tetraethyl orthosilicate (TEOS) was added dropwise and was left to stir overnight at room temperature. In the second step, to obtain disulphide-based B-PMO@SiOz core-shell particles, 100 mL of the as-prepared SiOj suspension was taken and placed in a new flask and further diluted with 200 mL distilled water. 30 mL of 0.12 M cetyltrimethylammonium bromide (CTABr) solution was added to the SiCh suspension, and the flask was left to stir for 30 minutes. Next, 400 pL bis(triethoxysilyl)ethylene precursor and 400 pL bis(3-triethoxysilylypropyl)disulphide precursor mixture was added dropwise and left to stir for 24 h (the flask was left at room temperature). The next day, the obtained powder was retrieved by centrifugation and was washed with water three times. The powder was next dried at 80 °C in an oven. The CTABr was extracted from the PMO pores by placing the powder in an acidified ethanol solution under reflux (1 mL 37% HCI in 100 mL ethanol) and the process was repeated 3 times. The resulting mixture was again centrifuged, washed with water, and left to dry in an oven at 80 °C. Finally, in a third step, to obtain the final material, 0.1 g of the dried B-PMO@SiO2 powder was placed in a flask where 0.636 g NajCOs and 20 mL distilled water was added. This mixture was heated for 1.5 h at 80 °C. The suspension was centrifuged and washed two times with water and dried at 80 °C in a drying oven. The obtained material is referred to as the B-PMO particles.

[0161] Example 2 loading of the hollow B-PMO particles with first and second dye

[0162] 15 mg of the as prepared dried B-PMO particles in Example 1 was weighed off and dispersed in 3 mL distilled water which contained a dissolved dye mixture (ratio of the specific dyes vary, but as standard a total weight amount of the sum of the two dyes is set as 2.5 mg). In case a poorly water soluble dye was used, a mixture of other solvents, e.g. watenethanol might be used for the loading process. The ratio of the dyes was dependent on the used dyes and was connected to their emission intensity.

[0163] An ultrasound bath was used for 5 minutes to ensure appropriate dispersion of the B-PMO in the solvent. The mixture was left to stir in the dark for 24 h at room temperature (flask was covered with aluminum foil to avoid any degradation of the dyes). The stirring was set to low speed (200-300 rpm). The solid particles were then separated by centrifugation, washed gently with water and followed by additional centrifugation. Centrifuge speed was set up at 4500 rpm and time of centrifugation was set as standard 5 minutes (in case of issues with sedimentation time can be prolonged). The product was dried in an oven at 80 °C.

[0164] In a final step a lipid bilayer coating was built on the B-PMO particles loaded with dye to reduce chances of dye leakage as well as enhance dispersibility of the particles with aqueous solution. For this first a solution of l,l-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was prepared (28 mg of DOPC was dissolved in 6 mL distilled water and 4 mL ethanol). For every 5 mg of B-PMO loaded with dye dried powder 500 pL of the DOPC solution was taken and added into a conical centrifuge tube. Ultrasounds were used to disperse the mixture (2-3 minutes). To this 4.5 mL of distilled water was added and quickly shaken vigorously (manually or using vortex). The mixture was centrifugated (4500 rpm, 5 min) and redispersed in water two times for purification purposes (redispersion in water using ultrasounds and sequential centrifugation). The final product was dried in an oven at 80 °C and stored in the dark in powder form. optimizing ratio between the first dye and the second dye

[0165] Selecting the ratios of the dyes is done through experimental optimization. A known molar amount (0.005 mmol) of first dye is encapsulated in 15 mg of the B-PMO (of Example 1), and after a lipid bilayer is built around it for water stability and prevention of dye leakage, the absorption spectrum and excitation and emission spectra are recorded after redispersing in distilled water. The same molar amount of second dye is separately encapsulated in B-PMO, a lipid bilayer is wrapped around the particles, and the absorption spectrum and excitation and emission spectra are recorded.

[0166] Based on this first data, the first and second dye excitation and emission range and brightness are evaluated in the B-PMO environment and in the presence of water quenching. Depending on the performance of the dyes in this environment different ratios of first dye to the second dye are proposed. E.g. if the first dye 1 and second dye both show comparable intensity in B-PMO environment ratios of 25:75, 50:50, 75:25 are investigated, to identify the highest sensitivity (Sr). A total amount of 2.5 mg of dye is aimed for in the synthesis of the mixed dye 15 mg of the B-PMO (of Example 1). If one of the dyes shows a very strong emission intensity compared to other dye, ratios such as 1:99, 5:95, 10:90 are the starting point, to identify the highest sensitivity. Feedback from experiments may lead to a further optimalisation of the dye ratio. layout of absorption bands and emission bands of the first and second dye

[0167] Figure 1 provides a sketch of a possible layout of absorption bands and emission bands of the first and second dye used in a thermometric composition according to an embodiment of the invention. The absorption band (Al) of the first dye is positioned at the lower wavelengths, and reaches an absorption peak (Alp). The emission band (El) of the first dye is shifted towards higher wavelength compared to Al, and reaches an emission peak (Elp). At yet higher wavelengths, one can find the absorption band (A2) of the second dye, reaching an absorption peak (A2p). The absorption band (A2) of the second dye overlaps (O) with the emission band (El), so that emission of the first dye can excite the second dye. Eventually, the emission band (E2) of the second dye is positioned at the higher end of the wavelengths and reached an emission peak (E2p). Preferably the thermometric composition comprising this first and second dye should be irradiated with light with a wavelength of Alp and monitoring the response should preferably be done around E2p.

[0168] 5 Temperature dependent fluorescence.

[0169] Figure 2 depicts various temperature dependent fluorescence of the first and second dye. In Figure 2A, the emission bands are depicted of the first dye (11) and the second dye (12). As the temperature increases the intensity of the fluorescence of the first dye decreases, whereas the fluorescence of the second dye also decreases.

[0170] In Figure 2B, the emission bands are depicted of the first dye (11) and the second dye (12). As the temperature increases the intensity of the fluorescence of the first dye decreases, whereas the fluorescence of the second dye remains the same.

[0171] In Figure 2C, the emission bands are depicted of the first dye (11) and the second dye (12). As the temperature increases the intensity of the fluorescence of the first dye decreases, whereas the fluorescence of the second dye increases.

[0172] Comparative Examples and Working Examples

[0173] As shown in Table 3, different thermometric compositions form the prior art (comparative examples) and according to embodiments of the invention (working examples) were tested. In the working examples, B-PMO as prepared in Example 1 is used, and the dyes as indicated in Table 3 are loading in line with Example 2. For all the examples in Table 3, the fluorescence of the thermometric compositions was investigated over a temperature range as indicated in the table. The emission range was determined in function of the temperature within the indicated temperature range.

[0174] For each thermometric composition, the relative sensitivity (Sr) was determined.

[0175] The relative sensitivity Srindicates the relative change of the thermometric parameters per degree of temperature change and is expressed in %°C-1. It is calculated using the equation below:

[0176] Where A= — and h and l2are the maximum intensities of the peaks (or integrated areas under the . peaks) at the selected wavelengths.

[0177] For each thermometric composition, the temperature uncertainty or resolution (6T) was determined, based on the signal to noise ratio.

[0178] Temperature uncertainty is calculated using the equation below:

[0179] 1 <5A 8T = — — SrA

[0180]

Claims

Claims1. A thermometric composition, comprising a plurality of biodegradable periodic mesoporous organosilica, B-PMO, particles, wherein:- each B-PMO particle is loaded with a first dye and a second dye,- the first dye and second dye are both fluorescent,- a fluorescent light emission by the first dye causes a luminescent excitation of the second dye,- the first dye and / or the second dye exhibit(s) a temperature-dependence fluorescence.

2. The thermometric composition according to claim 1, wherein the first dye and second dye are both organic.

3. The thermometric composition according to claim 1 or claim 2, wherein each B-PMO particle comprises multiple polymeric chains of alkoxysilane of the formula I- (RO)3Si-R' -, (formula I) wherein R is CH3-, or CH2CH3-; and, wherein R' is a substrate for an enzyme.

4. The thermometric composition according to claim 3, wherein, R' is [-(CH2)x-(S-S)n-(CH2)x-]; wherein x is equal to or greater than 1; and, wherein n equal to or greater than 1.

5. The thermometric composition according to any one of previous claims, wherein the first dye has an emission peak in the wavelength range 500 to 1200 nm, and wherein the second dye has an absorption peak in the in the wavelength range 500 to 1200 nm.

6. The thermometric composition according to any one of previous claims, wherein the absorption band, preferably the absorption peak, of the first dye is lower in wavelength compared to the emission band, preferably the emission peak, of the second dye.

7. The thermometric composition according to any one of previous claims, wherein the first dye is selected to have:an absorption peak between 500 and 750 nm; and, an emission peak of at least 600 nm: and, wherein the second dye is selected to have: an absorption spectrum overlapping with the emission spectrum of the first dye; an emission peak at least 20 nm removed from the emission peak of the first dye.

8. The thermometric composition according to any one of previous claims, wherein the emission peak of the second dye has a wavelength between 600 and 950 nm.

9. The thermometric composition according to any one of previous claims, wherein the first dye and the second dye are independently selected from the list comprising: a rhodamine derivative, a coumarin derivative, an acridine derivative, a carbopyronin derivative, an oxazine derivative, a cyanine dye, or a xanthene dye.

10. The thermometric composition according to any one of previous claims, wherein the ratio between the first dye and the second dye, equals the ratio between the intensities of their respective emission band ± 20%, preferably ± 15%, preferably ± 10%, preferably ± 5% of that ratio.

11. A method of preparing the thermometric composition according to any one of previous claims comprising the step of:-soaking B-PMO particles unloaded, with a combination of the first dye and the second dye.

12. The method according to claim 11, wherein the method involves applying a lipid bilayer on the surface of the loaded B-PMO particles.

13. A method for obtaining temperature data of a cell, a tissue or a subject comprising: obtaining, using a light sensor, light-emission data of the subject, where the light sensor is disposed external to a cell, a tissue or a subject;determining from the light-emission data, the temperature data of a subject, wherein the cell, the tissue or the subject been provided with the thermometric composition of any one of the previous claims, and the light sensor is configured for detection of light emissions by thethermometric composition.

14. The method according to claim 13, wherein the method involves irradiating the cell, the tissue or the subject with light with a wavelength within the absorption band of the first dye.

15. The method according to claim 13 or claim 14, wherein the method is performed in vitro.

Images