Delivery and measurement of fluorescent nanocrystals in biological tissues

Abstract

A microneedle assembly (10) for delivering fluorescent nanocrystals (FNC) into tissue (T) comprises one or more microneedles (11). Each microneedle (11) comprises a plurality of fluorescent nanocrystals (FNC) dispersed throughout a solid matrix material (11m). The tissue (T) may be penetrated by one or more microneedles (11), and at least part of the matrix material (11m) may be dissolved inside the tissue for delivery of the fluorescent nanocrystals (FNC) into the tissue (T). Various aspects of the tissue (T) can be measured by delivering light pulses to the tissue (T) and measuring the resulting fluorescence signal (Sf) from the fluorescent nanocrystals (FNC) inside the tissue (T). For example, a microneedle assembly (10) may be manufactured using a micro-forming technique using a liquid precursor of a matrix material (11m) containing a concentration of fluorescent nanocrystals (FNC) therein.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to methods, systems, and devices for delivering fluorescent nanocrystals (FNCs) to tissue and / or for making measurements in tissue with FNCs. The present disclosure further relates to a microneedle assembly and a method for manufacturing such an assembly. BACKGROUND

[0002] In biology, free radicals such as reactive oxygen species play a vital role. Free radicals are chemically reactive molecules that contain unpaired electrons in their outer shell, which makes them highly reactive and potentially damaging to biological molecules. While high levels of free radicals can be detrimental and lead to oxidative stress, an appropriate amount of free radicals has important physiological functions in a variety of biological processes. For example, psychological stress can trigger the production of free radicals, leading to oxidative stress. Chronic oxidative stress caused by increased free radicals can damage cells and lead to diseases. Detecting free radicals can be difficult due to their high reactivity and short lifetime, for example.

[0003] Sigaeva et al. recently published a paper [Small, 18(44), p.2105750; DOI: 10.1002 / smll.202105750] disclosing a nanoscale quantum relaxation measurement based on diamond for detecting the production of free radicals in cells. The contents of this paper are incorporated herein by reference. As explained in the paper, diamond magnetometry utilizes fluorescent defects in nanodiamonds to convert magnetic resonance signals into fluorescence signals. According to an overview of the experimental procedure, nanodiamonds containing NV centers are positioned in or near living cells and visualized by confocal microscopy. The NV centers are then pumped to a bright state using a sequence of laser pulses. The emission of the nanodiamond particles is then recorded within a fixed time window at the start of the next pulse after different dark times. The collected fluorescence intensity reveals whether the NV center is still in this prepared state or has relaxed to a darker equilibrium state. At lower levels of magnetic noise in the environment, the relaxation occurs more slowly. At higher levels of magnetic noise, the relaxation occurs more quickly and the fluorescence intensity recorded after a given dark time is lower. Optionally, a microwave pulse can be added before the light pulses to specifically target the spin resonance of the NV center. Subtracting the T1 of the microwave pulse from the full light T1 allows for the exclusion of competing effects that are not related to the NV center spin.

[0004] Fluorescent nanocrystals (FNCs, such as nanodiamonds with NV centers) have a limited range of detection of their environment, for example, the sensitivity is typically inversely proportional to the sixth power of the distance (about 1 / r 6The uptake of FNCs decreases. Therefore, for effective detection, it is desirable to place the FNC near the region of interest, for example, within 100 nm, preferably within 10 nm. However, suitable FNC sizes can be relatively large, for example ≥10 nm or even ≥100 nm, up to 200 nm, or larger. Relatively large sizes can make it difficult to deliver suitable FNCs to the region of interest in biological tissues. Theoretically, FNC uptake into a single cell can occur spontaneously or be promoted through cellular modification. However, uptake of FNCs into deeper regions of larger tissue samples (such as tissue sections) or even in vivo remains challenging.

[0005] Therefore, new methods, devices, and systems are still needed to improve the delivery of fluorescent nanocrystals, such as (relatively large) nanodiamonds with NV centers, into biological tissues. Summary of the Invention

[0006] This disclosure provides methods, apparatus, and systems for delivering fluorescent nanocrystals, such as nanodiamonds, to tissues. The microneedle assembly may be formed from a substrate having an array of microneedles for penetrating tissue. Each microneedle is formed from a solid matrix material in which multiple fluorescent nanocrystals are dispersed. For example, the substrate may include a patch configured to apply the microneedle assembly to a tissue surface while simultaneously penetrating the tissue with the array of microneedles. By penetrating the tissue with one or more microneedles and dissolving at least a portion of the matrix material forming the one or more microneedles, fluorescent nanocrystals dispersed in a portion of the dissolved matrix material can be efficiently delivered into the tissue.

[0007] By forming a solid matrix material that is biodegradable and / or biocompatible, delivery can be advantageously applied to tissues containing biological cells. For example, the biodegradable solid matrix material is configured to degrade within the tissue over a certain period of time. It is understood that one or more microneedles should be strong enough to penetrate a certain distance within the tissue for the delivery of fluorescent nanocrystals. For example, when using skin tissue, fluorescent nanocrystals can be delivered into the epidermis, typically less than one millimeter deep within the skin. Other types of biological tissues can also be used, allowing the delivery of fluorescent nanocrystals to any relevant depth. Further advantages can be obtained by conjugating the fluorescent nanocrystals to corresponding ligands. For example, when the matrix material dissolves, the ligands can remain in the tissue for targeting specific biological structures within the tissue after delivery. Other compounds or further compounds can also be used, for example, as part of the microneedles or used in other ways. For example, compounds such as collagenase can be used to loosen the tissue structure by increasing intercellular space, thereby facilitating the uptake of fluorescent nanocrystals into the tissue.

[0008] This disclosure further provides methods, apparatus, and systems for measuring tissue after delivery of fluorescent nanocrystals into tissue, as described herein. Typically, this involves measuring the fluorescence signal from the fluorescent nanocrystals in the tissue. In the measuring apparatus, a tissue fixator is configured to fix a slice of tissue. Preferably, the tissue is immersed in a liquid medium and / or placed in a controlled environment and temperature to maintain the cellular integrity of the tissue during measurement. The measuring apparatus (e.g., a microscope) may include or be used in conjunction with a light source configured to deliver pulses of source light to the tissue. A photodetector can be used to measure the fluorescence signal emitted by the fluorescent nanocrystals due to the pulses of source light. Based on the measured fluorescence signal, biological parameters of the tissue can be determined, for example, using an analyzer. When measuring larger tissue samples, a wavelength filter is preferably configured to filter out autofluorescence from the tissue slice at wavelengths below 650 nm or 700 nm, preventing autofluorescence from reaching the photodetector. Although this may reduce the overall signal intensity, the inventors have found that the filter improves the signal-to-noise ratio.

[0009] It is understood that the fluorescence signal of a corresponding fluorescent nanocrystal can depend on the local environment of the tissue adjacent to the nanocrystal. For example, local electric and / or magnetic fields can affect the fluorescence state of the color centers within the fluorescent nanocrystal. By measuring the fluorescence signal while applying variable stimuli to the tissue, further understanding of the effects of such stimuli on the tissue can be obtained. For example, the effects of UV irradiation, drugs, antioxidants, nutrients, and disease development on various tissue types can be studied in greater detail. In particular, the fluorescence signal of the fluorescent nanocrystal can be used as a local probe to determine how these stimuli can affect the presence and / or concentration of free radicals in the tissue. Furthermore, other effects, such as pH, pressure, temperature, etc., can also be measured.

[0010] This disclosure further provides methods, apparatus, and systems for manufacturing microneedle assemblies, such as those described herein for delivering fluorescent nanocrystals into tissues and / or measuring fluorescent nanocrystals in tissues. According to one method, fluorescent nanocrystals of a certain concentration are dispersed in a liquid precursor of a matrix material. The liquid precursor containing the fluorescent nanocrystals is cast into a mold, which forms a negative mold of an array of microneedles. The liquid precursor is solidified in the mold to form an array of microneedles comprising fluorescent nanocrystals dispersed in a solid matrix material. The array of microneedles can be removed from the mold to form a portion of a microneedle assembly. Attached Figure Description

[0011] These and other features, aspects, and advantages of the apparatus, system, and method of this disclosure will be better understood from the following description, appended claims, and drawings:

[0012] Figures 1A-1B This demonstrates the delivery of fluorescent nanocrystals into tissues;

[0013] Figures 2A-2C An image of the microneedle assembly is shown;

[0014] Figures 3A-3F The fabrication of the microneedle assembly and the insertion of the fabricated microneedles into tissue are illustrated.

[0015] Figure 4 This demonstrates the measurement of tissues using fluorescent nanocrystals;

[0016] Figures 5A-5C Images comparing the fluorescence signal of FNC with the background signal of tissue using different wavelength filters are shown;

[0017] Figure 6 and 7 The effects of collagenase on FNC uptake in various tissue and cell types were demonstrated. Detailed Implementation

[0018] The terminology used to describe particular embodiments is not intended to limit the invention. Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” used herein are intended to equally include the plural forms. The term “and / or” includes any and all combinations of one or more of the listed related items. It should be understood that the terms “comprising” and / or “including” specify the presence of the stated feature but do not exclude the presence or addition of one or more other features. It should be further understood that when a particular step in a method is described as following another step, unless otherwise specified, that step may be performed directly after said other step or may be performed before performing that particular step, with one or more intermediate steps. Similarly, unless otherwise specified, it should be understood that when describing a connection between structures or components, this connection may be established directly or through intermediate structures or components.

[0019] Fluorescence is the process by which a substance absorbs light or other electromagnetic radiation and re-emits it (usually at a longer emission wavelength). In some nanocrystals, such as color centers (like nitrogen vacancies (NVs) in diamond), fluorescence can occur due to specific defect structures within the crystal lattice. For example, an NV center consists of a nitrogen atom and a vacancy adjacent to each other, creating an electronic environment capable of absorbing and emitting photons. These, and other color centers formed in nanocrystals, can contribute to various properties, such as exhibiting stable and bright fluorescence. In other or further nanocrystals, such as nanoscale semiconductor particles, fluorescence can occur due to quantum confinement effects that alter the electronic and optical properties of the material. When these nanocrystals absorb photons, their electrons are excited to higher energy levels. These electrons then return to their ground state, resulting in the emission of light, the wavelength of which is tunable based on the size and composition of the nanocrystal.

[0020] The invention will now be described in more detail with reference to the accompanying drawings, which illustrate embodiments of the invention. For clarity, the absolute and relative dimensions of systems, components, layers, and regions in the drawings may be enlarged. Embodiments may be described with reference to schematic and / or cross-sectional diagrams of possibly idealized embodiments and intermediate structures of the invention. In the specification and drawings, the same numbers always refer to the same elements. Relative terms and their derivatives should be interpreted as referring to the orientation shown or described in the drawings under discussion. These relative terms are for convenience of description and, unless otherwise stated, do not require the system to be constructed or operated in a particular orientation.

[0021] Figures 1A-1B The delivery of fluorescent nanocrystals (FNCs) into tissue “T” is illustrated. In some embodiments, the FNCs are delivered using one or more microneedles 11. In one embodiment, each microneedle 11 contains multiple FNCs. In another or further embodiment, the FNCs are dispersed throughout a matrix material 11m. The matrix material is understood as a substance or medium in which another material or phase is embedded or dispersed. It acts as a carrier or support for the dispersed phase. The matrix material surrounds and contains the dispersed phase. As described herein, the matrix material can hold the FNCs dispersed therein together. The matrix material provides mechanical support and stability and generally defines the overall properties of the composite material.

[0022] Some implementations involve penetrating the tissue with one or more microneedles 11. Other or further implementations include, for example, dissolving or otherwise disintegrating the matrix material 11m as one or more microneedles 11 penetrate the tissue "T". In this way, the FNC can remain in a deeper layer of the tissue "T".

[0023] In some embodiments, the tissue "T" comprises biological cells C. For example, tissue "T" is human or animal skin tissue. Other biological tissues may also be used. In one embodiment, the microneedle 11 is configured to penetrate the epidermis of the skin tissue for delivering FNC into the epidermis. For example, the microneedle 11 is configured to penetrate the epidermis by at least 0.1 mm, preferably at least 0.2 mm, more preferably at least 0.5 mm, for example, to a depth of 1 mm or more. Therefore, it is understood that the matrix material 11m (in which FNC is dispersed) is suitable for forming microneedles capable of penetrating into (biological) tissues (such as skin tissue, organ tissue, etc.). For example, the microneedles have sufficient mechanical strength and rigidity to withstand the forces required for insertion into the tissue without bending or breaking. Thus, the microneedles can maintain their structural integrity during penetration. Preferably, the material exhibits a degree of flexibility and elasticity to accommodate bending and deformation that may occur during penetration. This property helps reduce the risk of microneedle breakage and / or enhances the ability to conform to irregular tissue surfaces.

[0024] Preferably, the matrix material 11m is capable of spontaneous dissolution or other dissociation. Most preferably, such spontaneous dissolution or dissociation occurs when the microneedle 11 comes into contact with the tissue. In one embodiment, the matrix material 11m comprises or is substantially composed of a biodegradable material. Biodegradable materials typically have the ability to undergo degradation through biological processes such as enzymatic or microbial action. These materials typically consist of organic compounds that can be recognized and metabolized by biological systems. Their molecular structure may contain easily broken chemical bonds or functional groups that facilitate enzymatic or microbial attack and subsequent degradation. Alternatively or additionally, biodegradable materials may dissolve or otherwise dissociate upon contact with a solvent such as water.

[0025] Preferably, the biodegradable material is also biocompatible, for example, producing non-toxic degradation products that can be absorbed into natural biological processes. Examples of suitable biodegradable and / or biocompatible materials for microneedle fabrication include polymers, such as those comprising or formed from compounds, including hyaluronic acid, lactic acid, glycolic acid, lactoglycolic acid copolymers, caprolactone, polyvinyl alcohol, gelatin, etc. Alternatively or additionally, suitable matrix materials may include sugar-based materials such as dextran, sucrose, trehalose; and / or protein-based materials such as collagen, silk fibroin. Other materials or compounds may also be used.

[0026] In some embodiments, the biodegradable material is configured to degrade (at least partially) within the tissue after insertion, for example, in less than ten hours, preferably less than one hour, more preferably less than half an hour, such as within a time period of ten minutes or less. On the one hand, the matrix material 11m is preferably capable of degrading within the tissue "T" within a reasonable time period to continue the experiment. On the other hand, the microneedles are preferably stable enough that they are not easily degraded before insertion. For example, the degradation rate can be controlled by adjusting factors such as polymer composition, molecular weight, and processing method. Alternatively or additionally, the microneedles can be stored in a controlled environment to prevent them from degrading before insertion. In principle, complete dissociation of the microneedles 11 is not required. For example, sufficient FNC can be released while some microneedles 11 dissociate; and the remaining microneedles 11 can be withdrawn from the tissue "T" before complete dissociation. Preferably, at least 10%, more preferably at least 50%, up to 90% or even 100% of the matrix material 11m forming the tissue-penetrating microneedles 11 dissolves within the said time period. Therefore, it should be understood that the microneedles 11 can dissolve partially or completely within the tissue "T".

[0027] In contrast to matrix material 11m, FNCs can be relatively stable, for example, not biodegradable or less biodegradable than matrix material 11m. This allows FNCs to be retained in tissue “T” for measurement after matrix material 11m has degraded. While FNCs may not be biodegradable, they are preferably nontoxic, for example, having no or minimal damaging effect on the delivered tissue “T”. For example, fluorescent nanodiamonds can be relatively inert and / or biocompatible (nontoxic) in several different types of cells. The biocompatibility of matrix material 11m and / or FNCs (such as nanodiamonds) allows this teaching to be advantageously used for measurements in tissues containing living cells (e.g., in vitro and / or in vivo). Furthermore, when the microneedles are relatively short, the delivery of FNCs can be restricted to, for example, the outer layer of frequently renewing skin (epidermis), causing the FNCs to detach even if they do not degrade.

[0028] In some embodiments, the FNCs forming portions of the microneedles 11 are linked to corresponding ligands (such as antibodies, aptamers, charged groups, lipids, and / or molecules recognized by receptors), wherein the FNCs linked to the corresponding ligands remain in the tissue “T” when the matrix material 11m dissolves, wherein the corresponding ligands are configured to bind to specific biological structures after delivery to the tissue “T”. Advantageously, this can facilitate further targeting of specific biological structures after the FNCs are delivered to the tissue.

[0029] In some embodiments, microneedles 11 are part of a microneedle assembly 10, wherein a plurality of microneedles 11 are arranged on a substrate 12. In one embodiment, the microneedle assembly 10 is configured to deliver FNC through a region "A" of tissue "T". Preferably, the microneedle assembly 10 contains at least four microneedles, more preferably at least ten, twenty, fifty, or one hundred, for example, up to one thousand or more microneedles. In another or further embodiment, the microneedles occupy an area of ​​at least one square millimeter, for example, up to one square centimeter or more. In another or further embodiment, the density of the microneedles is at least ten microneedles per square centimeter, preferably at least one hundred microneedles per square centimeter, for example, up to one thousand or more microneedles per square centimeter. For example, the microneedle assembly may form a 10×10 array of 100 microneedles in an area of ​​1 cm × 1 cm.

[0030] Figures 2A-2CImages are shown, for example, of a microneedle assembly 10 for delivering fluorescent nanocrystals (FNCs) as described herein. In one embodiment, each microneedle 11 has a length “L” of less than 1 mm (lateral or perpendicular to the substrate 12). For example, the microneedle 11 shown has a length L of 700 μm. Longer or shorter needles, for example, with a length of 0.1 to 10 mm, preferably 0.3 to 5 mm, and most preferably 0.5 to 1.5 mm, may also be used. The preferred length may depend on the tissue “T” to be penetrated. For example, when the needle is used to deliver fluorescent nanocrystals (FNCs) to the epidermis, the needle may have a length similar to or shorter than the thickness of the epidermis. For example, the needle may have a length shorter than 1.5 mm, shorter than 1 mm, or even shorter than 0.5 mm.

[0031] Preferably, the microneedles 11 are relatively narrow and / or sharp, for example, having at most half, preferably at most one-third, of the base width "B" of the corresponding needle length "L". For example, the microneedles 11 shown have a base width "B" of 200 μm. Wider or narrower needles can also be used. For example, narrower needles can be sharper but less strong; and vice versa. Preferably, adjacent microneedles 11 in the microneedle assembly 10 are relatively close, for example, separated by a distance "D" smaller than the corresponding length L of the microneedle 11, and / or having a distance "D" less than three times the base width "B". For example, in the image shown, the microneedles 11 are separated by a distance of 500 μm. Smaller or larger distances can also be used. The smaller the distance, the higher the density of needles that can be delivered per unit area and the higher the FNC concentration.

[0032] Preferably, the FNC is relatively small compared to the dimensions (L and / or B) of the microneedle 11, for example, at most one-hundredth, more preferably at most one-thousandth, of the needle length “L” and / or base width “B”. Typically, the FNC has a (maximum) diameter of 1–1000 nm, preferably 10–500 nm, and most preferably 100–250 nm. The inventors have found that using a relatively large (e.g., >100 nm) FNC can help eliminate problems arising from autofluorescence in tissue measurements. On the other hand, the FNC is still relatively small compared to the biological cells that form the tissue, for example, one-hundredth of the cell size. For example, the average diameter of human skin cells is about 30 μm, and the FNC is preferably <250 μm. Other types of tissue, and suitable FNCs and corresponding cells with possible other sizes, can also be used.

[0033] In a preferred embodiment, the FNC comprises fluorescent nanodiamonds (FND) or is substantially composed of FND. In principle, FNDs can be prepared using a variety of processes. Examples may include detonation nanodiamonds (DND) and nanodiamonds derived from a high-pressure, high-temperature (HPHT) source. Typically, DNDs are relatively small, for example, 5 nm, which may not be suitable for this application compared to larger nanodiamonds (e.g., nanodiamonds produced via HPHT). More preferably, each nanodiamond contains at least one or more NV centers. Generally, NV centers are defects in the lattice of a diamond crystal formed from carbon atoms, created when a nitrogen atom replaces a carbon atom in the diamond lattice while a vacancy exists in the lattice. Therefore, the amount of NV centers in each nanodiamond can be controlled by the nitrogen concentration. NV centers possess a unique electronic structure that endows them with special properties. They have a spin triplet ground state and a spin singlet excited state, which can be photoexcited between the two states. This allows them to be used as sensors for magnetic fields and other physical quantities such as pressure, temperature, and stress.

[0034] In addition to NV centers, or alternatively, other types of color centers can be used in nanodiamonds, such as silicon vacancy centers, aluminum vacancy centers, and double vacancy centers in diamond. In addition to nanodiamonds formed from crystalline carbon atoms, or alternatively, other crystalline materials can be used to form nanocrystals with corresponding (fluorescent) color centers. Examples may include nitrogen vacancy centers in silicon carbide, silicon vacancy centers in silicon carbide, carbon anti-centers in silicon carbide, aluminum vacancy centers in aluminum nitride, carbon anti-centers in aluminum nitride, carbon vacancy centers in aluminum nitride, and iron vacancy centers in aluminum nitride.

[0035] Figures 3A-3F The fabrication of the microneedle assembly 10 and the insertion of the fabricated microneedles 11 into a tissue “T” are illustrated. In some embodiments, the microneedles 11 are fabricated using microforming techniques. For example, a master mold with the desired microneedle geometry is first created using techniques such as photolithography or laser ablation. The mold is then filled with a solution or gel of a matrix material precursor that allows curing. After curing, the microneedle assembly is demolded to obtain individual hyaluronic acid microneedles.

[0036] like Figures 3A-3B As shown, one embodiment includes step "S", dispersing a certain concentration of FNC into the liquid precursor 11p of the matrix material 11m. For example, as... Figure 3C As shown, another or further embodiment includes step "C," casting the liquid precursor 11p having the FNC into the mold 20. For example, the mold 20 forms the negative mold of the microneedle assembly 10. In principle, other mold shapes can also be used to manufacture any desired shape of the matrix material 11m containing the FNC. For example, as... Figure 3DAs shown, another or further embodiment includes step "H," which involves hardening and / or solidifying the liquid precursor 11p containing FNCs in the mold 20 to form a microneedle assembly 10 comprising FNCs dispersed throughout the hardened and / or solidified matrix material 11m. For example, step "H" may include evaporating the solvent in the liquid precursor 11p and / or polymerizing the monomers to form the matrix material 11m. Furthermore, other curing steps and processes are conceivable. For example, as... Figure 3E As shown, another or further embodiment includes step "R", which involves removing the hardened matrix material 11m with FNC from the mold 20 to form the microneedle assembly 10.

[0037] In some embodiments, the liquid precursor of the matrix material 11m is formed from a solution containing the matrix material 11m dissolved in a suitable solvent, and a certain concentration of FND may be added to the solution. In one exemplary embodiment, an FND-loaded microneedle (MN) array (100MN / 1×1cm) is fabricated by solvent casting of a 10 μg / ml sonicated FND solution in deionized water, 3% hyaluronic acid, and a 10 w / v% polyvinylpyrrolidone (PVP) solution, followed by centrifugation and a degassing step in a vacuum chamber at 25°C to fill the cavity of the anion (PDMS) mold. 2 The MN patch loaded with FND was dried at 25°C for 48 hours, carefully peeled from the PDMS mold, and stored in a desiccator to maintain dryness. In this example, the matrix material 11m and / or its precursor contains hyaluronic acid. Hyaluronic acid is a glycosaminoglycan belonging to the long-chain, linear polysaccharide family, composed of repeating disaccharide units. The repeating units of hyaluronic acid may consist of D-glucuronic acid and N-acetyl-D-glucosamine. For example, this can form the cured matrix material 11m.

[0038] Some aspects of this disclosure can be embodied in a microneedle assembly 10, such as a microneedle assembly 10 manufactured according to the methods described herein or otherwise. In one embodiment, the microneedle assembly 10 includes a substrate 12 having a (one-dimensional or two-dimensional) array of microneedles 11 for penetrating tissue. In another or further embodiment, each microneedle 11 is formed of a matrix material 11m, in which a plurality of FNCs are dispersed. Preferably, the matrix material 11m is biodegradable and / or biocompatible. In some embodiments, for example, as shown in the figure below. Figure 3E As shown, substrate 12 includes patch 12p. In other or further embodiments, such as Figure 3FAs shown, patch 12p is configured to apply microneedle assembly 10 to a tissue surface while penetrating tissue “T” with an array of microneedles 11. For example, the patch may include an adhesive surface to adhere microneedle assembly 10 to the tissue surface while allowing the matrix material 11m forming the microneedles 11 to at least partially dissolve, thereby delivering FNC into tissue “T”.

[0039] Figure 4 The method of measuring tissue “T” using FNC is illustrated. In some embodiments, the method includes delivering FNC into tissue “T” using a microneedle assembly as described herein or otherwise. In one embodiment, a fluorescence signal “Sf” from the FNC in the tissue is measured. For example, the measured fluorescence signal “Sf” is generated by pulsed excitation of the FNC in tissue “T” by a source light “Ss”. In addition to applying the source light “Ss” or alternatively, other or further signals may be applied to the tissue. In one embodiment, tissue “T” is exposed to a microwave signal MW. For example, the microwave signal MW may affect the fluorescence state of tissue “T”. In another or further embodiment, a magnetic field (not shown here) is applied to tissue “T”.

[0040] In some embodiments, the measured fluorescence signal “Sf” of the corresponding FNC depends on or represents the local environment of the tissue “T” adjacent to the corresponding FNC. In one embodiment, the measured fluorescence signal “Sf” is used to determine the presence and / or concentration of free radicals in the tissue “T”. In another or further embodiment, the presence and / or concentration of free radicals in the tissue “T” and / or the fluorescence signal “Sf” are measured as a function of a stimulus applied to the tissue. For example, the stimulus applied to measure free radicals or other factors affecting the FNC may include one or more of the following: applied UV irradiation, applied drugs or antioxidants, applied nutrients, or certain disease development. For example, these or other stimuli may affect the oxidative state of the measurable tissue. In one embodiment, the fluorescence signal “Sf” is measured with and without stimulation and / or with different levels of stimulation (e.g., different amounts of UV irradiation). In other or further embodiments, the measured fluorescence signal is used to determine one or more of pH, pressure, temperature, and magnetic field in the tissue “T”.

[0041] As described herein, the inventors discovered significant differences in measuring FNC in cells compared to measurements in larger tissues. First, tissues are thicker and therefore may require deeper measurements within the material. Second, tissues are typically dependent on blood supply and have less direct contact with the culture medium, making them more fragile. Third, tissues exhibit stronger autofluorescence than cells. Furthermore, cells adhere to the bottom of the dish and thus remain stationary during measurements, while tissues do not and therefore require fixation. To eliminate the problems arising from autofluorescence, the inventors preferred to use larger nanodiamonds (e.g., 120 nm). Additionally, the inventors collected light at different wavelengths. While light can typically be collected above 600 nm in cells, the inventors found that collecting at higher wavelengths, such as above 650 nm or even 700 nm, is more advantageous in tissues. This, while potentially reducing the signal, further reduces the amount of background. Finally, while measurements at room temperature and ambient air may be sufficient for most cells, the inventors found that for tissue measurements, experiments are preferably conducted in a more controlled environment, such as at +37°C, and most preferably in the presence of 5% CO2.

[0042] Some aspects of this disclosure can be embodied in a measuring device 50 configured to measure the FNC delivered to a tissue “T”. In one embodiment, the measuring device includes or is otherwise configured to use a tissue fixator (not shown) configured to fix a slice of tissue “T”. Preferably, the tissue fixator is configured to hold the tissue in place and allow culture medium (e.g., containing nutrients) to approach the tissue. For example, to avoid stress on the tissue, the inventors have developed a custom-made stainless steel fixator that is reusable, easy to handle, and clean. Multiple metal wire meshes are present within the fixator to restrict tissue movement. To avoid excessive pressure, the height between the wire meshes and the dish can be adjusted according to the tissue thickness. Optionally, the fixator may also have one or more holes in its wall to allow culture medium to flow in and / or out of the fixator.

[0043] In some embodiments, the measuring device 50 includes or is otherwise configured to use a light source (not shown) configured to provide pulses of source light "Ss" to the tissue "T". In another or further embodiment, the measuring device 50 includes or is otherwise configured to use a photodetector (not shown) configured to measure a fluorescence signal "Sf" emitted by the FNC due to pulses of source light Sf. The device may also include other sources or detectors, such as sources and / or detectors for generating and / or measuring microwaves MW. Sources for generating constant and / or variable magnetic fields (e.g., gradient fields) may also be included as part of the device or otherwise coupled to the tissue T.

[0044] In some embodiments, the measuring device 50 includes an analyzer or is otherwise configured to use an analyzer to determine biological parameters of tissue "T" based on measured fluorescence signals "Sf". Preferably, the measuring device includes a wavelength filter (not shown) configured to filter out fluorescence with wavelengths below 650 nm, preferably below 700 nm. For example, Figures 5A-5C Measurements taken with different wavelength filters are shown, where it can be noted that the FNC signal is improved relative to tissue background at wavelength filters above 650 nm compared to wavelength filters above 600 nm, and further improved at wavelength filters above 700 nm.

[0045] In some embodiments, methods for delivering FNC include applying a compound, such as collagenase, configured to loosen the structure of the tissue “T”. This may facilitate the uptake of FNC into the tissue “T”. For example, the compound may break down portions of the cell wall, extracellular matrix, and / or intercellular junctions. In one embodiment, the compound forms part of the microneedle 11, for example, dispersed in matrix material 11m, or otherwise forms part of it. For example, the compound may be released along with the FNC when the matrix material 11m dissolves in the tissue “T”. In another or further embodiment, the compound may be applied before, after, or during the insertion of the microneedle 11. Applying the compound may also be envisioned as an alternative to delivering FNC into the tissue. For example, FNC may be applied to the surface of tissue treated with the compound, or the compound may be mixed with FNC and applied to the tissue. Using microneedles or otherwise treating tissue with the compound may be most suitable for in vitro tissue measurements, such as tissue sections.

[0046] Figure 6 Multiple measurements of fluorescent nanodiamond (FND) uptake in various tissue types, including kidney, liver, skin, and spleen sections, are shown. A comparison of uptake distribution with and without collagenase is presented. It can be understood that the use of collagenase improves uptake into deeper tissue regions. Figure 7 The figure further illustrates measurements of FND uptake in spleen cells. Specifically, it shows how collagenase use affects the percentage of different cell types carrying at least one FND. As shown, collagenase use promotes a relative increase in FND uptake by cell types other than macrophages.

[0047] For clarity and brevity, features are described herein as part of the same or separate embodiments. However, it should be appreciated that the scope of the invention may include embodiments having all or some of the features described. For example, while embodiments for specific tissue types under specific conditions are shown, those skilled in the art will envision alternative methods to achieve similar functionality and results after benefiting from this disclosure. For example, as an alternative to applying microneedle assemblies to skin tissue, assemblies or other methods for delivering FNCs may use, for example, tissue blocks, whole organs, or even living or dead organisms. Several elements of the embodiments discussed and shown offer certain advantages, such as improved FNC uptake in a relatively non-invasive manner and / or for tissues or cells that normally do not absorb FNCs at all. Of course, it should be appreciated that any of the above embodiments or processes may be combined with one or more other embodiments or processes to provide further improvements in the discovery and matching design and advantages. It should be appreciated that this disclosure provides particular advantages for delivering fluorescent nanodiamonds to skin tissue, particularly the epidermis, and can generally be applied to any application requiring the entry of FNCs or similar particles into any type of tissue.

[0048] In interpreting the appended claims, it should be understood that the word "comprising" does not exclude other elements or actions not listed in a given claim; the words "a" or "an" preceding an element do not exclude the presence of a plurality of such elements; any reference marks in the claims do not limit their scope; several "means" may be represented by the same or different items or implemented structures or functions; unless expressly stated otherwise, any disclosed means or portion thereof may be combined together or separated into further parts. When one claim references another claim, this may indicate a synergistic advantage achieved through a combination of their respective features. However, the fact that certain measures are referenced in mutually different claims does not in itself indicate that a combination of these measures cannot also be used to produce an advantage. Therefore, unless the context expressly excludes it, this embodiment may include all working combinations of the claims, wherein each claim may, in principle, reference any prior claim.

Claims

1. A method for delivering fluorescent nanocrystals (FNCs) into a tissue (T), comprising: providing one or more microneedles (11), wherein each microneedle (11) contains a plurality of fluorescent nanocrystals (FNCs) dispersed throughout a solid matrix material (11m); penetrating the tissue (11) with the one or more microneedles; and dissolving at least a portion of the matrix material (11m) of the one or more microneedles (11) that penetrated the tissue (T), thereby delivering the fluorescent nanocrystals (FNCs) dispersed in the dissolved portion of the matrix material (11m) into the tissue (T).

2. The method of claim 1, wherein the tissue (T) contains biological cells (C), and the solid matrix material (11m) is a biodegradable material, wherein the biodegradable solid matrix material (11m) of the one or more microneedles (11) that penetrated the tissue (T) is configured to degrade inside the tissue (T) within a period of less than one hour.

3. The method of claim 1, wherein the tissue (T) is a skin tissue, wherein the one or more microneedles (11) are configured to penetrate at least one-tenth of a millimeter into an epidermis of the skin tissue to deliver the fluorescent nanocrystals (FNCs) into the epidermis.

4. The method of claim 1, wherein the microneedle (11) is part of a microneedle assembly (10) that includes an array of the microneedles (11) arranged on a substrate (12), wherein each microneedle (11) has a maximum needle length (L) extending transverse to the substrate (12) of less than one millimeter; wherein the microneedle (11) has a maximum base width (B) parallel to the substrate (12) of at most one-third of the needle length (L); wherein adjacent microneedles (11) in the microneedle assembly (10) are separated by a distance (D) of less than the needle length (L).

5. The method of claim 1, wherein the fluorescent nanocrystals (FNCs) contain fluorescent nanodiamonds having an average diameter of 100-250 nm, wherein each nanodiamond contains at least one nitrogen-vacancy center.

6. The method of claim 1, wherein the fluorescent nanocrystals (FNCs) forming part of the one or more microneedles (11) are associated with respective ligands, wherein the fluorescent nanocrystals (FNCs) associated with respective ligands remain in the tissue (T) upon dissolution of the matrix material (11m), wherein the respective ligands are configured to bind to specific biological structures in the tissue (T) upon being delivered into the tissue (T).

7. The method of claim 1, comprising applying a compound collagenase configured to loosen cellular structures of the tissue (T) to facilitate uptake of the FNCs into the tissue (T).

8. The method according to claim 1, wherein the matrix material (11m) and the fluorescent nanocrystals (FNC) are biocompatible and non-toxic.

9. The method according to claim 1, wherein the tissue (T) is skin, and the penetration of the tissue with the one or more microneedles (11) is limited to the epidermis of the skin.

10. The method according to claim 1, wherein the tissue (T) is a tissue section and the method is performed in vitro.

11. A method for measuring a tissue (T), comprising: delivering fluorescent nanocrystals (FNC) into the tissue (T) according to the method of claim 1; and measuring a fluorescence signal (Sf) from the fluorescent nanocrystals (FNC) in the tissue.

12. The method according to claim 11, wherein the measured fluorescence signal (Sf) of a respective fluorescent nanocrystal (FNC) depends on the local environment of the tissue (T) adjacent to the respective fluorescent nanocrystal (FNC).

13. The method according to claim 12, wherein the fluorescence signal (Sf) is measured as a function of a variable stimulus applied to the tissue (T), wherein the variable stimulus comprises one or more of: an amount of UV irradiation applied to the tissue (T), an amount of a drug or antioxidant applied to the tissue (T), an amount and / or type of nutrients applied to the tissue (T), and a disease progression in the tissue (T).

14. The method according to claim 13, wherein the measured fluorescence signal (Sf) is used to determine the presence and / or concentration of free radicals in the tissue (T) as a function of the variable stimulus.

15. A method of manufacturing a microneedle assembly (10), comprising: dispersing a concentration of fluorescent nanocrystals (FNC) into a liquid precursor (11p) of a matrix material (11m); casting the liquid precursor (11p) with the fluorescent nanocrystals (FNC) into a mold (20) that forms a negative of an array of microneedles (11); solidifying (H) the liquid precursor (11p) in the mold (20) to form the array of microneedles (11) comprising the fluorescent nanocrystals (FNC) dispersed throughout a solid matrix material (11m); and removing the array of microneedles (11) from the mold (20) to form part of the microneedle assembly (10).

16. The method according to claim 15, wherein the fluorescent nanocrystals (FNC) comprise fluorescent color centers.

17. A microneedle assembly (10) comprising a substrate (12) having an array of microneedles (11) for penetrating a tissue, wherein each microneedle (11) is formed of a biodegradable solid matrix material (11m), and a plurality of fluorescent nanocrystals (FNC) are dispersed throughout the solid matrix material (11m).

18. The microneedle assembly (10) according to claim 17, wherein the fluorescent nanocrystals (FNC) comprise fluorescent color centers.

19. A measuring device (50) configured to measure fluorescent nanocrystals (FNC) delivered into a tissue (T), the measuring device comprising: a tissue holder configured to hold a section of the tissue (T) immersed in a liquid medium and exposed to controlled environment and temperature to maintain cellular integrity of the tissue (T) during the measurement; a light source configured to deliver pulses of source light (Ss) to the tissue (T); a light detector configured to measure fluorescent signals (Sf) emitted by the fluorescent nanocrystals (FNC) caused by the pulses of source light (Sf); an analyzer configured to determine biological parameters of the tissue (T) based on the measured fluorescent signals (Sf).

20. The device of claim 19, comprising a wavelength filter configured to filter out at least spontaneous fluorescence of the tissue section below 650 nm from reaching the light detector.

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