Compositions and articles comprising (NANO)diamond particles

Abstract

To provide compositions and articles comprising diamond particles, such as nanodiamond based pharmaceutical compositions.SOLUTION: In some embodiments, articles and methods comprising (nano)diamond particles may be useful for monitoring and / or treating a disease (e.g., in a subject).SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] Related Applications This application claims priority under 35 U.S.C. Section 119(e) to U.S. Provisional Application No. 62 / 862,802, filed June 18, 2019, and entitled “Compositions and Articles Comprising Nanodiamond Particles,” which is incorporated by reference in its entirety for all purposes.

[0002] Technical Field Briefly, compositions and articles including diamond particles, such as nanodiamond-based pharmaceutical compositions, are provided. [Background technology]

[0003] background The use of nanomaterials for novel diagnostic and therapeutic purposes is a rapidly evolving scientific field based on the bioengineering of biological and pharmaceutical entities in combination with physical materials. However, improved articles and methods are needed. Summary of the Invention [Problem to be solved by the invention]

[0004] Diamond particles and related devices and methods, e.g., nanodiamond particles (e.g., fluorescent nanodiamond particles), for administering therapeutic agents to a subject and / or monitoring disease progression in a subject The subject matter of the present invention may in some cases include interrelated products, alternative solutions to a particular problem, and / or a number of different uses of one or more systems and / or articles.

[0005] In one aspect, an article (e.g., configured for administration of a therapeutic agent for use with a subject) is provided. In some embodiments, the article comprises a plurality of fluorescent diamond particles and a therapeutic agent bound to at least a portion of the fluorescent diamond particles, the article being configured for long-term retention within an organ of the subject.

[0006] In some embodiments, the article comprises an injection component configured to administer a composition to a subject, and a reservoir associated with the injection component that contains the composition, the composition comprising a plurality of fluorescent diamond particles.

[0007] In another aspect, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises an intravenous carrier fluid and a plurality of fluorescent diamond particles suspended in the intravenous carrier fluid.

[0008] In yet another aspect, methods (e.g., methods of treating a disease, methods of monitoring disease progression in a subject suspected of having a disease) are provided. In some embodiments, the methods include intravenously administering to a subject a plurality of diamond particles and a therapeutic agent bound to at least a portion of the diamond particles, wherein the plurality of diamond particles are configured to remain within an organ of the subject for an extended period of time.

[0009] In some embodiments, the method includes administering a plurality of diamond particles to a subject, obtaining a first image of a location within the subject suspected of containing a plurality of diamond particles after the administering step, obtaining a second image of the location within the subject suspected of containing a plurality of diamond particles after a predetermined period of time, and then measuring a morphological change in the location within the subject between the first and second images of the plurality of diamond particles, where the morphological change is associated with disease progression.

[0010] In some embodiments there is provided the use of a plurality of fluorescent diamond particles in the manufacture of a medicament for the treatment of liver disease and / or liver cancer.In some embodiments there is provided the use of a plurality of fluorescent diamond particles in the manufacture of a medicament for monitoring disease progression.

[0011] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In the event that the present specification and any document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. [Brief description of the drawings]

[0012] BRIEF DESCRIPTION OF THE DRAWINGS Non-limiting embodiments of the present invention are described, by way of example, with reference to the accompanying drawings, which are schematic and not intended to be drawn to scale. In the drawings, each of the identical or nearly identical components shown in the drawings is typically represented by a single numeral. For clarity, not every component is labeled in every drawing, and not every component of each embodiment of the present invention is shown unless illustration is necessary to enable those skilled in the art to understand the invention. [Figure 1A] FIG. 1A is a schematic diagram of a system including fluorescent nanodiamond particles according to one set of embodiments. [Figure 1B] FIG. 1B is a schematic diagram of the method used to quantify FNDP-(NV) uptake into cells, according to one set of embodiments. [Figure 2A]2A-2B show fluorescence microscopy images of paraffin sections (5 μm) of livers obtained from rats treated or not with FNDP-(NV)-700 / 800 nm (FNDP-(NV)) according to one set of embodiments. In FIG. 2A, an image of the tissue section analyzed with a 10x objective with 1.6x extension is shown. In FIG. 2B, an image of the tissue section analyzed with an oil 40x objective is shown, with the image on the left showing an overlap of three colors: red (FNDP-(NV)), blue (DAPI-nuclei), and green (phalloidin-cytoskeleton), shown in different shades of grey. Meanwhile, the right figures show an overlap of two colors, red (FNDP-(NV)) and blue (DAPI-nuclei), shown in different shades of grey, with the top image in each panel representing a rat treated with FNDP-(NV) and the bottom image in each panel representing a control (PBS-treated rat), and the area occupied by the particles is indicated by a white arrow, according to some embodiments. [Figure 2B] 2A-2B show fluorescence microscopy images of paraffin sections (5 μm) of livers obtained from rats treated or not with FNDP-(NV)-700 / 800 nm (FNDP-(NV)) according to one set of embodiments. In FIG. 2A, an image of the tissue section analyzed with a 10x objective with 1.6x extension is shown. In FIG. 2B, an image of the tissue section analyzed with an oil 40x objective is shown, with the image on the left showing an overlap of three colors: red (FNDP-(NV)), blue (DAPI-nuclei), and green (phalloidin-cytoskeleton), shown in different shades of grey. Meanwhile, the right figures show an overlap of two colors, red (FNDP-(NV)) and blue (DAPI-nuclei), shown in different shades of grey, with the top image in each panel representing a rat treated with FNDP-(NV) and the bottom image in each panel representing a control (PBS-treated rat), and the area occupied by the particles is indicated by a white arrow, according to some embodiments. [Diagram 3]3A-3H show that, according to one set of embodiments, "panoramic" images of liver lobes demonstrate intralobular heterogeneity of particle distribution. Fig. 3A and (Fig. 3B) show full panoramic views of sagittal sections from representative liver lobules from two animals, constructed by "stitching" 4x images using an FSX100 microscope with phalloidin-stained sections (5 μm) imaged in the green channel (shown in shades of gray) and the presence of FNDP-(NV) imaged in the red (shown in gray) channel; particles in the images are enlarged by thresholding and repeated dilation to visualize at very low resolution; the figures show an example of a liver lobule with overlapping hexagons, the area shown in gold is enlarged in the other panels, and (Fig. 3C) shows the intralobular heterogeneity of particle distribution at the border of the "hexagonal" lobule format. Four liver lobules are shown demonstrating preferential particle distribution, (FIG. 3D) shows a 10x image of a single liver lobule demonstrating preferential FNDP-(NV) deposition; large FNDP-(NV) aggregates are seen distributed heterogeneously with the liver lobule indicated by the dashed hexagon (FIG. 3E), (FIG. 3F) presents a 10x image of a single liver lobule after thresholding and dilation to improve visibility of very small aggregates and demonstrate zonal deposition, (FIG. 3G), (FIG. 3H) provides a magnified image of a region of vasculature from panel (FIG. 3A) indicated by the gold dashed square, and (FIG. 3I) provides a schematic of a liver lobule demarcating various metabolic zones, according to some embodiments. [Figure 4] Figures 4A-4B show mathematical plots of size distribution of FNDP-(NV) aggregates in liver lobules; views of one complete liver lobule from two animals were stitched from 10x images of an FSX100 microscope; the stitched figures were thresholded in ImageJ using a maximum entropy criterion, resulting in the size and count of detected FNDP-(NV) assemblies; (Figure 4A) Distribution of FNDP-(NV) assembly sizes; (Figure 4B) Distribution of total particle mass estimated by the area of ​​each assembly, according to some embodiments. [Diagram 5]5A-5D show laser scanning confocal microscopy images of liver sections (50 μm) obtained from rats treated with FNDP-(NV) according to one set of embodiments. (FIG. 5A) Parenchymal area of ​​liver with cells shown in yellow circles with internalized particles. The insets at the bottom and right of the picture represent vertical projections of images performed along the yellow line. The yellow arrows indicate the location of the particles. (FIG. 5B) Parenchymal area of ​​liver with yellow circles suggesting aggregates of particles within hepatic sinusoids / venules. The insets at the bottom and right represent vertical projections of images performed along the yellow line. The yellow arrows indicate particles localized in sinusoids / venules. (FIG. 5C) Area of ​​a richly vascularized segment of liver lobule with white circle particles suggesting subendothelial and adventitial location of particles. Parenchymal cells with presumably internalized particles are shown in yellow circles. The insets at the bottom and right represent vertical projections of images performed along the yellow line. The yellow arrows indicate particles internalized in parenchymal cells. (Figure 5D) Area of ​​the liver hilum where the white circle indicates particles associated with adventitial cell elements. The insets at the bottom and right represent vertical projections of images performed along the yellow line. The yellow arrow indicates particles internalized in vascular cells. [Figure 6] Figure 6 shows confocal 3D reconstructions of hepatocytes with different amounts of FNDP-(NV) incorporated according to one set of embodiments. Confocal image stacks from 50 μm sections stained with DAPI (blue) and phalloidin (green) and incorporating nanodiamonds (red) were taken with a Fluoview F1000 confocal microscope and reconstructed using ImageJ's volume viewer. These intracellular particle inclusions (indicated by yellow arrows) include both sparse and dense FNDP-(NV) collections internalized within the cells. The left panel represents vehicle control; the middle panel represents low loading particles and the right panel represents high loading particles of two separate cells. [Figure 7]Figures 7A-7D show plots related to the internalization of different concentrations of FNDP-(NV) into HepG-2 and HUVEC cells over time according to one set of embodiments. (Figure 7A), (Figure 7B), (Figure 7C) show the dose- and time-dependent uptake of FNDP by HepG-2 cells and HUVEC exposed to various concentrations of FNDP-(NV). Exponential curves were fitted to all three doses of particles (high dose 0.1 mg / mL; medium dose 0.05 mg / mL, low dose 0.025 mg / mL). (Figure 7D) Total uptake of FNDP after 20 hours by HepG-2 cells and HUVEC exposed to various concentrations of FNDP. Error bars in all panels represent the SD of 4-fold samples. (*) P<0.001 vs. 0.025 mg / mL by two-tailed Student's test; (+) P<0.001 vs. 0.05 mg / mL by two-tailed Student's test. [Figure 8] 8A-8B show fluorescence microscopy images of HepG-2 cells and HUVECs obtained after 2 and 20 hours of incubation with FNDP-(NV) according to one set of embodiments. Images of HepG-2 cells (FIG. 8A) and HUVECs (FIG. 8B) obtained from fluorescence microscopy analysis using 160x and 400x magnification after 2 or 20 hours of exposure to FNDP-(NV). The 160x magnification images are displayed in overlapping three-color fluorescence (green- FITC-phalloidin, red-FNDP-(NV), blue-DAPI), and the 400x magnification images are displayed in overlapping three-color fluorescence (green, red, blue) (left panel), and two-color fluorescence (red and blue) (right panel). White arrows indicate examples of the cytoplasmic phase of particle transition. Grey arrows indicate perinuclear aggregation of multiple particles. [Figure 9] Figure 9 shows representative images showing the various stages of HUVEC division in the presence of FNDP-(NV) according to one set of embodiments. HUVEC were treated with 0.05 mg / mL FNDP-(NV) for 20 hours. Images at 400x or 640x magnification are displayed with overlapping three-color fluorescence (green-FITC-phalloidin, red-FNDP-(NV), blue-DAPI). The titles of the various phases described are visual images of the predicted cell replication mechanism. [Figure 10] Figures 10A-10B show the effect of passive adsorption of BSA on the aggregation and surface potential of FDP-NV functionalized with carboxyl groups, particles suspended in various dispersants, and applied to a capillary cuvette placed in a Zetasizer instrument (Malvern Inc.) to measure the Z-average, diameter size (Figure 10A) and zeta potential (Figure 10B). Error bars represent SD from triplicate measurements of independent samples. (*) P<0.01 and (**) P<0.001 for the difference between FDP-NV-BSA and native FDP-NV, especially for the dispersants, calculated using one-way ANOVA. [Figure 11A] 11A-11D show the effect of FDP-NV on cell proliferation determined by direct cell number assessment, where a graphical representation of the number of HepG-2 cells (FIG. 11A) and HUVECs (FIG. 11B) obtained after incubation or not with FDP-NV-BSA or vincristine, error bars represent SD from 5 independent wells applied to 7 observation fields per well. (*) P < 0.001 between control and treatment groups calculated using one-way ANOVA, and representative images of observation fields of HepG-2 cells (FIG. 11C) or HUVECs (FIG. 11D) applied to cell number determination using ImageJ software on images acquired using a fluorescent microscope (Olympus IX81) with an application 10x objective and DAPI (blue) and TRITC (red) filters (shown in various shades of grey), according to some embodiments, white arrows indicate internalized particles into neighboring cells of a HepG-2 colony. [Figure 11B]11A-11D show the effect of FDP-NV on cell proliferation determined by direct cell number assessment, where a graphical representation of the number of HepG-2 cells (FIG. 11A) and HUVECs (FIG. 11B) obtained after incubation or not with FDP-NV-BSA or vincristine, error bars represent SD from 5 independent wells applied to 7 observation fields per well. (*) P < 0.001 between control and treatment groups calculated using one-way ANOVA, and representative images of observation fields of HepG-2 cells (FIG. 11C) or HUVECs (FIG. 11D) applied to cell number determination using ImageJ software on images acquired using a fluorescent microscope (Olympus IX81) with an application 10x objective and DAPI (blue) and TRITC (red) filters (shown in various shades of grey), according to some embodiments, white arrows indicate internalized particles into neighboring cells of a HepG-2 colony. [Figure 11C] 11A-11D show the effect of FDP-NV on cell proliferation determined by direct cell number assessment, where a graphical representation of the number of HepG-2 cells (FIG. 11A) and HUVECs (FIG. 11B) obtained after incubation or not with FDP-NV-BSA or vincristine, error bars represent SD from 5 independent wells applied to 7 observation fields per well. (*) P < 0.001 between control and treatment groups calculated using one-way ANOVA, and representative images of observation fields of HepG-2 cells (FIG. 11C) or HUVECs (FIG. 11D) applied to cell number determination using ImageJ software on images acquired using a fluorescent microscope (Olympus IX81) with an application 10x objective and DAPI (blue) and TRITC (red) filters (shown in various shades of grey), according to some embodiments, white arrows indicate internalized particles into neighboring cells of a HepG-2 colony. [Figure 11D]11A-11D show the effect of FDP-NV on cell proliferation determined by direct cell number assessment, where a graphical representation of the number of HepG-2 cells (FIG. 11A) and HUVECs (FIG. 11B) obtained after incubation or not with FDP-NV-BSA or vincristine, error bars represent SD from 5 independent wells applied to 7 observation fields per well. (*) P < 0.001 between control and treatment groups calculated using one-way ANOVA, and representative images of observation fields of HepG-2 cells (FIG. 11C) or HUVECs (FIG. 11D) applied to cell number determination using ImageJ software on images acquired using a fluorescent microscope (Olympus IX81) with an application 10x objective and DAPI (blue) and TRITC (red) filters (shown in various shades of grey), according to some embodiments, white arrows indicate internalized particles into neighboring cells of a HepG-2 colony. [Figure 12] 12A-12B show the effect of FNDP-(NV) on HepG-2 (FIG. 12A) and HUVEC (FIG. 12B). According to some embodiments, (*) P<0.01 and (**) P<0.001 between the control group and the compound-treated group. [Figure 13] 13A-13B show the effect of FNDP-(NV) on HepG-2 (FIG. 13A) and HUVEC (FIG. 13B) esterase activity monitor calcein AM assays, where a graphical representation of the conversion of calcein AM to green fluorescent calcein by esterases present in living HepG-2 cells (FIG. 13A) and HUVEC (FIG. 13B) is shown with error bars representing SD from three independent experiments, where one-way ANOVA was calculated between control and compound-treated groups, according to some embodiments, (*) P<0.01 and (**) P<0.001. [Figure 14A]FIG. 14A shows the effect of FNDP-(NV) on migration of HUVEC stimulated by 2% FBS in a scratch assay, showing scratch closure stimulated with 2% FBS in the presence or absence of FNDP-NV-BSA with unstimulated cells treated with medium containing 0.1% FBS (negative control), error bars represent SD from three independent experiments, (*) P<0.001 versus control (2% FBS-treated) by one-way ANOVA according to one set of embodiments. [Figure 14B] Figure 14B shows the effect of FNDP-(NV) on migration of HUVEC stimulated by 2% FBS in a scratch assay, showing images of scratches obtained using a fluorescence microscope (Olympus IX81) at 20x magnification and showing DAPI (blue) and TRITC (red) filters, displayed in different shades of grey according to one set of embodiments; [Figure 15A] 15A-15B show the effect of FDP-NV on phosphorylation of MAPK Erk1 / 2 induced by FBS with serum-starved HepG-2 cells for 24 h (FIG. 15A) or HUVECs stimulated with 2% FBS for 10 and 20 min (FIG. 15B) and total MAPK Erk1 / 2 reprobed on PVDF membrane after stripping with anti-phosphorylation antibody, the plot bar on the right shows the ratio of the intensity of total protein band to phosphorylated protein band, the green bar shows the ratio of control (untreated cells), while the red bar shows the ratio of FDP-NV treated cells and the left panel shows representative blot images of each cell type, the error bars represent the SD of three independent experiments, according to one set of embodiments, (*)P<0.01 and (**)P<0.01 by one-way ANOVA for the contrast between treated or untreated cells with FDP-NV-BSA 0.1 mg / mL. [Figure 15B]15A-15B show the effect of FDP-NV on phosphorylation of MAPK Erk1 / 2 induced by FBS with serum-starved HepG-2 cells for 24 h (FIG. 15A) or HUVECs stimulated with 2% FBS for 10 and 20 min (FIG. 15B) and total MAPK Erk1 / 2 reprobed on PVDF membrane after stripping with anti-phosphorylation antibody, the plot bar on the right shows the ratio of the intensity of total protein band to phosphorylated protein band, the green bar shows the ratio of control (untreated cells), while the red bar shows the ratio of FDP-NV treated cells and the left panel shows representative blot images of each cell type, the error bars represent the SD of three independent experiments, according to one set of embodiments, (*)P<0.01 and (**)P<0.01 by one-way ANOVA for the contrast between treated or untreated cells with FDP-NV-BSA 0.1 mg / mL. [Figure 16A] Figures 16A-16B show the identification of phospho- and total MAPK Erk1 / 2 in the cytoplasm and nucleus of HepG-2 cells and HUVECs in the presence and absence of FDP-NV and TPA. HepG-2 cells (Figure 16A) or HUVECs (Figure 16B) were treated or not with FDP-NV-BSA (0.1 mg / mL) and stimulated or not with TPA after 24 h of serum starvation. Cells were lysed and fractionated into cytoplasmic and nuclear fractions and fractions subjected to WB using the indicated antibodies; Mek-1 was used as a marker for the cytoplasmic fraction and HDAC1 for the nuclear fraction. [Figure 16B] Figures 16A-16B show the identification of phospho- and total MAPK Erk1 / 2 in the cytoplasm and nucleus of HepG-2 cells and HUVECs in the presence and absence of FDP-NV and TPA. HepG-2 cells (Figure 16A) or HUVECs (Figure 16B) were treated or not with FDP-NV-BSA (0.1 mg / mL) and stimulated or not with TPA after 24 h of serum starvation. Cells were lysed and fractionated into cytoplasmic and nuclear fractions and fractions subjected to WB using the indicated antibodies; Mek-1 was used as a marker for the cytoplasmic fraction and HDAC1 for the nuclear fraction. [Figure 16C] Figures 16C-16D show the identification of phospho- and total MAPK Erk1 / 2 in the cytoplasm and nucleus of HepG-2 cells and HUVECs in the presence and absence of FDP-NV and TPA. HepG-2 cells (C) or HUVECs (D) were exposed to FDP-NV-BSA, then grown on chamber slides and serum-starved for 24 h. After treatment or not with TPA, cells were immunostained with anti-phosphorylated MAPK Erk 1 / 2 followed by goat anti-rabbit tagged with FITC. Slides were analyzed under a fluorescence microscope (Olympus IX81) with oil objectives at 400x magnification using FITC (green) and TRITC (red) filters, with overlapping areas of green and red shown in yellow and various shades of grey according to one set of embodiments, white arrows indicate greater particle accumulation in TPA-treated cells versus untreated cells, blue arrows indicate nuclei of cells not treated with TPA, and red arrows indicate nuclei of cells treated with TPA. [Figure 16D] Figures 16C-16D show the identification of phospho- and total MAPK Erk1 / 2 in the cytoplasm and nucleus of HepG-2 cells and HUVECs in the presence and absence of FDP-NV and TPA. HepG-2 cells (C) or HUVECs (D) were exposed to FDP-NV-BSA, then grown on chamber slides and serum-starved for 24 h. After treatment or not with TPA, cells were immunostained with anti-phosphorylated MAPK Erk 1 / 2 followed by goat anti-rabbit tagged with FITC. Slides were analyzed under a fluorescence microscope (Olympus IX81) with oil objectives at 400x magnification using FITC (green) and TRITC (red) filters, with overlapping areas of green and red shown in yellow and various shades of grey according to one set of embodiments, white arrows indicate greater particle accumulation in TPA-treated cells versus untreated cells, blue arrows indicate nuclei of cells not treated with TPA, and red arrows indicate nuclei of cells treated with TPA. [Figure 17]FIG. 17A shows the effect of FDP-NV on the induction of apoptosis and ER stress in HepG-2 cells and HUVECs by Western blot analysis of caspase 3 cleavage in the presence or absence of FDP-NV (0.1 mg / mL) in HepG-2 and HUVECs. Vincristine was used as a positive control for apoptosis. According to some embodiments, the localization of the molecular weight marker is indicated by the arrow on the left side of the image. FIG. 17B shows the effect of FDP-NV on the induction of apoptosis and ER stress in HepG-2 cells and HUVECs by Western blot analysis of the expression of chaperones in the ER in the presence or absence of FDP-NV (0.1 mg / mL) in HepG-2 and HUVECs. According to some embodiments, tunicamycin was used as a positive control for endoplasmic reticulum stress. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In general, compositions and articles including diamond particles, such as diamond-based pharmaceutical compositions, are provided. In some embodiments, articles and methods including diamond particles may be useful for monitoring and / or treating disease (e.g., in a subject). In some embodiments, the articles may be configured to administer a plurality of diamond particles (e.g., fluorescent (nano)diamond particles) that can be used to deliver a therapeutic agent bound to the (nano)diamond particles. For example, the plurality of (nano)diamond particles may be administered to a subject such that at least a portion of the plurality of (nano)diamond particles reside at an internal location of the subject (e.g., in an organ such as the liver). In some embodiments, the (nano)diamond particles may be used as a diagnostic tool. For example, in some embodiments, the plurality of (nano)diamond particles may be administered to a subject (e.g., via intravenous injection). In some such embodiments, an image of a location suspected of containing a plurality of (nano)diamond particles may be obtained, and after a diagnostically relevant period of time, a second image of the same location of the subject suspected of containing a plurality of (nano)diamond particles may be obtained. In some embodiments, the first image and / or the second image are based on near infrared and / or fluorescent emission (e.g., by the (nano)diamond particles). In some embodiments, the comparison of the first image and the second image may provide diagnostic information, including, for example, the progression of a disease state (e.g., cancer). For example, an area of ​​the second image that includes new tissue without the plurality of (nano)diamond particles may indicate malignant growth in some cases. Thus, in some embodiments, the (nano)diamond particles may be useful for monitoring disease progression. In some embodiments, the first image and the second image are obtained under similar (e.g., identical) conditions (e.g., the same wavelength of excitation and / or emission).

[0014] As used herein, "subject" refers to any animal, such as a mammal (e.g., human). Non-limiting examples of subjects include humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents such as mice, rats, hamsters, birds, fish, or guinea pigs. The embodiments described herein may be directed to human use in some cases. The embodiments described herein may be directed to veterinary use in some cases. In some embodiments, the subject may show health benefits, for example, upon administration of (nano)diamond particles.

[0015] In some embodiments, the (nano)diamond particles described herein can be configured to have an extended residence time in one or more organs of a subject, such as the liver. For example, the progression of tumor growth can be monitored by administering a plurality of (nano)diamond particles to a subject and imaging the organ suspected of tumor growth as described above.

[0016] In some embodiments, the (nano)diamond particles described herein may be configured to deliver a therapeutic agent (e.g., to an internal organ of a subject). In some embodiments, the therapeutic agent may be at least partially bound to the plurality of (nano)diamond particles. In some cases, the (nano)diamond particles bound to a therapeutic agent may be administered to a subject (e.g., to provide a therapeutic effect).

[0017] The (nano)diamond particles may be configured to have a relatively long retention within an internal location of a subject (e.g., an organ), so that therapeutic agents delivered using the (nano)diamond particles may advantageously deliver the therapeutic agent over an extended period of time. In some embodiments, the (nano)diamond particles are configured to have an extended retention within a subject or within an organ of a subject. In some embodiments, the (nano)diamond particles are configured to remain in the organ (e.g., have a size and / or shape that facilitates retention). In some embodiments, the (nano)diamond particles are configured to remain in the organ for 1 day or more, 3 days or more, 5 days or more, 7 days or more, 10 days or more, 2 weeks or more, 4 weeks or more, 6 weeks or more, 12 weeks or more, 26 weeks or more, or 52 weeks or more. In some embodiments, the (nano)diamond particles are configured to remain in the organ of the subject for 100 weeks or less, 52 weeks or less, 26 weeks or less, 12 weeks or less, 6 weeks or less, 4 weeks or less, 2 weeks or less, 10 days or less, 7 days or less, 5 days or less, or 3 days or less. Combinations of the above ranges are also possible (e.g., more than 1 day and less than 100 weeks, more than 5 days and less than 26 weeks, more than 6 weeks and less than 52 weeks). Other ranges are also possible. In some embodiments, the (nano)diamond particles can be configured to remain in the organ of the subject for the life of the subject. Advantageously, the (nano)diamond particles described herein may be retained in the subject's organs without toxic or adverse physiological effects.

[0018] In certain embodiments, the (nano)diamond particles may be captured by an internal organ of the subject. In some embodiments, the (nano)diamond particles may further organize or aggregate within the subject or within an organ of the subject. In some embodiments, the (nano)diamond particles may form aggregates within an organ, such as the liver, for example. In some embodiments, diamond nanoparticles (e.g., (nano)diamond particles) may form aggregates within, for example, the pancreas and / or pancreatic cells. In some cases, these aggregates may be useful for advantageously monitoring the progression of a condition or disease within a subject and / or for providing long-term delivery of a therapeutic agent.

[0019] As described herein, (nano)diamond particles can be administered to a subject. In some cases, (nano)diamond particles can be administered surgically (e.g., implanted) and / or injected (e.g., into the systemic circulation, into the eye, into the spinal cord or into bodily fluids, e.g., via a syringe). In certain embodiments, (nano)diamond particles can be administered orally, rectally, vaginally, nasally, or urinary (e.g., in a capsule).

[0020] In some embodiments, the administration of the (nano)diamond particles is via injection, such as intravenous injection. For example, an injection component associated with a reservoir containing (nano)diamond particles can be used. In some embodiments, the injection component is a needle and the associated reservoir is a syringe. The needle can be of any size or gauge suitable for administering the composition to a subject. The syringe can be of any size or capacity suitable for containing a particular amount of the composition to be administered to a subject. In some embodiments, the injection component is a pipette. Those skilled in the art will recognize other injection components suitable for administering the compositions described herein to a subject, as the present disclosure is not so limited.

[0021] In some embodiments, the reservoir comprises an intravenous carrier fluid and a plurality of (nano)diamond particles suspended in the intravenous carrier fluid.Non-limiting examples of suitable intravenous carrier fluids include saline (e.g., 9% saline, 45% saline), lactated Ringer's, and aqueous dextrose (e.g., 5% dextrose in water).

[0022] In some embodiments, (nano)diamond particles (e.g., a plurality of (nano)diamond particles comprising a therapeutic agent bound to the (nano)diamond particles) may be administered to a subject (e.g., for detection of an analyte (e.g., a physiological component of a pathological identity) suspected to be present in the subject). For example, in some cases, a plurality of (nano)diamond particles constituting a therapeutic agent are administered to a subject, and detection of the emission (e.g., fluorescence emission, near infrared emission, etc.) of the (nano)diamond particles confirms the presence of the therapeutic agent in the subject.

[0023] In some embodiments, the chemical species (e.g., therapeutic agent) is bound to the (nano)diamond particle or particles. In some embodiments, the (nano)diamond particle is associated (e.g., bound) with the chemical species through functionalization of the (nano)diamond particle. For example, in some embodiments, the (nano)diamond particle is associated with the chemical species through the formation of bonds such as ionic bonds, covalent bonds, hydrogen bonds, van der Waals interactions, etc. The covalent bonds can be, for example, carbon-carbon, carbon-oxygen, oxygen-silicon, sulfur-sulfur, phosphorus-nitrogen, carbon-nitrogen, metal-oxygen, or other covalent bonds. The hydrogen bonds can be, for example, between hydroxyl, amine, carboxyl, thiol, and / or similar functional groups. For example, the chemical species can further include functional groups such as thiol, aldehyde, ester, carboxylic acid, hydroxyl, etc., where the functional groups form bonds with the (nano)diamond particle. In some embodiments, the functional groups are bound to the (nano)diamond particle (e.g., can be bound to a therapeutic agent). In some cases, the chemical species may be electron rich or electron poor, where the interaction between the (nano)diamond particle and the chemical species involves electrostatic interactions.

[0024] In some embodiments, the chemical species (e.g., therapeutic agent) is associated with the functionalized (nano)diamond particle containing -COOH, -OH, -NH2, -SH, or -C=O functional groups by reacting the functionalized (nano)diamond particle with the chemical species in the presence of a crosslinking agent. Non-limiting examples of suitable crosslinking agents include carbodiimides such as 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC); amine-reactive compounds such as N-hydroxysuccinimide esters, imidoesters, and hydromethylphosphine; sulfhydryl-reactive compounds such as maleimides, pyridyl disulfides, and iodoacetyl; aldehyde-reactive compounds such as hydrazides and alkoxyamines; and photoreactive crosslinkers such as aryl azides and diazirines. Other crosslinking agents are possible. Those skilled in the art will be able to select the appropriate crosslinking agent based on the type of chemical species selected and the teachings herein.

[0025] Examples of suitable (nano)diamond particles are described in further detail in commonly owned International Patent Application No. PCT / US2017 / 050257, entitled "(Nano)Diamond Particles and Related Devices and Methods", filed September 6, 2017, which is incorporated herein by reference in its entirety for all purposes.

[0026] As described above and herein, in some embodiments, (nano)diamond particles can be used for imaging. For example, in some embodiments, (nano)diamond particles can emit characteristic luminescence (i.e., fluorescence) that can be detected by a detector. In some embodiments, the detector can be placed near the area of ​​the subject suspected to contain (nano)diamond particles. For example, a plurality of (fluorescent) (nano)diamond particles functionalized with chemical species can be administered to the subject, and a detector can be placed near the area of ​​the subject suspected to contain (nano)diamond particles to detect any (nano)diamond particles (e.g., by the luminescence of (nano)diamond particles).

[0027] Any suitable detector can be used in the devices and methods described herein. For example, in some embodiments, the detector can be an optical detector (e.g., a fluorescent detector, a visible light and / or UV detector, a near-infrared detector, a microscope, an MRI, a CT scanner, an X-ray detector).

[0028] As described herein, (nano)diamond particles are aggregates of carbon atoms, with a diamond cage at the core that is mainly composed of carbon atoms. (Nano)diamond particles contain diamond, but other carbon phases or allotropes may be present, such as graphite, graphene, fullerene, etc. A single (nano)diamond particle may contain a single form of carbon in some embodiments. In other embodiments, two or more forms of carbon may comprise the (nano)diamond particle.

[0029] In some embodiments, the diamond particles may have an average maximum cross-sectional dimension (e.g., diameter) of 2 μm or less. While much of the description herein generally relates to nanodiamond particles (i.e., diamond particles having a maximum cross-sectional dimension of less than 1000 nm), one of ordinary skill in the art will understand that diamond particles based on the teachings herein. Larger cross-sectional dimensions (e.g., 1000 nm or more) are also possible. For example, in some embodiments, the diamond particles may have an average maximum cross-sectional dimension of less than 2 μm (e.g., 1800 nm or less, 1600 nm or less, 1400 nm or less, 1200 nm or less, 1000 nm or less, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 400 nm or less, 200 nm or less, 180 nm or less, 160 nm or less, 140 nm or less, 120 nm or less, 100 nm or less, 80 nm or less, 60 nm or less, 40 nm or less, or 20 nm or less). In some cases, the diamond particles may have an average maximum cross-sectional dimension of 10 nm or more, 20 nm or more, 40 nm or more, 60 nm or more, 80 nm or more, 100 nm or more, 120 nm or more, 140 nm or more, 160 nm or more, 180 nm or more, 200 nm or more, 400 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, 1000 nm or more, 1200 nm or more, 1400 nm or more, 1600 nm or more, or 1800 nm or more. Combinations of the above ranges are also possible (e.g., 10 nm or more to less than 2 μm, 1000 nm or more to 1400 nm or less). Other ranges are also possible. Those skilled in the art can select a suitable method for determining the average cross-sectional dimension of the diamonds based on the teachings herein. In one exemplary set of embodiments, the diamond particles have an average maximum cross-sectional dimension of 900 nm or less and 700 nm or more. In some embodiments, the diamond particles may form aggregate structures with other diamond particles (eg, at locations within the object).In some embodiments, the aggregate of diamond particles may have a maximum cross-sectional dimension of 1 μm or more (e.g., 1 μm or more, 5 μm or more, 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or less, 90 μm or more) and 100 μm or less (e.g., 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less and 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, equal to 10 μm, 5 μm or less, 1 μm or less). Combinations of the above ranges are also possible (e.g., 1 micron or more and 50 microns or less, 1 micron or more and 100 microns or less). Other ranges are also possible.

[0030] In some embodiments, the (nano)diamond particles may form relatively large aggregate structures with other (nano)diamond particles (e.g., at a location within an object). For example, in some embodiments, the aggregates of (nano)diamond particles have a maximum cross-sectional dimension of 100 microns or more, 200 microns or more, 500 microns or more, 1000 microns or more, 2000 microns or more, 5000 microns or more, or 7500 microns or more. In some embodiments, the aggregates of (nano)diamond particles have a maximum cross-sectional dimension of 1000 microns or less, 7500 microns or less, 5000 microns or less, 2000 microns or less, 1000 microns or less, 500 microns or less, or 200 microns or less. Combinations of the above ranges are also possible (e.g., 1 micron or more and 10,000 microns or less, 100 microns or more and 10,000 microns or more and 500 microns or less and 5,000 microns or less, 1000 microns or more and 10,000 microns or more). Other ranges are also possible.

[0031] In some embodiments, the (nano)diamond particles may emit electromagnetic radiation. In some embodiments, the emission is fluorescent emission. In certain embodiments, the wavelength of the emission is 250 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, 450 nm or more, 500 nm or more, 550 nm or more, 600 nm or more, or 650 nm or more. In certain embodiments, the wavelength of the emission is 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, or 300 nm or less. Combinations of the above ranges are also possible (e.g., 250 nm or more and 700 nm or less). Other ranges are also possible.

[0032] In certain embodiments, the radiation is near infrared radiation. In some embodiments, the wavelength of the emitted light is 700 nm or more, 750 nm or more, 800 nm or more, 850 nm or more, 900 nm or more, 950 nm or more. In certain embodiments, the wavelength of the emitted light is 1000 nm or less, 950 nm or less, 900 nm or less, 850 nm or less, 800 nm or less, or 750 nm or less. Combinations of the above ranges are also possible (e.g., greater than 700 nm and less than 1000 nm). Other ranges are also possible.

[0033] In an exemplary embodiment, the (nano)diamond particles have a near infrared emission (e.g., 650 nm to 750 nm) and an average maximum cross-sectional dimension of about 700 to 900 nm. Other combinations of emission and cross-sectional dimensions are possible.

[0034] In some embodiments, (nano)diamond particles can emit fluorescence and / or near infrared emission when excited by electromagnetic radiation having a certain wavelength.For example, in some embodiments, (nano)diamond particles can be exposed to electromagnetic radiation having a wavelength of 250nm or more, 300nm or more, 350nm or more, 400nm or more, 450nm or more, 500nm or more, 550nm or more, 600nm or more, 650nm or more, 700nm, 750nm or more, 800nm ​​or more, 850nm or more, 900nm or more, or 950nm or more (e.g., (nano)diamond particles emit fluorescence and / or near infrared emission in one of the above ranges). In certain embodiments, the (nano) diamond particles can be exposed to electromagnetic radiation having a wavelength of 1000nm or less, 950nm or less, 900nm or less, 850nm or less, 800nm ​​or less, or 750nm or less, 700nm or less, 650nm or less, 600nm or less, 550nm or less, 500nm or less, 450nm or less, 400nm or less, 350nm or less, or 300nm or less. Combinations of the above ranges are also possible (e.g., 250nm or more and 1000nm or less, 550nm or more and 650nm or less). Other ranges are also possible.

[0035] Without wishing to be bound by theory, in some cases, the (nano)diamond particles described herein may be autofluorescent (e.g., the (nano)diamond particles fluoresce, for example, after absorbing electromagnetic radiation). In some cases, the (nano)diamond particles may contain one or more atomic type defects (e.g., point defects such as nitrogen-vacancy (NV) centers, point defects such as nitrogen-vacancy-nitrogen (NVN), combinations thereof), which result in near-infrared fluorescence and / or photoluminescence that can be detected and / or quantified. Other defects are also possible (e.g., gadolinium, europium, iron, Si-vacancy defects). In certain embodiments, the (nano)diamond particles fluoresce in response to applied electromagnetic radiation.

[0036] For example, in some embodiments, the (nano)diamond particles can be excited (e.g., by applying electromagnetic radiation having a first wavelength) so that the (nano)diamond particles emit detectable luminescence (e.g., electromagnetic radiation having a second wavelength different from the first wavelength). In a particular set of embodiments, if the analyte is present in the sample, the analyte binds to the (nano)diamond particles (e.g., binds to a chemical species bound to the (nano)diamond particles) and the emission from the (nano)diamond particles can be detected and / or quantified. In some cases, detection of the emission of the (nano)diamond particles in the subject may indicate that the (nano)diamond particles are bound to the suspected analyte. In some such cases, the emission can be quantified (e.g., to determine the relative amount of the analyte present in the subject).

[0037] As described herein, certain embodiments include a therapeutic agent bound to the (nano)diamond particles. According to some embodiments, the therapeutic agent may be one or a combination of therapeutic, diagnostic, and / or enhancing agents, such as drugs, nutrients, microorganisms, in vivo sensors, and tracers. In some embodiments, the therapeutic agent is a dietary supplement, a prophylactic, or a diagnostic agent. While much of the specification describes the use of therapeutic agents, other agents described herein are possible.

[0038] Agents include, but are not limited to, any synthetic or naturally occurring biologically active compound or composition that, when administered to a subject (e.g., a human or non-human animal), exerts a desired pharmacological, immunogenic, and / or physiological effect by local and / or systemic action. For example, useful or potentially useful in the context of certain embodiments are compounds or chemicals that are traditionally considered drugs, vaccines, and biopharmaceuticals. Certain such agents may include molecules such as proteins, peptides, hormones, nucleic acids, genetic constructs, and the like, that are useful for the medical or veterinary treatment, prevention, diagnosis, and / or mitigation of disease or illness (e.g., HMG, such as rosuvastatin).Co-A reductase inhibitors (statins), nonsteroidal anti-inflammatory drugs such as meloxicam, selective serotonin reuptake inhibitors such as escitalopram, blood thinners such as clopidogrel, steroids such as prednisone, antipsychotics such as aripiprazole and risperidone, painkillers such as buprenorphine, antagonists such as naloxone, montelukast and memantine, cardiac glycosides such as digoxin, alpha blockers such as tamsulosin, cholesterol blockers such as ezetimibe Resorptive inhibitors; metabolites such as colchicine; antihistamines such as loratadine and cetirizine; opioids such as loperamide; proton pump inhibitors such as omeprazole; anti-(retro)virals such as entecavir, dolutegravir, rilpivirine, and cabotegravir; antibiotics such as doxycycline, ciprofloxacin, and azithromycin; antimalarials; synthroid / levothyroxine; drug abuse treatment (e.g., methadone) (methadone and varenicline); family planning (e.g., hormonal contraceptives); performance enhancement (e.g., stimulants such as caffeine); nutrition and supplements (e.g., protein, folic acid, calcium, iodine, iron, zinc, thiamine, niacin, vitamin C, vitamin D, and other vitamin or mineral supplements). For use in the fields of treatment, diagnosis, and / or enhancement, including:

[0039] In certain embodiments, the therapeutic agent is one or more specific therapeutic agents.As used herein, the term "therapeutic agent" or the term "drug" refers to a drug administered to a subject for treating or preventing disease, disorder, or other clinically recognized conditions, and has a clinically significant effect on the subject's body to improve, treat, and / or prevent disease, disorder, or condition. Lists of examples of known therapeutic agents can be found, for example, in the United States Pharmacopeia (USP), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Ed., McGraw Hill, 2001; Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill / Appleton & Lange; 8th edition (September 21, 2000);Physician's Desk Reference (Thomson Publishing), and / or The Merck Manual of Diagnosis and Therapy, 17th ed. (1999), or the 18th ed (2006) thereafter published, Mark H. Beers and Robert Berkow (eds.), Merck Publishing Group, or, for animals, The Merck Veterinary Manual, 9th ed., Kahn, CA (ed.), Merck Publishing Group, 2005; and the United States Food and Drug Administration. (FDA) “Approved Drug Products with Therapeutic Equivalence and Evaluations,” (the “Orange Book”).Examples of pharmaceuticals approved for human use are listed by the FDA under 21 C.FR Sections 330.5, 331-361, and 440-460, and are incorporated herein by reference; veterinary pharmaceuticals are listed by the FDA under 21 C.FR Sections 500-589, and are incorporated herein by reference. In certain embodiments, the therapeutic agent is a small molecule. Exemplary classes of therapeutic agents include, but are not limited to, analgesics, anti-analgesics, anti-inflammatory agents, anti-pyretics, anti-depressants, anti-epileptics, anti-psychotics, neuroprotective agents, anti-proliferative agents such as anti-cancer agents, and anti-histamines. Anti-migraine drugs, hormones, prostaglandins, antimicrobials (including antibiotics, antifungals, antivirals, and antiparasitics), antimuscarinics, antidepressants, bacteriostatics, immunosuppressants, sedatives, hypnotics, antipsychotics, bronchodilators, antiasthmatics, cardio-coagulants, enzyme inhibitors, steroids, steroidal or nonsteroidal anti-inflammatory drugs, corticosteroids, dopamine agonists, electrolytes, gastrointestinal drugs, muscle relaxants, nutrients, vitamins, parasympathomimetics, stimulants, anorexics, and analgesics. Nutraceuticals can also be incorporated into the drug delivery device. These can be vitamins, supplements such as calcium or biotin, or natural ingredients such as plant extracts and plant hormones.

[0040] In some embodiments, the therapeutic agent is one or more anti-cancer agents (eg, chemotherapeutic agents).

[0041] Non-limiting examples of suitable anti-cancer therapeutic agents include alkylating agents (e.g., Cyclophosph, Busulfan, cisplatin), metabolic inhibitors (e.g., folic acid analogs-methotrexate), purine analogs (e.g., mercaptopurine, pentostatin), pyrimidine analogs (e.g., 5-fluorouracil), vinca alkaloids, camptothecins, proteasome inhibitors (e.g., Gefitinib), hormones (e.g., steroids), biologic adjuvant therapies (e.g., antibodies, Herceptin), adjuvant therapies (e.g., BRAF, Melanoma), dabrafenib / Tafinlar; These include trametinib / Mekinist, biospecific antibodies, blinatumomab / Blincyto, chemically labeled antibodies, and Brentuximab.

[0042] In another embodiment, the therapeutic agent is an immunosuppressant. Exemplary immunosuppressants include glucocorticoids, cytostatic agents (such as alkylating agents, antimetabolites, and cytotoxic antibodies), antibodies (such as those against the T cell receptor or the Il-2 receptor), drugs acting on immunophilins (cyclosporine, tacrolimus, and sirolimus), and other agents (such as interferons, opioids, TNF binding proteins, mycophenolate, and other small molecules such as fingolimod).

[0043] In certain embodiments, the therapeutic agent is a hormone or a derivative thereof. Non-limiting examples of hormones include insulin, growth hormones (e.g., human growth hormone), vasopressin, melatonin, thyroxine, thyrotropin-releasing hormone, glycoprotein hormones (e.g., luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, TSH), eicosanoids, estrogens, progestins, testosterone, estradiol, cortisol, adrenaline, and other steroids.

[0044] In some embodiments, the therapeutic agent is a small molecule drug with a molecular weight of less than about 2500 Daltons, less than about 2000 Daltons, less than about 1500 Daltons, less than about 1000 Daltons, less than about 750 Daltons, less than about 500 Daltons, or less than about 400 Daltons. In some cases, the therapeutic agent is a small molecule drug with a molecular weight between 200 and 400 Daltons, between 400 and 1000 Daltons, or between 500 and 2500 Daltons.

[0045] In some embodiments, the therapeutic agent is a nucleic acid, a peptide, a bacteriophage, DNA, mRNA, an aptamer, human growth hormone, a monoclonal antibody, adalimumab, epinephrine, a GLP-1 receptor agonist, an active agent such as semaglutide, liraglutide, dulaglitide, exenatide, factor VIII, a small molecule drug, a progestin, a vaccine, a subunit vaccine, a recombinant vaccine, a polysaccharide vaccine, a conjugate vaccine, a toxoid vaccine, an influenza vaccine, a single vaccine, a pre-pneumonia vaccine, an mmr vaccine, a tetanus vaccine, a hepatitis vaccine, an HIV vaccine Ad4-env clade C, an HIV vaccine Ad4-mGag, a DNA vaccine, an RNA vaccine, etanercept, infliximab, filgastrim, glatiramer acetate, acetate, rituximab, bevacizumab, any molecule encapsulated in a nanoparticle, epinephrine, lysozyme, glucose-6-phosphate dehydrogenase, other enzymes, certolizumab pegol, ustekinumab, ikekizumab, golimumab, brodalumab, gusellu, ab, secikinumab, omalizumab, TNF-alpha inhibitors, interleukin inhibitors, vedolizumab, octreotide, teriperatide, CRISPR cas9, oligonucleotides, and ondansetron.

[0046] Although much of the description herein is in the context of (fluorescent) (nano) diamond particles, those skilled in the art will understand that other particles are possible based on the teachings herein. For example, in some embodiments, the device may include particles such as nanoparticles (e.g., silica nanoparticles, sapphire nanoparticles, garnet nanoparticles, ruby ​​nanoparticles, quantum dots, quantum dot-polymer composites) that have an emission in one of the above reference ranges related to a chemical species (e.g., a chemical species that can bind to one or more target analytes). In some cases, the particles may be autofluorescent. In other cases, the particles may be functionalized (e.g., associated) with fluorescent molecules.

[0047] In exemplary embodiments, the fluorescent (nano)diamond particles administered to a subject gain access to liver cells (e.g., hepatocytes, Kupffer cells) as well as other cells (e.g., endothelium), where deposition of the (nano)diamond particles in the liver is substantially immediate (upon injection of the (nano)diamond particles). In some such embodiments, the presence of the (nano)diamond particles in the liver is extended, e.g., a single injection may provide a sustained presence of the particles for at least 12 weeks. In some embodiments, the (nano)diamond particles present in the liver do not adversely affect normal liver cells (e.g., measured after at least 3 months). The (nano)diamond particles and / or related chemical species (e.g., chemical and / or organic additives functionalized with (nano)diamond particles) may, in some cases, find enhanced entry and increased accumulation within cancer cells (on normal liver). In some embodiments, therapeutic agents with anti-cancer properties, when tagged to the fluorescent (nano)diamond particles, may inhibit the growth of cancer cells (e.g., reduce metastatic scale and progression). In some such embodiments, (nano)diamond particles (e.g., bound to a therapeutic agent) may advantageously provide longer "progression-free survival" periods and reduced mortality. In some embodiments, without wishing to be bound by theory, reducing metastatic burden in the liver may advantageously contribute to improved liver function (itself a significant cause of morbidity). EXAMPLES

[0048] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0049] Example 1 The rodent POC (validity) study raised an important caveat: anatomical considerations suggest that recording NIR from locations within the body will result in challenges related to opacity and autofluorescence in an organ- and tissue-dependent manner. To optimize the NIR signal captured from within the body at locations such as the carotid artery (in humans, 15-20 mm from the skin surface), a large nanodiamond particle was chosen: FNDP-(NV)-Z-average ~800 nm (fluorescent nanodiamond particles with nitrogen-vacancies and an average maximum cross-sectional area of ​​~800 nm), a strain of particles with superior NIR emission (650-720 nm) to smaller particles of the same strain as well as the NVN "color center" strain, given the rapid clearance of FNDP-(NV) from the circulation (50% clearance from serum within 4 min) immediately after injection (assuming a targeted clinical procedure), the particles "homed" to the target (intravascular clot). The loading of FDNP-(NV) was investigated, possibly via rapid uptake by the hepatic reticuloendothelial system, allowing targeted imaging.

[0050] Direct administration of particles of the selected size (Z-average ~800 nm) into the systemic circulation may result in the particles remaining within organs, as excretion routes (urinary or hepatobiliary) are unlikely. Such concerns are supported by in vitro studies in which prolonged retention of other similarly sized particles within cells (in culture) suggested interference with biological function and viability.

[0051] The examples herein, designed to investigate FDNP-(NV) distribution in rat organs during both short-term and long-term exposure, showed a primary deposition of particles in the liver and secondary deposition in the spleen, while other organs shared only a small proportion. Interestingly, the massive deposition of FDNP-(NV) in the liver after 5 days of exposure did not change in studies at 14 days and 12 weeks after exposure.

[0052] The present investigation on the intrahepatic topological distribution of FNDP-(NV) was carried out by conventional fluorescence microscopy (FM) and confocal fluorescence microscopy (CFM) of liver slices (5-50 μm) in outline. Furthermore, in vitro investigations were performed on the kinetics of FNDP-(NV) uptake into cells such as human hepatoma cells (HepG2) and human umbilical vein endothelial cells (HUVEC), which are commonly used as proxies for hepatocytes and vascular endothelium, respectively. The in vitro results demonstrated the ability of hepatocytes to uptake FDNP-(NV), as well as the intracellular distribution of the engulfed particles.

[0053] Materials and Methods FNDP-(NV)-Z-average ~800nm: origin and functionalization FNDP-(NV)-Z-average ~800 nm functionalized with carboxyl moieties was purchased from ADAMAS Nanotechnologies (Raleigh, NC, USA). The physical properties of FNDP-(NV) were determined by dynamic light scattering on a ZetasizerNano (Malvern) with an average diameter of 858 ± 47 nm and a Z-potential of -56 mV as previously reported. Sterile and BSA-blocked FNDP-(NV) was used in cell-based studies.

[0054] Liver specimens were obtained as follows: Briefly, Sprague-Dawley rats were injected into the femoral vein over 2–3 min with 60 mg / Kg FNDP-(NV) suspension in 2 mL of PBS. After 12 weeks, animals were sacrificed by exsanguination under deep (5% isoflurane) anesthesia and perfusion with 10 mL of sterile saline to minimize residual blood in the organ vasculature, followed by perfusion with 4% paraformaldehyde in saline for further organ preservation. Organs were carefully dissected and suspended in excess 10% neutral buffered formaldehyde (10% NBF). Liver specimens were then processed, embedded in paraffin, and cut into 5 or 50 μm slices for analysis by fluorescence microscopy (FM) or confocal fluorescence microscopy (SCM), respectively. Liver specimens evaluated in this study were discrete and whole lobes dissected after whole organ imaging by IVIS. For histopathological examination, 5 μm sections of liver specimens were stained with hematoxylin and eosin (H&E) and Masson's trichrome by independent histopathological evaluation.

[0055] Rat liver specimens were embedded in paraffin and sectioned at 5 or 50 μm thickness as previously described. Briefly, slides were deparaffinized by three successive rinses in xylene (5 min each), followed by two successive rinses in 100%, 95%, 70%, and 50% ethanol (10 min each), and finally two washes in deionized water. Cellular actin filaments were stained with FITC-phalloidin. Briefly, slides were permeabilized by incubation with 0.4% TritonX-100 in PBS on ice for 10 min. Slides were then washed three times with PBS at room temperature and immersed in FITC-phalloidin (6 μm in PBS) for 1 h. Slides were washed three times with PBS and mounted with mounting buffer containing DAPI to stain nuclei. 5 μm thick slices were analyzed under a fluorescent microscope using 10× and 40× (oil immersion) objectives. A green fluorescent filter set was used to detect FITC-phalloidin stained microfilaments, a red fluorescent filter was used to detect FNDP-(NV), and a blue fluorescent filter was used to detect DAPI stained cell nuclei.

[0056] A global panoramic view of the sagittal section of the liver was constructed by "stitching" images at 4x magnification using an FSX100 microscope. 50 μm sections were stained with FITC-phalloidin to visualize actin filaments imaged in the green channel, and sections were imaged in the red channel to visualize FNDP-(NV). Images were digitally collected and further processed in ImageJ 1.51e (NIH, Bethesda MD, USA). To improve visualization of FNDP-(NV), which were only a few pixels in size at ultra-low magnification, we thresholded the red channel using the maximum entropy method and enlarged the particles by magnifying the result 3x.

[0057] The presence of FNDP-(NV) within cells after image thresholding was also quantified, but not expanded, using the AnalyzeParticles function in ImageJ. Groups of FNDP-(NV) detected as a single continuous mass (aggregates) at 4x were counted and sized. A size distribution by number histogram was created to show the distribution of FNDP-(NV) aggregate sizes detected within the micrographs. Here, the height of the line corresponds to the fraction of particles detected by diameter. Since a large number of small aggregates may account for a small number of total particle masses, the size distribution by number can be considered biased to expand the prevalence of small particle sizes. To reduce this bias, a second histogram of size distribution by cross-sectional area was also created. Here, the height of the line correlates with the fraction of the total NIR fluorescent area.

[0058] Confocal images of liver slices (10–50 μm) were taken using an FV1000 scanning confocal microscope and imaged using a 60x oil immersion objective in Fluoview software (v4.2.2.9 Olympus). For 3D reconstructions, confocal stacks were taken using images every 0.5 μm throughout the thickness of the tissue. Nuclei were visualized by DAPI staining with 405 nm excitation and 425–460 nm emission. The actin cytoskeleton was visualized by FITC-phalloidin with 488 nm excitation and 400–500 nm emission. NIR fluorescence emitted from FNDP-(NV) was visualized with 543 nm excitation and 655–755 nm emission. 2D maximum intensity projections and cross sections were created in Fluoview. Three-dimensional views were reconstructed in ImageJ via the VolumeViewer plugin.

[0059] HepG-2 (human hepatocellular carcinoma) cell line was purchased from American Type Culture Collection (ATTC) (Manassas, VA, USA) and cultured in Eagle's minimum essential medium (EMEM, ThermoFisher Scientific) containing 10% fetal bovine serum (FBS). Primary human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Basel, Switzerland) and cultured in EGM-2MV medium. HUVECs were used for experiments for passages 5–8. FNDP-(NV) uptake by either cell line was performed according to a previously published protocol with some modifications, as shown in Figure 1. Briefly, cells were seeded (2 x 10 per well) in two 96-well plates. 4Live samples were grown to 90% confluence. The medium was removed from one plate (background control) and 100 μL of 4% paraformaldehyde (PFA) in PBS was added to fix the cells. The control plate was then incubated for 20 min at room temperature and washed three times with cell culture medium. The medium was then removed from each well of both plates (fixed control and live samples) and replaced with 100 μL of medium containing 0.025, 0.05, and 0.1 mg / mL of FNDP-(NV) as indicated and incubated for 0.5–20 h. Both plates were washed three times with Hanks' balanced salt solution with calcium and magnesium (HBSS, ThermoFisher, and Waltham, MA, USA) to remove excess particles. The cells were then lysed by adding 100 μL of 0.5% Triton X-100 and incubating overnight at room temperature on an orbital shaker. Plates were read for FNDP-(NV)-associated NIR signal (excitation 570 nm, emission 670 nm) using a spectrophotometer (Infinite M200 Pro, Tecan AG, Mannedorf, Switzerland). Fluorescence obtained from FNDP-(NV) mounted control plates with PFA-fixed cells was subtracted from the fluorescence measured from live (active) cells.

[0060] Cells were grown in 8-well chamber slides (ThermoFisher) to a maximum of 70% confluence. Cells were treated with 0.05 mg / mL FNDP-(NV), incubated for 2 or 20 h, and fixed in 4% PFA as above. Following cell fixation and permeabilization, cells were stained with FITC-phalloidin as above. The chambers were removed from the slides and mounting was completed using a buffer containing DAPI (Vectashield) and coverslips fixed with nail polish. Slides were then analyzed at 10x or 40x on an FM Olympus IX81 using green, red, and blue filter cubes as described in Fluorescence microscopy of preserved liver slices.

[0061] Data are presented as mean ± SD. Statistical analysis was performed using ANOVA (where appropriate) and SigmaPlot software (SigmaPlot (R) 12 Analysis was performed by Student's t test using SPSS; Systat Software Inc., San Jose CA, USA). Statistical significance was established at P < 0.05 for the number of independent studies performed.

[0062] result Fluorescence microscopy (FM) and panoramic analysis of preserved liver slices Figure 2A shows the distribution of FNDP-(NV) within 5 µm slices of liver tissue imaged at 160x and 400x magnification. Two representative regions are selected; the first (Figure 2A) where vascular elements are present and the second (Figure 2B) an area of ​​only parenchymal cells. The upper panel represents tissue obtained from an animal 12 weeks after intravenous (i.v.) administration of FNDP-(NV), while the lower panel represents tissue obtained from a vehicle (PBS) control animal. FNDP-(NV) (imaged in red, shown in grey shading) can be visualized with DAPI counterstain in the right panel, as identified by white arrows. Large aggregates of 5-10 µm as well as particles of very small size are clearly shown. To assess intra- or intercellular distribution, sections were stained with FITC-phalloidin, as shown in the left panel. A corresponding yellow color (red-over-green) can be visualized for larger aggregates indicating possible endocytosis of the particles (left panel (Figure 2A and 2B)). In Figure 2A, the red fluorescence of very small aggregates is seen close to the nuclei, possibly representing the portal vein (PV) endothelium, but mostly distributed in the parenchyma where it is rather difficult to discern the venous space from the location of the parenchymal cells.

[0063] Figures 3A-3H present the analysis at multiple magnifications of complete sagittal sections from two different FNDP-(NV)-treated rats. Due to the very low effective magnification of the "stitched" images, the red channel, sensitive to the NIR fluorescence of FNDP-(NV), has been expanded by binarization thresholding and dilation, as indicated in the methods. This allows a qualitative visualization even of single pixels of FNDP-(NV). Figures 3A and 3B show particles scattered throughout the complete "panoramic landscape", but with distinct densities evident in their distribution within the core hepatic lobule units. To facilitate visualization, a selected number of hepatic lobules are shown in "hexagons". Particles can also be easily found in the venous system (yellow box). A magnified view suitable for visualization without enhancement, of a set of four hepatic lobules (area indicated by the blue dotted rectangle in Figure 3B), is presented in Figure 3C, showing the obvious heterogeneity of particle distribution within the hepatic lobule. A higher magnification of a single lobule (yellow hexagon from Figure 3A) is shown in Figure 3D. To enhance visualization, higher magnifications of one representative lobule from each animal (indicated by the yellow hexagon in Figure 3A and B) are shown in Figure 3E and F. After thresholding and dilation, a better view of the heterogeneous distribution of particles throughout the "hexagon" formation of the hepatic lobule is easily seen. Although the presence of particles seems concentrated at the periphery of the "hexagon" (in the case of landmarks, red arrows indicate the central vein), some particles are also clearly present next to the central vein. Figure 3G and H show a venous system (yellow square in Figure 3A) with large aggregations of particles (white arrows) that are attached to the wall but protrude significantly into the vessel lumen (visualized by the yellow-red transition), which in two examples occupy 35% and 48% of the vessel cross-sectional area, respectively. Figure 3I provides a scheme of the general orientation of the structure of the hepatic lobule, including the primary metabolic zone.

[0064] The size of FNDP-NV-positive areas in the "panoramic" view of the liver varies greatly, as mentioned above. To quantify this distribution, histograms of FNDP-NV-positive areas are shown in Figure 4. In Figure 4A, the distribution of areas by number shows numerous FNDP-NV-positive areas, ranging from single pixels to areas of 20 µm in diameter. Although few in number and barely visible in Figure 4A, the large aggregates (Figure 3G and H) would represent a disproportionate mass of the total particles detected in the liver section. To represent the percentage of the total particle mass, the distribution of the total FNDP-(NV)-positive area is shown in Figure 4B. In terms of area, the modal diameter of the particle aggregates is approximately 14 µm. In one animal, large aggregates with diameters of 40-100 µm were found in the venous system, accounting for 20% of the FNDP-(NV)-positive area, whereas no aggregates of this size were found in the second animal.

[0065] Confocal fluorescence microscopy (CFM) of preserved liver slices Figures 5A-5D show four different topographical segments studied by CFM in liver sections (10-50 µm). In Figure 5A, several aggregates of perinuclear particles of about 5-10 µm can be seen (yellow circles and arrows), but a clear intracellular location cannot be established. Figure 5B shows intercellular spaces that likely represent portal sinusoids, some of which contain large aggregates of FNDP-(NV) at 10-30 µm (yellow circles and arrows). The dark red color suggests a location well away from the internal environment of parenchymal cells (stained green), but yellow is also present indicating possible at least partial internalization. Figures 5C and 5D show several non-parenchymal structures (surrounded by parenchymal cells), such as veins, arteries, portal veins, and possibly bile ducts. In Figures 5C and 5D, several small particle aggregates (white circles) are located in the subendothelial zone of the vascular intima, while some aggregates present within parenchymal cells (yellow circles) are also noted.

[0066] Figure 6 shows confocal 3D reconstructions of hepatocytes with different amounts of incorporated FNDP-(NV). Two areas with different masses of particles are shown. The cells in the middle panel were barely acquired, while the cells in the right panel appear to be aggregated with very large particles. The left panel represents a rat treated with vehicle, where no particles are visible. In all examples provided, the nuclei and nucleoli of these cells show the same normal phenotype.

[0067] Kinetics of FNDP-(NV) uptake into cultured HUVEC and HepG-2 cells Figures 7A-7D show the kinetics of FNDP-(NV) uptake into HUVEC and HEPG-2 cells under various concentration and time course conditions. Figures 7A-7C represent the time course at three different exposure levels of FNDP-(NV). Each of the exposed doses showed the same pattern of rapid uptake of particles into the cell body. The rapid uptake phase decays within 1-2 h, reaching a plateau proportional to the FNDP-(NV) exposure dose. Figure 7D represents the quantitative accumulation of FNDP-(NV) monitored by NIR fluorescence for each cell line at three concentrations of FNDP-(NV). The difference in the total accumulated FNDP-(NV) is statistically significant between exposure levels but similar between cell lines.

[0068] Figures 8A and 8B are FM micrographs of FNDP-(NV) accumulation at early and late stages of the in vitro experiment. Early stages (up to 2 h) show particles mainly in the cytoplasm, while the final time point (20 h) shows heavy aggregation in the form of a perinuclear corona. Such a pattern was also recorded in preserved liver slices (12 weeks after exposure), as shown in Figure 6C.

[0069] Images of HUVECs in early mitosis through the end of cytokinesis are shown in Figure 9A for cells treated with FNDP-(NV) for 20 hours and in Figure 9B for untreated control cells. All treated cells show heavy perinuclear FNDP-(NV) accumulation, including late cytokinesis and cell separation. Similar observations were made in the control group.

[0070] Consideration The total distribution of high doses (60mg / Kg) of FNDP-(NV) injected into intact rats was characterized and trends followed over acute (90 min), subacute (5 or 14 days), and chronic (12 weeks) periods following FNDP-(NV) exposure. Analysis of particle distribution across six organs (liver, spleen, lungs, kidneys, heart, and brain) confirmed that the liver was the primary storage organ for these particles. Evaluation of organ histology revealed no gross or histopathological adverse effects associated with FNDP-(NV). The lack of adverse effects associated with FNDP-(NV) is consistent with reported normal liver function tests.

[0071] As mentioned above, persistence of large numbers of FNDP-(NV) particles in the liver raises concerns about potential adverse effects, especially in the context of long-term residence, and possibly indefinite presence. Such a prospect could raise regulatory hurdles and impact GLP (Good Laboratory Practice) preclinical development for human use. Although the aforementioned certified pathology report confirmed the lack of histopathological findings, this study aimed to address three main objectives: 1. Comprehensive investigation of FNDP-(NV) distribution in various hepatocytes, including their subcellular location; 2. Localization of FNDP-(NV) in the microvasculature of hepatic lobules; 3. Exploring the kinetics of FNDP-(NV) particle uptake into cultured hepatocytes and their subcellular distribution, using surrogate cell cultures such as HUVEC (endothelial) and HepG-2 (human hepatoma) cells.

[0072] The main outcomes of this study are: 1. Definition of a unique pattern of spatial distribution of FNDP-(NV) in hepatic lobules including parenchymal cells (hepatocytes), non-parenchymal cells (vascular endothelial and adventitial cells) and venous supplying (portal vein) and draining (central venous) systems. 2. Demonstration of intracellular uptake and compartmentalization of FNDP-(NV) in hepatocytes in vivo and in vitro. 3. Confirmation of preservation of normal macro- and micro-morphological phenotype of hepatocytes including cells with large corona of particles in the perinuclear space. 4. Preservation of viable cytokinesis process from anaphase to completion of cytokinesis and cell replication including cells with extensive perinuclear corona.

[0073] The distribution of FNDP-(NV) throughout the complete "panoramic" display (Figures 3A-3B) revealed a repetitive pattern found throughout the "hexagonal" lobules of the liver (see Figures 3D, 3E, and 3F). Particle aggregates were found in greater abundance at the periphery of the hepatic lobule surrounding the "portal triad" (PT), but were considerably scarcer in more proximal regions near the CV. Although the mechanism for such distribution is currently unclear, it is hypothesized that this kind of spatial distribution of FNDP-(NV) throughout the hepatic lobule may be the result of several converging factors.

[0074] First, delivery of FNDP-(NV) via the PV frequently presented aggregated particles with sizes barely fitting or exceeding the diameter of the sinusoids. As shown in Figure 4A, 30-40% of the detected FNDP-(NV) aggregates were larger than 7 µm, so, without wishing to be bound by theory, they tended to be mechanically trapped in the more proximal parts of the sinusoids. Although this did not represent the majority of the FNDP-(NV)-positive area by number, these particles accounted for 75-85% of the total FNDP-(NV)-positive area (Figure 4B), which may explain the strong fluorescence bias within the lobule, even though a substantial number of small aggregates (individual or limited replicates) migrate further down the sinusoids, cross the sinusoids, and recirculate in the systemic circulation. The presence of small particles at the entrance of the sinusoids to the central vein (CV) supports this possibility (see Figures 3D and 3G).

[0075] Second, Kupffer cells, which provide the scavenging function of the liver (reticuloendothelial system, RES), are generally abundant in the sinusoids and even more abundant in the proximal zone of the sinusoids exiting the PV. These macrophage-like cells rapidly remove particles with preferential kinetics for larger particles over smaller ones, enhancing their deposition more proximal to the PV on the CV zone in the case of FNDP-(NV) as shown by the data.

[0076] Third, the sinusoidal / venular terminal zones are generally more “spacious” than the sinusoidal exit from the PV. Such an anatomy supports hemodynamic conditions and facilitates clearance of particles into the CV and further into the systemic circulation, thereby contributing to the relative paucity of particles near the CV.

[0077] Fourth, the venous microcirculation is a key element in ensuring the most delicate biochemical functions of the hepatic lobule. The data presented herein clearly show the presence of large particle aggregates in the PV and possibly the CV, together with their enhanced presence at the periphery of the hepatic lobule (peri-PT), and few but notable small particles throughout the lobule (see Figure 3E). Particles within these spaces may disturb the delicate balance of blood flow in the sinusoids, causing hemodynamic disturbances (e.g., turbulence) or congestion that impedes flow. Interruptions in flow may affect the supply of oxygen and the distribution of nutrients to parenchymal cells, thereby negatively affecting the synthetic and catabolic functions of the liver. Although we cannot directly exclude microhemodynamic disturbances of the sinusoids, in a detailed histological analysis (Supplementary Material) we could not observe areas of blood congestion (due to partial blood flow interruption), thrombosis (due to stagnation), or ischemic consequences at the microscopic level.

[0078] Nevertheless, the topographical heterogeneity of FNDP-(NV) distribution may still have physiological consequences. Due to particle mass or size, subcellular localization, and immature microhemodynamic factors (at the end of the study), we show aberrant consequences on the anatomy and physiology of the entire liver unit. The peripheral zone of the liver lobule, where larger aggregates of particles were most common (see zone 1 in Figure 3H), is the location of many important biochemical and cell survival functions in the liver (e.g., fatty acid oxidation, gluconeogenesis, bile production, xenobiotic metabolism, and regenerative cell recruitment).

[0079] Support for possible preservation of liver morphology in the microenvironment is shown in Figures 6A and 6B. Topological surveys over panoramic fields of the entire liver surface suggest that the percentage of particles and the area they occupy are only a small (or moderate) fraction of the total. As the data presented in this manuscript evaluates a situation that occurred 12 weeks prior, acute exposure after FNDP-(NV) cannot be rejected.

[0080] Finally, cell culture studies were performed to gain insight into the direct interaction of liver cells with FNDP-(NV) in an isolated system and to investigate the kinetics of particle internalization, compartmentalization, and viability of cytokinesis competence in the presence of particles. Two different cell types used as surrogates for human hepatocytes and endothelial cells, respectively, showed a rapid initial uptake of FNDP-(NV) into cells in a time- and concentration-dependent manner. This in vitro study supports the in vivo observations of cellular uptake of FNDP-(NV) into non-scavenging liver cells (hepatocytes).

[0081] summary In this work, we investigated the interaction of FNDP-(NV)-Z-800nm ​​with liver cells in vitro and in vivo. These studies addressed the magnitude and extent of FNDP-(NV) deposition in terms of cellular and subcellular resolution, parenchymal and non-parenchymal cells, as well as their presence in the microcirculation. The in vivo data were complemented by studies performed in vitro (HUVEC, HepG-2 cells), where direct kinetic studies of particle uptake and assembly in these surrogate hepatocytes corroborated the results obtained from whole animal exposure studies. Taken together, the above data strongly suggest the hepatic biocompatibility of FNDP-(NV). This is because we were unable to identify any aberrant outcomes in terms of preservation of cell phenotype, cytoskeleton, and nuclear architecture, as well as reduced cytokinesis and cell replication. Thus, FNDP-(NV) may be well tolerated by humans exposed to FNDP-(NV) by the intravenous exposure route.

[0082] Example 2 The following example shows the effect of FDP-NV~800 on the pharmacokinetics of cultured human umbilical vein endothelial cells (HUVEC) and human hepatoma cell line (HepG-2) exposed in vitro to FDP-NV~800 at exposure levels within the pharmacokinetics (Cmax and nadir) reported in vivo. Describe the cellular and biochemical functions.

[0083] Nanomedicine is a rapidly growing medical field characterized by strong preclinical research and emerging clinical discovery research, as evidenced by over 25,000 articles listed in PubMed in the past decade. Nanomedicine offers a "third leg" of pharmaceutical technology beyond synthetic organic molecules and engineered organisms. Nanomedicine is built on diverse materials combined with additives aimed at targeting biologically active nanoparticles to specific cells, organs, or pathological processes.

[0084] Of primary contemporary interest are particles engineered to emit near-infrared light in response to electromagnetic stimuli (excitation light) producing fluorescence, either through intrinsic properties (e.g., "color centers") or through coating with organic fluorescent additives. The ability to emit in the NIR opens up the possibility of imaging of body structures themselves, or as an adjunct to cutting-edge imaging techniques (e.g., MRI / magnetic resonance imaging, ultrasound, etc.), as well as targeted delivery of therapeutic agents.

[0085] Of particular interest are diamond particles, such as nanodiamond particles and fluorescent diamond particles, that possess nitrogen-vacancies (FDP-NV-), making them fluorescent upon excitation at 580-620 nm and capable of producing near-infrared (NIR) emission in the peak range of 720-740 nm. The NIR light emission of such particles exhibits exceptional stability and negligible interference by biological elements such as water and oxyhemoglobin. Furthermore, the surface of these particles can be functionalized with a variety of chemical groups (carboxyl, amine, etc.) that offer a variety of conjugation opportunities, ranging from small organic molecules to polymers, proteins, and nucleic acids.

[0086] Within the context of this disclosure, a bioengineered fluorescent diamond particle-NV-Z~800nm ​​(FDP-NV) conjugated with the snake venom disintegrin, bitistatin (Bit), was discovered and FDP-NV~800nm / Bit was shown (in vitro and ex vivo) to specifically bind to the platelet fibrinogen receptor αIIbβ3 integrin. In vivo studies then demonstrated the binding of FDP-NV-Bit to acutely generated (iatrogenic) clots in rat carotid arteries. Taken together, FDP-NV~800nm / Bit demonstrated target homing in vivo and thus potentially served as a diagnostic tool for high-risk vascular thrombosis.

[0087] The study was followed by three safety and biocompatibility studies in which a high dose (60 mg / Kg, delivered as a single intravenous bolus) of FDP-NV-800 nm blocked by BSA (FDP-NV) was injected into intact rats to establish the pharmacokinetic profile, organ distribution, and evaluate a comprehensive panel of hematological, metabolic, and biochemical safety biomarkers. These studies found that within a follow-up period of 5 days to 12 weeks, FDP-NV was distributed primarily to the liver and spleen, with little evidence found in the lungs, heart, and kidneys. Furthermore, no specific histopathological observations associated with FDP-NV particles were observed. However, so far, no studies have addressed the potential acute safety or toxicological effects in endothelial or hepatic parenchymal cells exposed to FDP-NV-800 nm.

[0088] In this example, we investigated the search for possible direct toxic effects associated with FDP-NV-800nm ​​using two different cell types, HUVECs and HepG-2. These cells were chosen because endothelial cells are the first exposure to FDP-NV when injected into the systemic circulation, and hepatocytes are the main reservoir of circulating FDP-NV. FDP-NV exposure levels were chosen according to the acute pharmacokinetic levels observed in vivo, including the maximum blood level and its nadir at 90 min after exposure. Considering that no acute biocompatibility studies with FDP-NV-800nm ​​have yet been reported in the published literature, we hope that the work presented here provides new information and insights into the acute biocompatibility of FDP-NV, supporting its intended clinical development in humans.

[0089] Overall, the data indicate that C max This supports the reasonable biocompatibility of FDP-NV-800nm ​​with respect to proliferation over a short period of exposure. HepG-2 was unaffected with the same exposure and time.

[0090] method Diverse cellular and biochemical functions were monitored, which, in summary, provide insight into cellular integrity and vital functions. Cell proliferation, migration, and regeneration were assessed by quantitative microscopy. Mitochondrial (oxidative) function was tested by MTT redox reaction and cytosolic esterase activity was studied by calcein AM assay. ER-stress biomarkers were examined by chaperones CHOP and BiP, and apoptosis was examined by caspase-3 activation using Western blot (WB). MAPK Erk1 / 2 signaling was assessed by detection of protein phosphorylation (P-Erk 1 / 2) and translocation to the cell nucleus.

[0091] result Exposure of HUVEC cells to 100 μg / mL FDP-NV (Cmax) suggested potential adverse effects on cell proliferation, cytosolic esterase activity, and oxidative function. Cell signaling (MAPK Erk1 / 2) and ER-stress biomarkers remained intact as did activation of pro-apoptotic pathways (caspase 3 activation). At similar exposure and time frame, no abnormal tests were observed in HepG-2 cells, which showed resilience in several studies at several exposure levels.

[0092] Materials and Methods Preparation of nanoparticles Carboxyl-functionalized FDP-NV~800 nm (FDP-NV) was purchased from ADAMAS Nanotechnologies (Raleigh, NC, USA). FDP-NV was sterilized by suspending in 70% ethanol at room temperature (RT) for 15 min, followed by centrifugation at 2900 x g for 7 min at RT to isolate the particles. Passive blocking of potential nonspecific protein binding sites on the particles was performed by incubation at 37 °C for 1 h in PBS (phosphate buffer dosarin, pH = 7.4, ThermoFisher Sci., Waltham, MA, USA) containing 3% BSA (bovine serum albumin, Sigma, St Louis, MO, USA). FDP-NV-BSA was isolated by centrifugation as described above, and the particles were stored at 40 °C as a stock solution of 1 mg / mL in PBS.

[0093] Analysis of Z-average and Zeta potential of FDP-NV-800nm ​​in different dispersants Particles blocked with BSA (FDP-NV-BSA) or "naive" (FDP-NV-COOH, pre-BSA blocking) according to various protocols used in cell experiments (see below). The particles were suspended in deionized (DI) water, PBS, or culture medium. HepG-2 (human hepatoma) cells were cultured in Eagle's minimum essential medium (EMEM, ThermoFisher Sci.) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher Sci.) and penicillin / streptomycin (ThermoFisher Sci.), and HUVECs were cultured in EGM-2MV medium (Lonza, Basel, Switzerland). The particles were suspended in each medium as a dispersant at a density of 0.5 mg / mL and placed in a dual-purpose capillary cuvette (total volume 1 mL). Samples were tested on a Zetasizer Ver. 7.11 (Malvern Panalytical Ltd., Malvern, UK).

[0094] Cell counting assay Cell counts were performed. The HepG-2 cell line was purchased from American Type Culture Collection (ATTC) (Manassas, VA, USA). Primary HUVECs were purchased from Lonza and used for experiments between passages 5 and 8. Cells were maintained in their respective media (37 °C in a 5% CO2 atmosphere) as described above. HepG-2 and HUVECs were "seeded" into 96-well plates (2 x 10 per well in 100 μL media). 3 ), FDP-NV-BSA, or left untreated for 24 h. In each experiment, vincristine (50 μg / mL), a cell cycle proliferation inhibitor, was added as a positive control. After 24 h, the medium was removed, cells were fixed with 4% paraformaldehyde (PFA, ThermoFisher Sci.), and nuclei were stained using DAPI (4',6-diamino-2-phenylindole, dihydrochloride, ThermoFisher Sci.). Plates were analyzed by imaging seven observation fields per well under a fluorescence microscope (Olympus IX81, Olympus, Tokyo, Japan) using 100x magnification and DAPI (blue filter) for visualization of nuclei and TRITC (red filter) for visualization of FNDP-NV-BSA. The number of live cells in each field was determined by analysis of DAPI-stained nuclei using ImageJ software (National Institutes of Health, Bethesda, MD, USA) with a cell counter with digital settings.

[0095] Cell metabolic activity monitored by MTT assay The MTT assay was performed as a colorimetric assay using a cell proliferation assay kit (ThermoFisher Sci.) consisting of component A (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) and component B (SDS (sodium dodecyl sulfate)) in accordance with the manufacturer's protocol. Briefly, HepG-2 cells and HUVECs were plated in 96-well plates at 1x10 cells per well in the medium described above for each cell type. 4Cells were seeded at a density of 1000 x g / mL. Cells were treated with FDP-NV-BSA or vincristine (50 μg / mL) for 24 h or left untreated. The medium (100 μL) was changed to phenol red-free DMEM (Dulbecco's modified Eagle's medium) containing MTT component A (ThermoFisher Sci.). Plates were incubated for 4 h and cells were lysed by adding an equal volume of 10% SDS (kit component B). Plates were incubated overnight and read at a wavelength of 562 nm using an ELISA plate reader ELx800 (BioTek, Winooski, VT, USA).

[0096] Calcein AM cell cytosolic esterase assay HepG-2 cells and HUVECs were seeded and treated as described above for the MTT assay. Cells were treated with 5 μg / mL calcein AM (ThermoFisher Sci.) in serum-free medium and incubated for 30 min at 37°C. Plates were read using a fluorescence microplate reader FLx800 (BioTek) with excitation at 485 nm and emission wavelengths at 530 nm.

[0097] "Wound Healing" (WH) In Vitro Assay HUVECs were seeded in 12-well plates and maintained for 1–2 days until 80–90% confluency, then treated with FNDP-NV-BSA for 24 h or left untreated. Confluent HUVEC cells (monolayer) were subjected to "gentle scraping" across the plate using a plastic spatula tip, resulting in a gap area (free of cells) approximately 1 mm wide. FNDP-NV-BSA-treated cells were stimulated for 24 h migration time by replacing the medium with one containing 2% FBS. Control cells (not exposed to FNDP-NV-BSA) were divided into positive, stimulated by 2% FBS, and negative, in which the stimulator of migration was minimized to 0.1% FBS (HUVECs are sensitive to the complete removal of FBS and detach from the surface). Cells were fixed with 4% PFA and stained with DAPI as described above. Imaging of the scratches was performed under a fluorescence microscope (Olympus IX81) at 20x magnification with DAPI (blue filter) for visualization of nuclei and TRITC (red filter) for visualization of FNBDP-NV. Control plates contained confluent cells that had received the same scratch immediately before PFA exposure. Migration index was estimated by measuring the total surface area of ​​the cell-free region of the images using ImageJ software.

[0098] Cell signaling assays expressed by phosphorylation of MAPK Erk1 / 2 MAPK Erk1 / 2 HepG-2 cells and HUVECs were cultured in 6 cm diameter Petri dishes to 90% confluence and treated with FDP-NV-BSA (density 0.1 mg / mL) as above or left untreated. Cells were serum starved for 24 h and then stimulated with 2% FBS for 0, 10, and 20 min. Cells were lysed in ice-cold RIPA (radioimmunoprecipitation assay) buffer (Teknova Inc., Hollister, CA, USA) containing "cocktail" of protease inhibitors (Sigma Inc.) and "Halt" phosphatase inhibitor cocktail (ThermoFisher Sci.).

[0099] Protein lysates (20 μg) were applied to SDS-PAGE (sodium dodecyl sulfate, polyacrylamide gel electrophoresis) using Mini-PROTEAN precast gradient (4-20%) gels (Bio-Rad Inc., Hercules, CA, USA) and transferred to PVDF (polyvinylidene difluoride) membranes (Sigma Inc.) using a semi-dry blotting system (Bio-Rad Inc.). The presence of phospho- and total Erk1 / 2 (after "stripping" the membrane) was detected using a polyclonal antibody (Cell Signaling Techn., Danvers, MA, USA). Visualization of protein bands on the membrane was performed using a C-DiGit Blot Scanner (LI-COR Biosci., Lincoln, NE, USA). Band intensity was quantified using UN-Scan-It software (Silk Scientific Corp., Orem, UT, USA), and the ratio of phosphor-Erk1 / 2 to total Erk1 / 2 was calculated.

[0100] Nuclear translocation of phospho-Erk1 / 2 Fractionation of cell lysates HepG-2 cells and HUVECs were grown in 6 cm diameter dishes and treated with FDP-NV (0.1 mg / mL) or left untreated and "starved" under the same conditions as described above for MAPK Erk1 / 2 cell signaling. Cells were stimulated by exposure to 250 nm TPA (tetradecanoylphorbol acetate, Sigma Inc.) for 15 min. Cell fractionation was performed using the Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher Sci.) according to the protocol provided by the manufacturer. Cytoplasmic and nuclear extracts of the fractions were analyzed by WB using phospho- and total MAPK Erk1 / 2 as described above. Validation of the cytoplasmic and nuclear fractions was performed by WB analysis using anti-Mek and anti-HDAC1 polyclonal antibodies (Cell Signaling Techn.), respectively.

[0101] Immunocytochemistry for detection of phosphorylated MAPK Erk1 / 2 in the cytoplasm and nucleus Immunostaining of cells cultured on 8-well glass slides was performed as described previously. Cells were treated with 0.1 mg / mL FDP-NV-BSA in the presence or absence of TPA or left untreated. Polyclonal anti-phosphorylated Erk1 / 2 antibody (Cell Sign. Techn.) was used in combination with FITC-tagged goat anti-rabbit IgG (Vector Labs Inc., Burlingame CA, USA). Slides were analyzed using a fluorescence microscope (Olympus IX81) at 400x magnification with an oil objective and FITC (green filter) for the detection of phosphorylated MAPK Erk 1 / 2 and TRITC (red filter) for the detection of FNDP-NV.

[0102] Cell apoptosis assay (caspase-3) and endoplasmic reticulum (ER)-stress biomarkers HepG-2 cells and HUVECs were treated with 0.1 mg / mL FDP-NV-BSA (or left untreated) as described above. Treatment with vincristine (200 μg / mL) was used as a positive control for apoptosis, and treatment with tunicamycin (25 μg / mL) was used as a positive control for endoplasmic reticulum stress. A rabbit polyclonal antibody against caspase 3 (Cell Sign. Techn.), which recognizes both cleaved and uncleaved proteins, was used to detect apoptosis. A rabbit mAb against BiP (clone C50B12) and a mouse mAb against Chop (clone L63F7) (both from Cell Sign. Techn.) were used to detect endoplasmic reticulum stress. Equal loading of proteins was verified by membrane stripping and reprobing with anti-actin mouse monoclonal antibody (Sigma Inc.).

[0103] result Physical properties of FDP-NV~800nm ​​suspended in various seed media FDP-NV ~800 nm was suspended in various dispersants known to alter particle size (Z-average) and surface zeta potential. Figure 10 shows the change in diameter (Z-average) of FDP-NV-COOH (native particles without passive absorption of BSA) or FDP-NV-BSA suspended in DI water, PBS (pH = 7.4), or the media used for cell culture, respectively. A substantial and statistically significant increase in Z-average was observed when FDP-NV-COOH was suspended in PBS; particle size increased from 778 nm (DI water suspension) to 1488 nm (PBS), 1215 nm (HUVEC media), and 1403 nm (HepG-2 cell media), respectively. Passive absorption of BSA at the surface of the particles (FDP-NV-BSA) neutralized the increase observed in the respective solutions. Zeta potential was substantially affected (e.g., Figure 10B) with a trend toward a more positive charge toward the isoelectric point. The zeta potential of FDP-NV~800 nm-COOH dispersed in DI water was -47.9 mV, but increased to -21.9 mV when the particles were immersed in PBS, -9.9 mV in HUVEC medium, and -10.9 mV in HepG-2 cell medium. However, unlike its effect on the Z-average, passive coating of FDP-NV with BSA had only a minimal effect on the zeta potential of FDP-NV-COOH. It is noteworthy that the medium used for HUVEC or HepG-2 made no difference in its effect on either the Z-average or zeta potential.

[0104] Effect of FDP-NV on HUVEC or HepG-2 cell proliferation The HepG-2 cell line was not affected by the presence of FDP-NV-BSA (up to 0.1 mg / mL) as inferred from the increase in cell number over 24 h (Figure 11A). In contrast, HUVECs exposed to high concentrations of FDP-NV (0.1 mg / mL FDP-NV-BSA) showed a statistically significant decrease in cell number to approximately 60%. No effect was observed after exposure of HUVECs to lower concentrations (1 / 10) of particles (Figure 11). As expected, vincristine inhibited proliferation to 50% and 80% of control in HepG-2 and HUVECs, respectively. Representative images of cells treated with 0.1 mg / mL FDP-NV-BSA for 24 h confirmed particle accumulation in cells and perinuclear aggregation, especially in HUVECs (Figure 11D). Similarly, although less pronounced, HepG-2 cells also showed accumulation of FDP-NV-BSA in the cytoplasm and formation of a perinuclear corona (e.g., Figure S11C), an observation consistent with recent studies in both cell types.

[0105] Effect of FDP-NV-BSA on the redox intensity of cultured HUVEC or HepG-2 cells The redox status of cultured HepG-2 cells, assessed by MTT, was resilient to the presence of FDP-NV-BSA at several concentrations, including Cmax (0.1 mg / mL, Figure 12A). In contrast, HUVECs showed reduced oxidative capacity at exposure levels within the Cmax and nadir of pharmacokinetic plasma concentrations (0.01 mg / mL). However, at the lower tested concentration of FDP-NV-BSA (0.001 mg / mL), MTT activity was indistinguishable from that of untreated controls (Figure 12B). The positive control, vincristine, reduced redox activity to approximately 25-30% of normal controls for both cell types.

[0106] Effect of FDP-NV-BSA on cytosolic esterase activity in HUVEC or HepG-2 cells The calcein AM assay provided information on cytosolic nonspecific esterase activity. Figure 13 shows no deviations from this test in HepG-2 cells (Figure 13A), whereas HUVECs (Figure 13A) showed a reduction of about 30% at a concentration of 0.1 mg / mL of FDP-NV and no interference at the nadir level of exposure (0.01 mg / mL).

[0107] Effect of FDP-NV-BSA on HUVEC migration stimulated by FBS in an in vitro "scratch" injury model The effect of FDP-NV-BSA on cell migration was investigated in an in vitro model of “wound healing” (“scratch assay”, Figure 14). This assay was applied only to HUVECs, as the growth pattern of HepG-2 (forming clusters of colonies) was not suitable for this test. Quantification of cell migration across an artificially generated cell-free area (area of ​​the scratch) revealed no differences between control, untreated, and FDP-NV-BSA-exposed cells. HUVECs treated with 2% FBS migrated readily even when exposed to a high concentration of particles (0.1 mg / mL, Figure 14A). Interestingly, fluorescence microscopy images revealed visually similar particle loading of internalized FDP-NV-BSA (indicated by different shades of grey, with overlapping blue and red colors) in active cells (migrating into the “scratch zone”) and in “quiescent” cells located outside the scratch zone (Figure 14B).

[0108] Effect of FDP-NV-BSA on activation of MAPK Erk1 / 2 in HUVEC and HepG-2 cells Figure 15 shows no significant difference in FBS-induced activation of MAPK Erk1 / 2 between HUVEC and HepG-2 cells exposed or not to 0.1 mg / μL FDP-NV-BSA at two time points (10 and 20 min) after stimulation. Interestingly, HepG-2 cells reached a plateau of FBS stimulation at 10 min (Figure 15A), whereas HUVEC reached maximum phosphorylation of MAPK Erk1 / 2 at 20 min (Figure 15B).

[0109] Protein translocation from the cytosol to the nucleus is one of the paradigms that could be affected by the strong perinuclear accumulation of FNDP-NV. Therefore, translocation of phosphorylated MAPK Erk 1 / 2 to the nucleus was tested using TPA, an applied stimulator of this process. For this, cells were fractionated and phosphorylated and total MAPK Erk1 / 2 in the cytoplasmic and nuclear fractions was evaluated by WB (Figure 16A-16B) and fluorescence microscopy (Figure 16C-16D). HepG-2 cells (Figure 16A) and HUVEC (Figure 16B) showed no difference between FND-NV-BSA-exposed and control (no exposure) cells in the amount of phospho / total MAPK Erk1 / 2 in the nucleus or cytoplasm, respectively. Note that exposure to TPA enhanced the internalization and perinuclear accumulation of FDP-NV-BSA, which can be observed in fluorescence microscopy images as an intense yellow color (overlap of red and green (Figures 16C-16D)).

[0110] Effect of FDP-NV-BSA on the induction of apoptosis and endoplasmic reticulum stress The internalization into the cytoplasm and perinuclear accumulation of FDP-NV-BSA may suggest a possible interference with the essential traffic between the nucleus and the cytosol, leading to a stress state manifested by activation of apoptosis or endoplasmic reticulum stress. Therefore, using WB, both HepG-2 cells and HUVEC biomarkers were assessed for stress conditions, such as activation of caspase 3 and the expressed chaperone proteins CHOP and BiP (Figure 17). Exposure to FDP-NV-BSA (0.1 mg / mL) did not result in activation of caspase 3 in either cell, in contrast to vincristine (positive control, Figure 17A). The strong perinuclear accumulation of FDP-NV seems to persist without any impact within the experimental time. The expression of the two chaperone proteins CHOP and BiP was also not affected by the presence of FDP-NV. Moreover, there was no obvious difference between HepG-2 and HUVEC (Figure 17B). Both cell types were sensitive to tunicamycin, which served as a standard control for activation of endoplasmic reticulum stress proteins.

[0111] Consideration The current series of experiments aimed to investigate the safety of FDP-NV(800nm) and constitutes part of the preclinical evaluation of these particles before initiating Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP) for Phase I clinical trials. The safety, tolerability, and high biocompatibility of FDP-NV-800nm ​​administered intravenously at relatively high doses to intact rats was seen as inferred from the lack of morbidity and mortality monitored for 5 days, 14 days, or 12 weeks after exposure. In all of these studies, no abnormal hematological and biochemical functions, including blood cell counts and differentials or histopathological observations in the liver, kidney, and lungs, were detected. Furthermore, the pharmacokinetic range after acute injection of high doses of FDP-NV-800nm ​​showed rapid clearance from the systemic circulation and rapid rise to the liver and spleen. It is important to note that the particles deposited in the liver and spleen were retained over the 12-week follow-up period.

[0112] The current experiments were designed to address the potential adverse effects of FDP-NV-800 nm in an acute in vitro setting to gain deeper insight into potential biochemical outcomes that could not be identified by histological and histochemical biomarkers in vivo. Such studies are justified as we could not find any record of published data investigating the same FDP-NV size (~800 nm). Furthermore, acute safety biomarkers may not be visible in subacute or chronic dosing studies due to compensatory mechanisms following exposure.

[0113] Adverse interactions of nanodiamond particles with cellular functions have already been reported, despite the use of different particle sizes, shapes, and adjuvants. These reports highlight the importance of investigating the effects of FDP-NV~800nm ​​on cellular functions, especially those of cells exposed to maximum blood concentrations (Cmax) during and immediately after particle injection. Not surprisingly, endothelial cells and circulating blood cells are the primary targets of acute high-dose exposure, as are liver cells, which serve as the immediate repository of FDP-NV~800nm. Indeed, pilot studies with FDP-NV~800nm ​​using HUVEC and HepG-2 cells revealed the uptake of particles into each of these cells' cytoplasm (over 1-2 hours) and the eventual assembly of a perinuclear zone to form a "corona" over 20-24 hours after exposure. However, cytogenesis and cell division of these target cells were maintained, suggesting that the cell cycle and transport into and out of the nucleus remained intact.

[0114] The present study extends the observations to probe additional important cellular functions and biochemical processes, including cell proliferation, migration, signaling endoplasmic reticulum stress, and apoptosis, which are keystones of cellular integrity. The present study followed pharmacokinetic data obtained after high dose (60mg / Kg) injection in in vivo (rat) experiments. In the study described herein, cultured cells were exposed to Cmax levels (immediately after injection, 0.1mg / mL) or nadir (0.01μg / mL, 90 minutes after injection) over a 24 hour period.

[0115] We addressed the well-known issue of size changes of FDP-NV-COOH when suspended in solutions containing electrolytes, proteins, and various organic additives. Indeed, monitoring the Z-average and zeta potential of FDP-NV~800 nm-COOH in DI water (native product provided by the manufacturer) revealed a close similarity with the manufacturer's information (778 nm for Z-average and -48 mV for zeta potential). The pronounced shift in Z-average produced by dispersing the particles in PBS or medium was abolished in the presence of 3% BSA, while there was only a minor (or negligible) effect on the zeta potential shift (Figures 10A-10B). The potential impact of persistent zeta potential changes on the experimental results needs to be investigated in detail.

[0116] The cellular effects of nanodiamond particles (NDPs) have been intensively investigated in vitro using various cultured cell types, mainly in terms of cell viability, as reported by the MTT assay. In general, NDPs are well tolerated by most cell types when cultured in complete medium. The mitochondrial-dependent respiratory chain is not affected by NDPs, even at very relatively high concentrations of 1 mg / mL. Capping exposure at a Cmax concentration of 0.1 mg / mL suggested no interference with the redox status of the HepG-2 cell line (Figure 11), in agreement with previous reports on other cancer cell lines. In contrast, a significant inhibitory effect was observed in HUVECs (Figure 11B), in agreement with previous data using the immortalized HUVEC-ST cell line. Direct cell counting and calcein AM assays also suggested interference of the particles with cytosolic esterase activity in HUVECs at 0.1 mg / mL, but in contrast to the MTT, no effect was observed at the lower (nadir) level of 0.01 mg / mL. These data suggest that primary cells, in contrast to cell lines, may be more susceptible to FDP-NV in terms of important biochemical processes and overall cellular functions.

[0117] The proliferation-promoting cell signaling pathway MAPK Erk 1 / 2 was not affected by exposure to the Cmax dose of FDP-NV in either cell type (Figure 15). The extensive perinuclear accumulation of particles suggested a potential interference by this "corona" in cytosol-nuclear cross-transport. This possibility was investigated by following the translocation of phospho-Erk1 / 2 to the nucleus. Figure 16 shows the presence of P~phospho-Erk1 / 2 in the nucleus following activation of this signaling pathway by the potent agonist TPA. Similarly, FDP-NV did not activate the central pathway of apoptosis (caspase-3) at either FDP-NV concentration (Figure 17). Taken together, the data suggest that the physical presence of FDP-NV-BSA appears to expose HUVECs to some stress conditions at Cmax, but not at nadir exposure levels. However, the ability of HUVECs to fully migrate even at the highest FDP-NV exposure (see Figure 14) suggests a discrepancy in functional sensitivity to intracellular particle loading.

[0118] HepG-2 cells appear to be generally more resilient in several tests compared to HUVECs. In some cases, a negative effect of NDPs on HepG-2 cell migration was observed at the same exposure levels used in the HUVEC "wound healing" model ("scratch assay"). For example, exposure of HepG-2 cells to 50-100 μg / mL of FNDs resulted in 25-50% inhibition of migration, with further inhibition (90%) at 200 μg / mL over the same time frame (24 h). It is interesting to note that these exposure levels did not interfere with HepG-2 cell proliferation, consistent with the data reported herein (Figure 10). The differences between the two data sets could represent differences in particle size (100 nm vs. 800 nm) and physical properties of the non-functionalized NDPs of some existing systems vs. the carboxy-functionalized FDP-NV used in this disclosure.

[0119] In vitro studies showed that either cell type was exposed to the particles over a 24-hour period. However, pharmacokinetic data from some existing systems indicate that in vivo endothelial cells are exposed to Cmax levels of FDP-NV within 15–30 minutes, as blood levels decline to <10 μg / mL within 90 minutes after particle injection due to rapid clearance into the liver. In this light, this indicated that FNDP-NV ~800 nm is observed in the cytosol of hepatocytes within 1–2 hours after cell exposure. However, within the relevant short in vivo exposure time, intracellular levels of the particles are several-fold lower compared to the long-term (24 hours) in vitro fixed FDP-NV concentrations. The resilience of the HepG-2 strain across all conditions of stress lends credibility to the in vivo observations of hepatocyte health after high-dose FDP-NV-800 nm injection into rats over a 12-week period.

[0120] In summary, FDP-NV-800 nm had no adverse effects when injected in vivo into intact rats18-20, nor in the cultured HepG-2 cell line across the seven "stress tests" that these cells underwent in vitro. In some cases, we could not exclude abnormal outcomes related to immune-inflammatory or other cells / organs, especially those with high volumes of phagocytosis or priming effects that may exacerbate highlighting pathological conditions. The results obtained in this study indicate that further development of FDP-NV-800 nm for in vivo imaging and as a vehicle for the delivery of drugs and therapeutics may be justified.

[0121] summary This example shows the biocompatibility of FDP-NV~800nm ​​on endothelial (HUVEC) and hepatic (HepG-2) cells in vitro. This study seems to differ from existing systems in that it investigates the biocompatibility within the realm of the particle's pharmacokinetics in vivo (rat model). It can be concluded that HUVECs are more sensitive to FDP-NV~800nm ​​accumulation than HepG-2 cells; this observation is not described for a negative response at the highest exposure level (Cmax). Considering the mild to moderate interference in certain biochemical functions of HUVECs, it is plausible to predict limited abnormal consequences on the endothelium in light of the pharmacokinetic profile that the particles show in vivo. The resilience of HepG-2 cells in all of the biochemical tests at the highest dose of FDP-NV~800nm ​​supports the in vivo data on normal liver function, despite the long-term retention of the particles in this organ. Overall, the results obtained in this example indicate that FDP-NV-800 nm may be useful for in vivo imaging and / or as a vehicle for the delivery of drugs and therapeutics.

[0122] While several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize, without more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Many equivalents equivalent to the specific embodiments of the invention described herein will be recognized, or can be ascertained using no more than routine experimentation. Thus, the foregoing embodiments have been presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more of such features, systems, articles, materials, and / or methods is included within the scope of the present invention, if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0123] As used herein, in the specification and claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless clearly indicated to the contrary.

[0124] In the present specification and claims, the term "and / or" as used herein should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements than the elements specifically identified by the "and / or" clause may be optionally present, whether related to the elements specifically identified or not, unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and / or B", when used in combination with open-ended language such as "comprises", can refer to, in one embodiment, A without B (optionally including other elements); in another embodiment, B without A (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements); and so forth.

[0125] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one of a number or list of elements, but including a plurality and optionally further unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element from a number or list of elements. In general, the term "or" as used herein refers to exclusive alternatives (i.e., "one or both, but not both") when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0126] As used in this specification and claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in said list of elements, but not necessarily including at least one and every element specifically listed in said list of elements, nor excluding any combination of elements in said list of elements. This definition also allows that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") means, in one embodiment, at least one, optionally more than one A, and no B (and optionally including elements other than B); in another embodiment, at least one, optionally more than one B, and no A (and optionally including elements other than A); in yet another embodiment, at least one, optionally more than one A, and at least one, optionally more than one B (and optionally including other elements); and so forth.

[0127] Some embodiments may be embodied as a method, various examples of which are described. The acts performed as part of the method may be ordered in any suitable manner. Thus, embodiments may be constructed in which acts are performed in an order different from that shown, which may include acts different (e.g., more or less) than those described, and / or may include performing some acts simultaneously, even though the acts have been shown in the above embodiments as being performed sequentially.

[0128] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claimed elements does not, by itself, imply a priority, precedence, or order or chronological sequence of one claimed element relative to another in which acts of the method are performed, but is used solely as a marker to distinguish one claimed element having a particular name from another element having the same name (but for common terminology purposes) to distinguish the claimed elements.

[0129] In the claims and the above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," etc., are understood to be open-ended, i.e., meaning inclusive, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedure, Section 2111.03.

Claims

1. 1. An article configured for administration of a therapeutic agent, comprising: A plurality of fluorescent diamond particles; and A therapeutic agent bound to at least a portion of the fluorescent diamond particles. and configured to reside within the organ of a subject for an extended period of time.

2. 1. An article for use with a subject, comprising: an injection component configured to administer the composition to the subject; and A reservoir associated with an injection component containing the composition wherein the composition comprises a plurality of fluorescent diamond particles.

3. 2. The article of any preceding claim, wherein the plurality of diamond particles have an average particle size of 100 nm or more and 2000 nm or less.

4. 20. The article of any preceding claim, wherein the plurality of diamond particles are configured to agglomerate within an organ of the subject.

5. 10. An article as claimed in any preceding claim, wherein the plurality of diamond particles are adapted to be captured by an organ of the subject.

6. 2. The article of any preceding claim, wherein the agglomerates of diamond particles have an average diameter of from 1 micron to 100 microns.

7. 10. The article of any preceding claim, wherein the plurality of diamond particles have a characteristic near infrared emission.

8. 20. The article of any preceding claim, wherein the organ is the liver of the subject.

9. 10. The article of any preceding claim, wherein the extended residence is 5 days or more.

10. 1. A pharmaceutical composition comprising: Intravenous carrier fluid; and a plurality of fluorescent diamond particles suspended in said intravenous carrier fluid; 13. A pharmaceutical composition comprising:

11. 11. The pharmaceutical composition of claim 10, comprising a therapeutic agent bound to at least a portion of the fluorescent diamond particles.

12. 11. A pharmaceutical composition according to claim 10, comprising a diagnostic agent which is bound to at least a portion of the fluorescent diamond particles.

13. 2. A pharmaceutical composition according to any preceding claim, wherein the plurality of diamond particles have an average particle size of 100 nm or more and 2000 nm or less.

14. 10. A pharmaceutical composition according to any preceding claim, wherein the plurality of diamond particles are configured to agglomerate within an organ of the subject.

15. 10. A pharmaceutical composition according to any preceding claim, wherein the plurality of diamond particles are adapted to be captured by an organ of the subject.

16. 2. A pharmaceutical composition according to any preceding claim, wherein the agglomerates of diamond particles have an average diameter of at least 1 micron and not more than 10,000 microns.

17. 10. A pharmaceutical composition according to any preceding claim, wherein the plurality of diamond particles have a characteristic near infrared emission.

18. 2. The pharmaceutical composition of any preceding claim, wherein the organ is the liver of the subject.

19. 2. The pharmaceutical composition of any preceding claim, wherein the extended residence is for 5 days or more.

20. 2. A pharmaceutical composition according to any preceding claim, wherein the extended residence time is up to at least 12 days.

21. 1. A method of treating a disease, comprising:

1. A method comprising intravenously administering to a subject a plurality of diamond particles and a therapeutic agent bound to at least a portion of said plurality of diamonds, wherein said plurality of diamond particles are configured to reside within an organ of the subject.

22. 22. The method of claim 21 , wherein the plurality of diamond particles are present in the liver of the subject.

23. 1. A method for monitoring disease progression in a subject suspected of having a disease, comprising: administering to said subject a plurality of diamond particles; obtaining a first image of a location within the subject suspected of containing said plurality of diamond particles after the administering step; obtaining a second image of a location within the object suspected of containing said plurality of diamond particles after a predetermined period of time; Measuring morphological changes at locations within the object between the first and second images for the plurality of diamond particles. wherein the morphological changes are associated with progression of relaxation.

24. 24. The method of claim 23, wherein the morphological change is determined by measuring an increase in area of ​​the internal organ of the subject in the second image versus the first image for the portion of the internal organ containing the plurality of diamond particles.

25. Use of a plurality of fluorescent diamond particles in the manufacture of a medicament for the treatment of liver disease and / or liver cancer.

26. 26. The use of claim 25, comprising a therapeutic agent bound to the plurality of fluorescent diamonds.

27. Use of a plurality of fluorescent diamond particles in the manufacture of a medicament for monitoring disease progression.

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