FLUORESCENT PROBES FOR DETECTION OF CALCIFICATIONS

Abstract

A fluorescent probe includes one or more metal binding functional group, such as phosphonic acid group and an arsonic acid group, in which the functional group is covalently linked to a fluorescent core via a sp.sup.2-carbon atom of the fluorescent core. In embodiments, the fluorescent core is an organic fluorescent compound/moiety, that can be a tetrapyrrole derivative, such as porphyrin or phthalocyanine, acridine, BODIPY, cyanine or cyanine derivatives, carbazole, coumarine or coumarine derivatives, xanthene or xanthene derivatives such as fluorescein or rhodamine. The fluorescent probe can bind to calcium and/or a calcification, such as hydroxyapatite (HAP). In a further aspect, a fluorescent probe is used in a method of detecting calcium, such as a calcification or HAP, in a bodily tissue. The use of the fluorescent probe is also provided for detecting calcium, a calcification and/or HAP, such as calcium depositions in a bodily tissue.

Claims

1. A fluorescent probe comprising one or more metal binding functional group, preferably selected from the group comprising phosphonic acid group and arsonic acid group, and a fluorescent core, wherein the one or more functional group is covalently linked to a sp.sup.2-carbon atom of the fluorescent core.

2. The fluorescent probe according to claim 1, wherein the fluorescent core is an organic fluorescent compound/moiety.

3. The fluorescent probe according to claim 1, wherein the one or more metal binding functional group is a phosphonic acid group.

4. The fluorescent probe according to claim 1, wherein the probe comprises at least two, three, four, five, six, seven, eight, nine or ten or more metal binding functional groups.

5. The fluorescent probe according to claim 1, wherein the compound is selected from the group consisting of ((4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-414,514-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-10-yl)phenyl)phosphonic acid) (BODIPY-PPA), 5,10,15,20-tetrakis[p-phenylphosphonic acid] porphyrin (p-H.sub.8TPPA), and 5,10,15,20-tetrakis[m-phenylphosphonic acid] porphyrin (m-H.sub.8TPPA).

6. The fluorescent probe according to claim 1, wherein the probe can bind to calcium and/or a calcification.

7. The fluorescent probe according to claim 1, wherein binding of the probe to calcium, a calcification or HAP leads to an increase in fluorescence.

8. A contrast agent, comprising a fluorescent probe according to claim 1.

9. The fluorescent probe according to claim 1 for use in a method of detecting calcium in a bodily tissue.

10. The fluorescent probe for use according to claim 9, wherein the method is a diagnostic method.

11. The fluorescent probe for use according to claim 9, wherein the method is for detecting bone growth and/or bone resorption, a soft tissue calcification, and/or for early diagnosis of breast cancer.

12. The fluorescent probe for use according to claim 9, wherein the method is for imaging tissue, lumens, or cells, the method comprising contacting the tissue, lumen or cell with a fluorescent probe according to any one of claims 1-7, irradiating the tissue, lumen, or cells at a wavelength absorbed by the compound; and detecting a signal from the fluorescent probe, thereby imaging the tissue, lumen, or cells.

13. The fluorescent probe for use in a method according to claim 12, wherein the fluorescent probe is administered to a subject comprising the tissue, lumen, or cells.

14. Use of a fluorescent probe according to claim 1, for detecting calcium, a calcification and/or HAP.

15. The use of a fluorescent probe according to claim 14, wherein the calcium, calcification of HAP is deposited in a bodily tissue.

16. The fluorescent probe according to claim 2, wherein the organic fluorescent compound/moiety is a tetrapyrrole derivative.

17. The fluorescent probe according to claim 16, wherein the tetrapyrrole derivative is one or more of a porphyrin or phthalocyanine, an acridine, BODIPY, a cyanine or cyanine derivative, a carbazole, a coumarine or coumarine derivative, or a xanthene or xanthene derivative.

18. The fluorescent probe according to claim 17, wherein the xanthene or xanthene derivative is fluorescein or rhodamine.

19. The fluorescent probe according to claim 6, wherein the calcium and/or a calcification is hydroxyapatite (HAP).

20. The fluorescent probe for use according to claim 9, wherein the diagnostic method comprises administering the fluorescent probe to a subject, or exposing an isolated sample to the fluorescent probe in vitro.

Description

DESCRIPTION OF THE FIGURES

[0157] FIG. 1: The fluorescent probes used in this study a) BODIPY-PPA b) p-H.sub.8TPPA c) m-H.sub.8TPPA d) crystal structure of BODIPY-PPA-2Et.sub.2 e) p-TBr3PPA-iPr.sub.2 f) p-H.sub.8TPPA-iPr.sub.8.

[0158] FIG. 2: Mouse ribs incubated with p-H.sub.8TPPA (λ.sub.ex/em 595/613 nm; exposure time—200 ms) diluted to 1 mg/mL (A), 0.1 mg/mL (B) and 0.01 mg/mL (C) and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—200 ms); scalebar=100 μm (microscope, objective, filters or cubes, camera, or are details elsewhere).

[0159] FIG. 3: Mouse ribs incubated with p-H.sub.8TPPA (λ.sub.ex/em 595/613 nm; exposure time—200 ms) at 1 mg/mL in HEPES (A), PBS (B), TBS (C) and dH.sub.2O (D) and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—200 ms); scalebar=100 μm.

[0160] FIG. 4: Mouse ribs incubated with p-H.sub.8TPPA (λ.sub.ex/em 595/613 nm; exposure time—200 ms) at 1 mg/mL for 120, 60, 30, 20 and 10 mins (A-E, respectively) in HEPES buffer and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—200 ms). A negative control of mouse ribs incubated with HEPES buffer alone is also included (F). Scalebar=100 μm.

[0161] FIG. 5: Mouse ribs incubated with p-H.sub.8TPPA (λ.sub.ex/em 595/613 nm; exposure time—200 ms) diluted to 1 mg/mL in HEPES, PBS, TBS and dH.sub.2O (A-D, respectively) and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—200 ms). Images were captured on the day of staining (A-D), as well as after 4 days (E-H), 7 days (I-L) and 14 days (M-P). A negative control of mouse ribs incubated with each buffer alone is also included (Q-T, respectively). Scalebar=100 μm.

[0162] FIG. 6: Mouse ribs incubated with m-H.sub.8TPPA (λ.sub.ex/em 578/603 nm; exposure time—100 ms) diluted to 1 mg/mL (A), 0.1 mg/mL (B) and 0.01 mg/mL (C) and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—100 ms); scalebar=100 μm.

[0163] FIG. 7: Mouse ribs incubated with m-H.sub.8TPPA (λ.sub.ex/em 578/603 nm; exposure time—100 ms) at 1 mg/mL for 120, 60, 30, 20 and 10 mins (A-E, respectively) in HEPES buffer and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—100 ms). A negative control of mouse ribs incubated with HEPES buffer alone is also included (F). Scalebar=100 μm.

[0164] FIG. 8: Mouse ribs incubated with m-H.sub.8TPPA (λ.sub.ex/em 578/603 nm; exposure time—100 ms) at 1 mg/mL for 120 mins in HEPES and imaged at day 1 (A), day 4 (B), day 7 (C) and day 14 (D) and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—100 ms). A negative control of mouse ribs incubated with HEPES buffer alone is also included (E). Scalebar=100 μm.

[0165] FIG. 9: Mouse ribs incubated with BODIPY-PPA (λ.sub.ex/em 578/603 nm; exposure time—400 ms) diluted to 1 mg/mL (A), 0.1 mg/mL (B) and 0.01 mg/mL (C) in HEPES and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—100 ms); scalebar=100 μm.

[0166] FIG. 10: Mouse ribs incubated BODIPY-PPA (λ.sub.ex/em 578/603 nm; exposure time—400 ms) at 1 mg/mL buffer for 120, 60, 30, 20 and 10 mins (A-E, respectively) in HEPES buffer and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—100 ms). A negative control of mouse ribs incubated with HEPES buffer alone is also included (F). Scalebar=100 μm.

[0167] FIG. 11: Mouse ribs incubated with BODIPY-PPA (λ.sub.ex/em 578/603 nm; exposure time—400 ms) at 1 mg/mL for 120 mins in HEPES and imaged at day 1 (A), day 4 (B), day 7 (C) and day 14 (D) and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—100 ms). A negative control of mouse ribs incubated with HEPES buffer alone is also included (E). Scalebar=100 μm.

[0168] FIG. 12: Mouse ribs incubated with p-TBr3PPA-iPr.sub.2 (λ.sub.ex/em 595/613 nm; exposure time—400 ms) at 1 mg/mL in HEPES, PBS, TBS, dH.sub.2O and DMSO (A-E, respectively) and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—100 ms). A negative control of mouse ribs incubated with each buffer alone is also included (F-J, respectively). Scalebar=100 μm.

[0169] FIG. 13: Mouse ribs incubated with p-H.sub.8TPPA-iPr.sub.8 (λ.sub.ex/em 595/613 nm; exposure time—400 ms) at 1 mg/mL in HEPES, PBS, TBS, dH.sub.2O and DMSO (A-E, respectively) and counter-stained with DAPI (λ.sub.ex/em 359/461 nm; exposure time—100 ms). A negative control of mouse ribs incubated with each buffer alone is also included (F-J, respectively). Scalebar=100 μm.

[0170] FIG. 14: FIG. 13 shows the fluorescent intensity increase of p-H.sub.8TPPA upon hydroxyapatite (HAP) binding in comparison with the fluorescent intensity in the absence of HAP. The absorbance (A) and fluorescence (B) spectra of 0.01 mg mL.sup.−1p-H.sub.8TPPA in the presence (dashed lines) and absence (solid lines) of HAP. The dye was diluted to 0.01 mg mL.sup.−1 in PBS (pH 7.4, green), TBS (pH 7.4, orange), HEPES (pH 7.4, purple) and d H.sub.2O with absorbance and fluorescence spectra obtained using the Flexstation 3 microplate reader.

[0171] FIG. 15: Fluorescence spectra of THP-1 monocytes cells incubated with p-H.sub.8TPPA.

EXAMPLES

[0172] The invention is further described by the following examples. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

SUMMARY OF THE EXAMPLES

[0173] Recent studies revealed the presence of microscopic particles of bone mineral within deposits in the retina (the best known are called drusen), and they seem to be associated with the development of these deposits in AMD, and in the retinal periphery, with Alzheimer Disease. We have synthesized a group of chemical probes for use in studying the deposition of the bone mineral by microscopy and other means. Using these novel methods, we seek to understand the biology of deposit formation and growth and use this knowledge to develop new diagnostics and potential treatments for these diseases.

[0174] Recently, it has emerged that mineralization in the retina with hydroxyapatite (HAP) and other species is correlated with the development of AMD and Alzheimer disease. While we have used legacy stains including classic tetracycline antibiotics as selective fluorescent stains for HAP in the retina, these compounds have some limitations and novel HAP-specific fluorescent stains may offer useful new properties including fluorescence imaging contrast using new mechanisms. These properties may further elucidate the HAP deposition process mechanism(s) and suggest novel treatments. Herein, the following compounds of the invention are used: [0175] ((4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-414,514-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-10-yl)phenyl)phosphonic acid) (BODIPY-PPA) [0176] 5,10,15,20-tetrakis[p-phenylphosphonic acid] porphyrin (p-H.sub.8TPPA) [0177] 5,10,15,20-tetrakis[m-phenylphosphonic acid] porphyrin (m-H.sub.8TPPA)

[0178] Characterization of Probes: The structure and purity of the probes were characterized by liquid chromatography, infrared, NMR, and/or mass spectrometry. Their optical properties were characterized by absorption and fluorescence spectrophotometry in the presence and absence of authentic hydroxyapatite, whitlockite, and other doped apatites. The dyes were also tested on mouse ribs as an example of hydroxyapatite in tissue.

[0179] We found that several of the synthesized probes bound HAP tightly, in some cases with alterations of their fluorescence properties, which we attribute to synergistic interactions arising from conjugation of the sp2 carbons of the aryl fluorophore moiety with the phosphonate moiety. Staining a cross section of mouse rib with meso-tetra(4-phosphorylphenyl)porphyrin (red) and DAPI (blue), together with autofluorescence (green) is depicted in the figure (A), with a DAPI-stained control section from the same mouse (B). Based on these preliminary results, staining appears specific for hydroxyapatite.

[0180] We conclude that these new fluorescent labels offer novel features that will be of use in elucidating the biology of retinal mineralization and its relationship to macular and peripheral deposit formation seen in AMD and Alzheimer disease, respectively.

METHODS OF THE EXAMPLES

[0181] Mouse ribs (C57BL/6) were fixed in 4% paraformaldehyde (PFA). Ribs were processed for wax embedding through sequential immersion in increasing concentrations of ethanol followed by immersion in xylene/clearene and paraffin wax embedding. Rib sections (6 μm thickness) were dewaxed with xylene and rehydrated with decreasing concentrations of ethanol and distilled water (dH.sub.2O). Rib sections were incubated with a variety of either namely((4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-414,514-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-10-yl)phenyl)phosphonic acid) (BODIPY-PPA), 5,10,15,20-tetrakis[p-phenylphosphonicacid] porphyrin (p-H.sub.8TPPA) and 5,10,15,20-tetrakis[m-phenylphosphonic acid] porphyrin (m-H.sub.8TPPA) dyes. These are 5,10,15,20-tetrakis[p-phenylphosphonic acid] porphyrin (p-H.sub.8TPPA), as well as its diester form (p-H.sub.8TPPA-iPr.sub.8); 5,10,15-tris[p-bromophenyl]-20-[p-phenyl-, bis(1-methylethyl) ester phosphonic acid] porphyrin (p-TBr.sub.3PPA-iPr.sub.2) with bulkyisopropyl groups, and BODIPY-PPA-2Et.sub.2 with two ethyl groups. p-H.sub.8TPPA was incubated for 120 mins at 1 mg/mL in 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, pH 7.4), phosphate-buffered saline (PBS, pH 7.4), tris-buffered saline (TBS, 7.4; Trizma base) and dH.sub.2O. Meso-tetra(4-phosphorylphenyl)porphine, porph-tetra-meta-phenyl phosphonate, BODIPY-PPA; and BODIPY-PPA-2Et.sub.2 were all incubated for 120 mins when diluted to 1, 0.1 and 0.01 mg/mL in HEPES, and incubated at 1 mg/mL for 120, 60, 30, 20 and 10 mins. Images were obtained for up to two weeks after staining. p-H.sub.8TPPA-iPr.sub.8 and p-TBr.sub.3PPA-iPr.sub.2 were only incubated for 120 mins at 1 mg/mL, including an incubation with dimethyl sulfoxide (DMSO). Negative controls were included by incubating ribs with the buffers alone. Following incubation with the respective dyes, slides were washed with their respective buffers (3×5 mins) and counter stained with DAPI, diluted in PBS, for 20 mins. After washing with PBS (3×5 mins), slides were mounted with 70% glycerol, diluted in PBS. Images were acquired on a Leica DM5500 epifluorescent microscope, using a 20× objective. The exposure times used to capture these images were kept the same throughout imaging. Images were processed using the FIJI software.

DESCRIPTION OF THE EXAMPLES

[0182] For this study, as seen in FIG. 1, we have synthesized 6 fluorescent probes. Three of them incorporate phenylphosphonic acid as HAP binding unit with highly conjugated BODIPY, and porphyrin cores, namely((4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-414,514-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-10-yl)phenyl)phosphonic acid) (BODIPY-PPA), 5,10,15,20-tetrakis[p-phenylphosphonicacid] porphyrin (p-H.sub.8TPPA) and 5,10,15,20-tetrakis[m-phenylphosphonic acid] porphyrin (m-H.sub.8TPPA) (FIG. 1).

[0183] Furthermore, all fluorophores listed in Table 1 were synthetized via previously known state of the art from the corresponding brominated precursors. These methods include Ni and Pd catalyzed synthesis of arylphosphonic acids, and Suzuki Cross Coupling Methods (see Schütrumpf, A. et al. “Tetrahedral Tetraphosphonic Acids. New Building Blocks in Supramolecular Chemistry.” Crystal Growth & Design 2015, 15 (10), 4925-4931; Schütrumpf, A. et al. “Synthesis of Some Di- and Tetraphosphonic Acids by Suzuki Cross-Coupling.” Zeitschrift für anorganische and allgemeine Chemie 2018, 644 (19), 1134-1142).

[0184] p-H.sub.8TPPA has four para positioned PPA units and BODIPY-PPA has one para positioned PPA promoting direct conjugation between the HAP and the fluorescent core. On the other hand, m-H.sub.8TPPA has four meta positioned PPA units creating a more protective environment between the fluorescent porphyrin core and the HAP. We then explored their potential for binding hydroxyapatite. As a control group we used three different phosphonate diesters p-H.sub.8TPPA-iPr.sub.8, 5,10,15-tris[p-bromophenyl]-20-[p-phenyl-, bis(1-methylethyl) ester phosphonic acid] porphyrin (p-TBr3PPA-iPr.sub.2) with bulkyisopropyl groups, and BODIPY-PPA-2Et.sub.2 with two ethyl groups to block the metal-phosphonate interactions. To the best of our knowledge, all of the fluorescent probes except the p-H.sub.8TPPA and its ester form are novel, and have not previously appeared in the literature, and this study reports the first synthesis of a phosphonic acid with a BODIPY fluorescent core. [10, 19] Previous synthesis of p-H.sub.8TPPA relied on Ni catalyzed Arbuzov reaction. Therefore, p-H.sub.8TPPA that is synthesized using this route usually had Ni in the imidazole ring the porphyrin core. In this study, we have used an alternative method using Pd catalyzed Arbuzov reaction to obtain metal free p-H.sub.8TPPA. Likewise, we provide two alternative methods to synthesize p-H.sub.8TPPA and m-H.sub.8TPPA. BODIPY-PPA was synthesized using diethyl (4-formylphenyl)phosphonate and and 2,4-Dimethylpyrrole.

[0185] To further probe their HAP binding properties, we have used laterally sectioned mouse ribs as an example of HAP, which were incubated with p-H.sub.8TPPA at concentrations ranging from 0.01 to 1.0 mg/mL in HEPES buffer for varying times at room temperature and imaged by fluorescence microscopy (for details see description of the figures), to assess HAP binding capability. Ribs incubated with 1 mg/mL meso-tetra(4-phosphorylphenyl)porphine showed a greater fluorescent intensity than both 0.1 mg/mL and 0.01 mg/mL (FIG. 2A-C, respectively), whilst still showing clear fluorescent signal at a concentration of 0.1 mg/mL. This indicates that the dye binds to HAP with a concentration dependence in the range tested.

[0186] When the mouse ribs were incubated with 1 mg/mL para-tetra(4-phosphorylphenyl)porphine in HEPES for differing times, 10 to 120 mins, there was a clear increase in fluorescence intensity with an increasing incubation time (FIG. 4A-E). Although there was an increase in fluorescence intensity, there was still a clear fluorescent signal when the ribs were incubated for 10 mins compared to the HEPES only negative control (FIG. 4E compared to FIG. 4F).

[0187] When ribs were incubated with p-H.sub.8TPPA diluted to 1 mg/mL in different buffers, there was no clear difference between the fluorescence intensity of HEPES, PBS, TBS (FIG. 3A-C, respectively). However, when p-H.sub.8TPPA was diluted in dH.sub.2O (FIG. 3D), there was no fluorescent signal. Images were also obtained for the two weeks following the original staining.

[0188] There was a noticeable decrease in fluorescence intensity between ribs incubated with 1 mg/mL p-H.sub.8TPPA in HEPES buffer and imaged on the day of staining compared with an image taken after 7 days later (FIG. 5A and FIG. 51, respectively). Mouse ribs stained with p-H.sub.8TPPA diluted in PBS and TBS both showed fluorescent signal above background signal one week after staining (PBS, FIG. 5B and FIG. 5J; TBS, FIG. 5C and FIG. 5K, respectively). After two weeks, there was still a weak fluorescent signal observed for p-H.sub.8TPPA diluted in PBS, but not for the other solvents. (FIG. 5N compared to FIGS. 5M, O and P). No signal was associated with incubating the ribs with the solvents as a negative control (FIG. 5Q-T).

[0189] Phosphonic acid moieties in m-H.sub.8TPPA are more protected compared to the para positioned p-H.sub.8TPPA. Mouse ribs were incubated with m-H.sub.8TPPA at different concentrations, 1 to 0.01 mg/mL in HEPES buffer. Ribs incubated with each concentration of m-H.sub.8TPPA showed little to no difference in the fluorescent intensity between 1, 0.1 and 0.01 mg/mL (FIG. 6A-C, respectively).

[0190] When the mouse ribs were incubated with 1 mg/mL m-H.sub.8TPPA in HEPES for differing times, 10 to 120 mins, there no clear difference in fluorescence intensity with increasing incubation times (FIG. 7A-E, respectively). No fluorescent signal was observed when the ribs were incubated with HEPES alone (FIG. 7F).

[0191] Images were also obtained for the two weeks following the original staining. However, unlike the para isomer (FIG. 5), there was no noticeable decrease in fluorescence intensity between ribs incubated with 1 mg/mL m-H.sub.8TPPA in HEPES buffer and imaged on the day of staining compared with images taken 4, 7 and 14 days later (FIG. 8A-D, respectively). This could be associated with the more protected HAP phosphonic acid interaction in the meta position limiting the interactions with the surrounding molecules solvents etc. Again, no signal was associated with incubating the ribs with HEPES only as a negative control (FIG. 8E).

[0192] The red filter (λ.sub.ex/em 578/603 nm) has been used to visualize the fluorescent signal of the dye bound to HAP in this channel (FIGS. 9, 10 and 11). The dye shows a strong concentration dependency of staining, with a noticeable increase in fluorescence intensity when incubating with 1 mg/mL BODIPY-PPA compared to either 0.1 mg/mL or 0.01 mg/mL (FIG. 9A-C, respectively).

[0193] A similar decrease in fluorescence intensity is observed for decreasing incubation times from 120 to 10 mins (FIG. 10A-E, respectively). No fluorescent signal was observed under these conditions when the slices were incubated with HEPES alone (FIG. 10F).

[0194] There appeared to be only a very slight decrease in fluorescence intensity of dye up to two weeks following staining, with no signal was associated with incubating the ribs with HEPES only as a negative control (FIG. 11A-E, respectively).

[0195] As seen in FIG. 3, the fluorescence intensity is increased upon HAP binding of the fluorescent probe with p-H.sub.8TPPA having four phosphonic acid groups bonded to the sp2 carbons of the fluorescent core.

[0196] Comparable experiments were conducted using the phosphonateesters BODIPY-PPA-2Et.sub.2, p-TBr3PPA-iPr.sub.2 (see FIG. 12), p-H.sub.8TPPA-iPr.sub.8 (see FIG. 13) as stains. As seen by the lack of HAP-associated staining, it seems evident that the interaction of phosphonates with HAP was reduced by the isopropylester groups on the phosphonates, preventing binding and interaction of the fluorescent porphyrin core with the HAP. On the other hand, the phosphonatediester of the BODIPY-PPA showed some interaction with the HAP, perhaps because phosphonate oxygens were more available to interact with the HAP (See the crystal structure FIG. 1d).

[0197] Mouse ribs were incubated for 120 mins with p-TBr3PPA-iPr.sub.2 (5,10,15-tris[p-bromophenyl]-20-[p-phenyl-, bis(1-methylethyl) ester phosphonic acid] porphyrin) diluted to 1 mg/mL in different buffers. It was observed that the dye precipitated in HEPES, PBS, TBS and dH.sub.2O (FIG. 12A-D) during the incubation period. This resulted in a fluorescent signal from the precipitated dye crystals, with no binding to HAP occurring. An additional incubation with the dye in an apolar solvent, DMSO, was attempted but this also resulted in the precipitation of the dye (FIG. 12E). Negative controls showed the fluorescent signal excited with λ.sub.ex 595 nm was due to dye precipitation and not the buffer itself (FIG. 12F-J).

[0198] Mouse ribs were incubated for 120 mins with p-H.sub.8TPPA-iPr.sub.8 phosphonate diester diluted to 1 mg/mL in different buffers. Similar to the tribromoporphine dye, it was observed that the dye precipitated in HEPES, PBS, TBS and dH.sub.2O (FIG. 13A-D) during the incubation period. This resulted in a fluorescent signal from the precipitated dye crystals, with no binding to HAP occurring. An additional incubation with the dye in an apolar solvent, DMSO, was attempted but this also resulted in the precipitation of the dye (FIG. 13E). Negative controls showed the fluorescent signal excited with Aex 595 nm was due to dye precipitation and not the buffer itself (FIG. 13F-J).

[0199] As seen in FIG. 14, the absorbance peak (FIG. 14A) of p-H.sub.8TTPA in PBS, TBS and HEPES with the addition of HAP corresponds with the absorbance of the dye in d-DMSO. There is a noticeable shift in the absorbance peak maximum when diluted in d H.sub.2O, from 415 nm to 435 nm, which suggests a shift in the fluorescent properties of the dye due to protonation. This is also reflected in the maximal peak of the fluorescence intensity (FIG. 14 B), with a shift from λ.sub.em 646-648 nm for PBS, TBS and HEPES to λ.sub.em 675 nm when diluted in dH.sub.2O. The fluorescence intensity of p-H.sub.8TPPA is also substantially increased by 1.8-, 2.0-, 2.0-, and 1.6-fold upon HAP binding of the fluorescent probe p-H.sub.8TPPA in PBS, TBS, HEPES and d H.sub.2O, respectively (FIG. 14 B, solid vs. dashed lines). Previously reported bisphosphonate fluorophores had spa aliphatic carbons between the fluorescent core and HAP binding, therefore, such systems merely functioned as fluorescent labels with no change in emission due to HAP binding since they lacked synergistic interaction between the HAP and the fluorescent core. Phosphonic acids that have direct sp.sup.2 bonds to the fluorescent core could extend the conjugation of the fluorescent core to the HAP and thereby could initiate changes in the ground and excited states resulting in quenching or enhancing the fluorescence emission. As seen in FIG. 14 (B), upon binding of HAP, p-H.sub.8TPPA produces increased fluorescence supporting this hypothesis; p-H.sub.8TPPA is thus the first example of a single sp.sup.2 bonded phosphonic acid unit targeting HAP in the literature.

[0200] Comparable experiments with mouse rib sections were conducted using the isopropyldiester forms p-H.sub.8TPPA-iPr.sub.8 and m-H.sub.8TPPA-OEt.sub.8 as stains. We found a complete lack of HAP-associated staining with these compounds suggesting that the interaction of phosphonates with HAP was reduced by the isopropylester groups on the phosphonates, preventing binding and interaction of the fluorescent porphyrin core with the HAP. We also noted that the presence of hydrophobic isopropyl groups dramatically reduced their solubilities in the aqueous.

[0201] In addition to the application on bone sections, the phenylphosphonic acid/esterified porphyrins' propensity for cellular uptake was tested on proliferating human cells (FIG. 15). Aiming to use phenylphosphonic acid functionalized porphyrins in in vivo applications, the negative charge on the deprotonated phosphonate might be a handicap by limiting cell permeability. Optional demasking of phosphonate esters in metabolic surroundings might occur, recreating the metal binding PhPO.sub.3.sup.2-. This possibility led us to examine p-H.sub.8TPPA and p-H.sub.8TPPA-iPr.sub.8 suitability for in vivo cellular experiments by testing their permeability on proliferating human THP-1 monocytes.

[0202] We have treated THP-1 monocytes with the Phosphorylphenyl-modified porphyrins in loading buffer (10 mM HEPES, pH 7.35, 120 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1.3 mM CaCl.sub.2, 1 mM MgCl.sub.2, 1 mM NaH.sub.2PO.sub.4, 0.3% bovine serum albumin) for 60 min. Excess fluorescence dye was removed by multiple washing steps before fluorescence emission scanning using 420 nm excitation (Tecan Infinite M200 reader; Tecan, Germany). (Haase et al. 2006) p-H.sub.8TPPA was much more efficient in cell labeling compared to the isopropyl-modified molecule. We hypothesize that the phosphonates, being somewhat amphipathic, were able to penetrate the cell membrane, whereas the more nonpolar esters were not as efficient.

CONCLUSION OF THE EXAMPLES

[0203] Herein, we report the synthesis of novel fluorescent probes namely BODIPY-PPA, p-H.sub.8TPPA, m-H.sub.8TPPA, BODIPY-PPA-2Et.sub.2, p-TBr3PPA-iPr.sub.2, p-H.sub.8TPPA-iPr.sub.8 in which the phosphonic acid metal binding unit(s) are exclusively bonded to sp2 carbon atoms of the fluorescent core. Sp2 bonding of the metal binding unit to the fluorescent core further extended the conjugation of the fluorescent core to the HAP leading significant increase in fluorescence upon HAP binding. We further used these fluorescent probes to target the HAP in mouse ribs and observed their interaction with human cells. The more protected metal binding nature of the m-H.sub.8TPPA resulted in longer fluorescence period compared to the p-H.sub.8TPPA. As a control, we have also synthesized phosphonatediesters of the synthesized fluorescent probes, which showed no HAP binding. p-H.sub.8TPPA can travel through the cellular membrane, whereas the presence of bulky and hydrophobic isopropyl groups in p-H.sub.8TPPA-iPr.sub.8 hindered their passage through the cellular membrane. This study shows that compact fluorescent probes with phosphonic acid metal binding group(s) may be used to monitor microcalcifications, calcifications. In addition, their easy acceptance into the cellular matrix indicate that they could be used to target organelle specific calcifications such as mitochondrial calcifications. Reported low toxicity of arylphosphonic acid fluorophores and the new synthetic methods indicate that they can be used in targeting wide range of calcifications in vivo. We are currently developing our library to target organelle specific calcifications.

REFERENCES

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