WO2011011646A1 - Luminescent diketonate polymers - Google Patents

Abstract

The present invention provides in one aspect polymeric luminescent dye compounds having fluorescent properties, phosphorescent properties, or both fluorescent and phosphorescent properties. The luminescent dye compounds and compositions can be used for oxygen sensing that can be conducted without the need for invasive techniques.

Description

LUMINESCENT DIKETONATE POLYMERS

Government Funding

[0001] This invention was made with United States Government support under Grant No. CHE-0350121 awarded by the National Science Foundation. The United States Government has certain rights in this invention.

Priority

[0002] This application claims priority from U.S. Provisional Application No. 61/227,601, filed July 22, 2009, the disclosure of which is incorporated by reference.

Background

[0003] There are many optical oxygen sensing systems based on Ru, Pt, or Ir heavy metal luminophores. However, oxygen sensors based on non-heavy metal Iuminophores with unusual dual emissive properties (e.g., based on boron), particularly single component systems with biocompatible "green" polymers are very rare. Fluorescent boron difluoride dyes such as "bodipy" and boron diketonates possess large molar extinction coefficients and two-photon absorption cross-sections, high emission quantum yields and sensitivity to the surrounding medium. These features have been exploited in lasers, imaging agents, molecular probes, and photosensitizers. As two-photon absorbers, they are compatible with optical imaging technologies employing tunable Ti:sapphire lasers (700-1100 nm). Focused, longer wavelength IR excitation corresponds with greater tissue penetration, and reduced cell damage and interference from biological absorbers. Boron difluoride diketonate dyes possess large dipole moments and their emission wavelength sensitive to the polarity of the surrounding medium. Thus, solvatochromic boron complexes serve as probes of their local environments. [0004] Luminescent materials are widely used for imaging and sensing due to their high sensitivity, rapid response, and facile detection by many optical technologies. See, e.g., Yuste, R. Fluorescence microscopy today. Nat. Methods. 2, 902-904 (2005). Typically materials must be chemically tailored to achieve intense, photostable fluorescence (F), oxygen sensitive phosphorescence (P), or dual emission for ratiometric sensing (F/P), often by blending two dyes in a matrix. Dual-emissive materials combining all of these features in one easily tunable molecular platform are desirable, but when fluorescence and phosphorescence originate from the same dye, it can be challenging to vary relative F/P intensities for practical sensing applications. Heavy atom substitution alone is reported to increase phosphorescence by a given, not variable amount.

[0005] A major drawback of two dye-component/matrix systems is difficulties in controlling the homogeneity of the doped matrix. Blended dyes can be prone to aggregation and can photodegrade due to local temperature buildup. These problems may be addressed by covalent attachment of dye molecules to supports such as polymers. Higher relative dye concentrations may be achieved without dye precipitation, and for solvatochromic dyes, color may be tuned across a broader range.

[0006] Boron dye fluorescence is well known, however, phosphorescence is usually only observed in the presence of toxic heavy atom substituents or additives (e.g., Pb, Tl, or halogens such as I, Br), at low temperatures, or in rigid, solid matrices, which can be difficult to process and often are not biocompatible and biodegradable. Phosphorescence is quenched by oxygen, which at room temperature more accurately and conveniently may serve as the basis for quantitative optical oxygen sensing. Single component, readily processable systems exhibiting both fluorescence and phosphorescence are rare and may be adapted for imaging and ratiometric sensing. Fluorescence (short emission lifetimes) serves as an invariant feature providing information to quantify and locate the dye/emitter, whereas phosphorescence (long emission lifetimes) is quenched to variable extents depending upon the amount of oxygen that is present. Phosphorescent materials with long emission lifetimes are more sensitive to oxygen, and may serve as highly sensitive oxygen sensors in low oxygen environments (food and tamper resistant packaging, hypoxic tumor or cardiovascular tissues, tissue engineering matrices, the environment e.g. eutrophication in lakes, streams, low-oxygen soils, etc.) Luminescent materials can also be used as photosensitizers, transferring energy to other molecules, and generating reactive species by light activation. For example, this feature is exploited in photodynamic therapy, generating reactive singlet oxygen to selectively damage tumor tissue, and in lithography with two-photon dyes.

[0007] Given the importance of oxygenation for understanding biology and health, determining prognosis, treatment and response to a therapy, the ability to measure pθ₂ and image hypoxia is important. Methods have been developed that range from histological staining with reducible nitroimidazole reagents, protein markers, and GFP or luciferase reporter genes, to electrochemical probe methods, and optical, nuclear medicine, and magnetic resonance techniques. Some methods are only suitable for the laboratory, while others have been translated to the clinic. All have their advantages and drawbacks. Oxygen electrodes are often considered the standard of comparison, however, only single-point or line measurements are possible with this invasive oxygen sensing technique. Due to the heterogeneous and dynamic nature of hypoxia, serial methods with good spatial and temporal resolution would be particularly informative. Magnetic resonance methods such as blood oxygen level-dependent (BOLD) MRI are powerful and non-invasive; however, these are indirect indicators of hypoxia and require expensive specialized instrumentation and careful interpretation. Optical methods such as hemoglobin saturation, redox imaging, and phosphorescence quenching are attractive. The latter method, for instance, offers the advantages of high sensitivity, specificity and accuracy, and direct, absolute pθ₂

measurements in hypoxic environments over time. However, challenges remain due to the tissue penetration of light, requiring light guides, fiber optic probes, or specialized reagents and 2-photon excitation protocols that operate in the near IR up to cm depth for certain uses.

[0008] Currently, there is a need for methods for oxygen sensing that can be conducted without the need for invasive techniques. In addition, there is a need for compounds that have the ability to both fluoresce and phosphoresce, which can be used for such imaging and diagnostic techniques. There is also a need for stable oxygen sensors that can be used to detect oxygen at low levels in tumors, cardiovascular tissue, wounds, and a great many other biological contexts, including cells and tissues.

Summary

[0009] The present invention provides in one aspect polymeric luminescent dye compounds having both fluorescent and phosphorescent properties. Accordingly, the invention provides compounds having formula I:

wherein R¹ is (C₆-C₂₂)aryl or (C₅-C₂ Oheteroaryl and R¹ is substituted with at least one substituent which is a heavy atom or an ionizable group and optionally substituted with additional substituent groups. R² is (C6-C₂₂)aryl, (C₅-C₂₁)heteroaryl, or (C₁-C₁s)alkyl and R² is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substituent groups.

The substituent groups on R¹ or R² are halo, (d-C₁₂)alkyl, hydroxy(C_!-C₁₂)alkyl, ImIo(C₁- C_I2)alkyl, R¹⁵O(C,-C₁₂)alkyl, R¹⁵O(C₁ -Ci₂)alkyl-O-, (C₂-C₁₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅- C₁₃)heteroaryl, -OR¹⁵, oxo (>C=O), -CN, -NO₂, -CO₂R¹⁵, -OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, - N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, -SO₂R²⁰; -OSi(R²⁵)₃, -Si(R²⁵)₃ -Si(R²⁵)i(OR²⁵)_j, - P(OR²⁵)₃, -P(R²⁵)₃, isocyanate, isothiocyanate, urea, or thiourea; or two substituent groups can form a ring together with the atom to which they are attached optionally having from 3 to 8 ring atoms and optionally having 1, 2, or 3 heteroatoms; each R²⁵ is independently hydrogen, alkyl or aryl; and i and j are independently integers from 1-3 wherein the sum of i and j is 3;

Y¹ is B; X¹ and X² are independently a bond, alkyl, alkenyl, alkynyl or aryl, optionally substituted with 1, 2, 3, 4, 5, or 6 substituent groups; where the substituent groups are halo, (Ci-C₁₂)alkyl, hydroxytQ-C^alkyl, hak^Q-C^alkyl, R^Q-C^alkyl, R¹⁵O(C₁- Ci₂)alkyl-O-, (C₂-C i₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅-C₁₃)heteroaryl, -OR¹⁵, oxo (>C=O), -CN, - NO₂, -CO₂ R¹⁵, -OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, -N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, - SO₂R²⁰, -OSi(R²⁵)₃, -Si(R²⁵)₃, -Si(R²⁵)i(OR²⁵)j, -P(OR²⁵)₃, -P(R²⁵)₃, isocyanate, isothiocyanate, urea, thiourea or two substituent groups can form a ring together with the atom to which they are attached optionally having from 3 to 8 ring atoms and optionally having 1, 2, or 3 heteroatoms; where i and j are independently 1, 2, or 3 and the sum of i and j is 3;

R³ and R⁴ are independently, halo, hydroxy, R¹⁵O(C₁-Ci₂)alkyl, R¹⁵O(Ci-Ci₂)alkyl, (C₆- C₂₂)aryl, or -OR¹⁵; or R³ and R⁴ taken together form a bidentate chelate, such as deprotonated acid or diacid group, HOC(=O)CH₂C(=O)OH (malonic acid) or HOC(=O)CH₂C(=O)OH (oxalic acid), or chelating group such as acid-alcohol, acid-ether, or SiO groups, with two donor groups, or R³ and R⁴ taken together with the boron atom form a ring having the formula:

R⁵ is hydrogen, halo, (Ci-C₁₂)alkyl, hydroxy(C₁-Ci₂)alkyl, halo(Cj-Ci₂)alkyl, (C₂- C₁₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅-C₁₃)heteroaryl, -OR¹⁵, -CN, -NO₂, -CO₂ R¹⁵, -OC(O)R¹⁶, - C(O)R¹⁶, -NR¹³R¹⁴, -N(R²³)C(O)R²⁴, -C(O)NR¹⁷ R¹⁸, -SR¹⁹, -SO₂R²⁰ or -SO₃H; or wherein R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²³ and R²⁴ are independently hydrogen, alkyl, alkenyl, (C₃-C₁₂)cycloalkyl, aryl, aralkyl or haloalkyl; each Q is a polymer chain where each chain is conjugated directly to the compound (e.g., via a covalent, coordinate, ionic, or hydrogen bond) through one of R¹, R², R⁵, X¹, X² or to a substituent attached to R¹, R², R⁵, X¹, or X²; wherein the polymer chain optionally includes a targeting group; and n is 1, 2, 3, 4, 5, 6, 7 ,8, 9, 11, 12, 13, 14, or 15; or a pharmaceutically acceptable salt thereof.

[0010] In another aspect the invention provides luminescent dye compositions having a polymeric compound in combination with a luminescent dye compound formula II:

II

wherein the composition has both fluorescent and phosphorescent properties. In the luminescent dye compounds R¹ is (C₆-C₂₂)aryl or (C₅-C₂i)heteroaryl and R¹ is substituted with at least one substituent which is a heavy atom or an ionizable group and optionally substituted with additional substituent groups. R² is (C₆-C₂₂)aryl, (C₅-C₂₁)heteroaryl or (C₁- C₁₈)alkyl and R² is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substituent groups;

The substituent groups on R¹ or R² are halo, (C₁-C₁₂)alkyl, hydroxy(C₁-C₁₂)alkyl, ImIo(C₁- Ci₂)alkyl, R¹⁵O(Ci-C₁₂)alkyl, R¹⁵O(Ci-Ci₂)alkyl-O-, (C₂-C ₁₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅- C₁₃)heteroaryl, -OR¹⁵, oxo (>C=O), -CN, -NO₂, -CO₂R¹⁵, -OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, - N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, -SO₂R²⁰; -OSi(R²⁵)₃, -Si(R²⁵)₃ -Si(R²⁵),(OR²⁵)_J; - P(OR²⁵)₃, -P(R²⁵)₃, isocyanate, isothiocyanate, urea, or thiourea; or two substituent groups can form a ring together with the atom to which they are attached optionally having from 3 to 8 ring atoms and optionally having 1, 2, or 3 heteroatoms; each R is independently hydrogen, alkyl or aryl; and i and j are independently integers from 1-3 wherein the sum of i and j is 3;

Y¹ is B; X¹ and X² are independently a bond, alkyl, alkenyl, alkynyl or aryl, optionally substituted with 1, 2, 3, 4, 5, or 6 substituent groups; where the substituent groups are halo, (Ci-C₁₂)alkyl, hydroxy(C₁-C₁₂)alkyl, halo(Ci-C_I2)alkyl, R¹⁵O(C₁-Ci₂)alkyl, R¹⁵O(C,- Ci₂)alkyl-O-, (C₂-C₁₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅-C₁₃)heteroaryl, -OR¹⁵, oxo (>C=0), -CN, - NO₂, -CO₂ R¹⁵, -OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, -N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, -SO₂R²⁰; -OSi(R²⁵)₃, -Si(R²⁵)₃, -Si(R²⁵)₁(OR²⁵)_J, -P(OR²⁵)₃, -P(R²⁵)₃, isocyanate,

isothiocyanate, urea, or thiourea; or two substituent groups can form a ring together with the atom to which they are attached optionally having from 3 to 8 ring atoms and optionally having 1, 2, or 3 heteroatoms; each R²⁵ is independently hydrogen, alkyl or aryl; where i and j are independently 1, 2, or 3 and the sum of i and j is 3; R³ and R⁴ are independently, halo, hydroxy, R¹⁵O(C_rC₁₂)alkyl, R¹⁵O(Ci-C₁₂)alkyl, or - OR¹⁵; or R³ and R⁴ taken together form a bidentate chelate, such as deprotonated acid or diacid group, HOC(=O)CH₂C(=O)OH (malonic acid) or HOC(=O)CH₂C(=O)OH (oxalic acid), or chelating group such as acid-alcohol, acid-ether, with two donor groups, or R³ and R⁴ taken together with the boron atom form a ring having the formula:

R⁵ is hydrogen, halo,

hydroxy(C₁-C₁₂)alkyl, halo(d-C₁₂)alkyl, (C₂- Ci₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅-Ci₃)heteroaryl, -OR¹⁵, -CN, -NO₂, -CO₂ R¹⁵, -OC(O)R¹⁶, - C(O)R¹⁶, -NR¹³R¹⁴, -N(R²³)C(O)R²⁴, -C(O)NR¹⁷ R¹⁸, -SR¹⁹, -SO₂R²⁰ or -SO₃H; or wherein R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²³ and R²⁴ are independently hydrogen, alkyl, alkenyl, (C₃-Ci₂)cycloalkyl, aryl, aralkyl or haloalkyl; or a pharmaceutically acceptable salt thereof.

[0011] In another aspect, the invention provides luminescent dyes of formula I or compositions having a compound of formula II that are color tunable, e.g., the color from the fluorescence or phosphorescence of the luminescent dye may be altered by changing the groups, e.g., R¹, R²; R⁵, X¹ and X² attached to diketone core, or by varying the polymer molecular weight (for polymer-dye conjugates of formula I) and concentration or loading of the dye compound of formula II in the polymer (for blends).

[0012] In another aspect, the invention provides a method for modulating the fluorescence or phosphorescence intensities or ratios of the luminescent dye compounds of formula I comprising modifying the molecular weight of the polymer chain to provide a suitable balance between the phosphorescence and fluorescence intensities. The invention provides a method for modulating the fluorescence or phosphorescence intensities or ratios of the luminescent dye compositions having a polymeric compound in combination with a luminescent dye compound formula II by varying the dye loading to provide a suitable balance between the phosphorescence and fluorescence intensities.

[0013] In another aspect, the invention provides a method for tuning the phosphorescence and fluorescence intensities of a luminescent dye compound or the luminescent dye composition by modifying the molecular weight of the polymer chain to provide a suitable balance between the phosphorescence and fluorescence intensities.

[0014] In another aspect, the invention provides a method for tuning the phosphorescence and the composition to provide a suitable balance between the phosphorescence and fluorescence intensities.

[0015] In another aspect, the invention provides a method for tuning relative fluorescence and room-temperature phosphorescence intensities in a dual-emissive boron biomaterial. The method includes manipulation of both spin-orbit coupling and singlet-triplet energy splitting. This provides wide-range fluorescence/phosphorescence (F/P) tunability of the disclosed biomaterials and allows for application in lifetime, time-gated intensity, ratiometric, intensity based or "turn on" sensing modes, optical imaging, oxygen sensing and fluid and

aerodynamics applications.

[0016] In another aspect, the invention provides luminescent dyes of formula I or compositions having a compound of formula II that are suitable as oxygen sensors for diagnostic purposes and for monitoring the effectiveness of therapy (e.g. hypoxia as a marker for cancer chemotherapy or radiation therapy effectiveness). The disclosed compounds and compositions are suitable for imaging and quantifying hypoxia and anoxia in cell, tissue and in vivo contexts. Examples include tumors, vasculature, wounds, brain imaging, high altitude drug testing, monitoring effectiveness of drugs that deliver oxygen to tissues, organ transplantation or tissue transplantation, or cell transplantation, tissue engineering, stem cells, or contexts where the measurement of oxygen is important

[0017] In another aspect, the luminescent dye polymers and compositions can be readily processed into powders, films, particles (including e.g., nanoparticles), fibers (including e.g., nanofibers), coatings, bulk materials, gels, networks, assemblies, suspensions, composites, and the like.

[0018] In another aspect, the invention provides luminescent dyes of formula I or compositions having a compound of formula II that can serve as "turn on" sensors that light up in anaerobic or low oxygen environments such as in ischemia, damaged or blocked vasculature, or are used e.g., in fluid or gas flow and aerodynamics applications. [0019] In another aspect, the invention provides luminescent dyes that are more stable and homogeneous. The dyes provide improved device performance and are less prone to dye matrix (e.g. polymer) phase separation or leaching. The disclosed dyes are better protected from chemical degradation, color fading is minimized, and ambiguity in imaging and sensing schemes due to dye derealization is reduced.

[0020] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0021] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Brief Description of the Figures

[0022] For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

[0023] Fig. 1 is an illustration of the synthesis of an iodo containing boron polylactide polymer, BF₂dbm(I)PLA, (P1-P3). b-e, Steady-state emission spectra of polymers P1-P3 as powders (b and c) and spin-cast films (d and e) under air (b and d) and under N₂ (c and e). f- g, Simple-cast P1-P3 (right to left) films in vials under air (f) and N₂ (g) (UV excitation: X_6x = 365 nm).

[0024] Fig. 2a is an illustration of emission spectra of the spin-cast film (Pl) under increasing oxygen levels (indicated by arrow, 0-1%) normalized to the fluorescence band. Fig.2b, is an image showing the yellow phosphorescence emission under an N₂ gas stream for a spin-cast Pl film under UV excitation. (Yellow phosphorescence turns on immediately upon gas contact. Blue green background = weak Pl fluorescence.) Fig. 2c, illustrates the linear relationship between oxygen level and the F/P intensity ratio at two fixed wavelengths (450 nm and 525 nm).

[0025] Fig. 3 is in vivo imaging of the breast cancer 4Tl mammary carcinoma tumor region in a mouse window chamber model showing the brightfield (3a), and boron nanoparticle F/P ratio while breathing carbogen-95% O₂ (3b), room air-21% O₂ (3c), and nitrogen-0% O₂ (3d). Fig. 4 is an illustration of the absorption spectrum of BF₂dbm(I)PL A (1) in CH₂Cl₂.

[0026] Fig. 5. is an illustration of the emission spectra of BF₂dbm(I)OH (1) and

BF2bm(I)PLA (P1-P3) in CH₂Cl₂. (Note: Spectra for P1-P3 overlap.)

[0027] Fig. 6 is an illustration of the oxygen sensitivity calibration for the Pl film.

Relationship between the oxygen level (0-21%) and the F/P emission intensity ratio at two fixed wavelengths (450 nm and 525 nm).

[0028] Fig. 7 is an illustration of emission spectra of P2 boron nanoparticles in aqueous suspension under increasing oxygen levels (0-21%, indicated by the arrow) normalized to the fluorescence band.

[0029] Fig. 8 is an illustration of oxygen sensitivity calibration for the P2 nanoparticles.

[0030] Fig. 9 is an illustration of the chemical structures of BF₂dbm(I)PLLA (1) and mPEG-ό-PDLA (2) used to prepare pegylated BNPs, shown schematically. The NP corona is composed of mPEG chains (red lines), while the core contains PLLA (dashed lines) and PDLA (black lines). BF₂dbm(I) dye molecules are shown as blue spheres within the stereocomplex core.

[0031] Fig. 10 is an illustration of the UV-vis absorption spectra for compound 1 (Fig. 9) (-3.7 μM) and freeze-dried nanoparticles (BNP, -3.4 μM) dissolved in CH₂Cl₂ (top) and for the stereocomplexed nanoparticles in aqueous suspension (-0.13 mg/mL; bottom).

[0032] Fig. 11 is an illustration of normalized emission spectra for freeze-dried BNPs in CH₂Cl₂ solution (black) and as an aqueous BNP suspension (dashed: fluorescence; gray:

phosphorescence). The phosphorescence spectrum was collected under N₂. The fluorescence λex was 369 nm, while the phosphorescence λ_eX was 391 nm. [0033] Fig. 12 is an illustration of the emission spectra of aqueous BNP solution under increasing oxygen levels (indicated by the arrow, 0-2.6% in N₂) normalized to the

fluorescence λp.

[0034] Fig. 13 is an illustration of the linear relationship between the

fluorescence/phosphorescence (F/P) intensity ratio at two fixed wavelengths (450 nm and 530 nm) and oxygen concentration.

[0035] Fig. 14 is an illustration of a plot of BF₂dbm(I) dye degradation over time as measured by UV -Vis, ¹H-NMR, and fluorescence spectroscopy.

[0036] Fig. 15 is an illustration of fluorescence images showing BNP uptake (a) prior to injection and (b) 10 minutes after injection, (c) An overlay of fluorescence with transmission image, (d) A plot of fluorescence emission over time for vascular arid surrounding tissue. All images are 1.55 x 1.55 mm.

[0037] Fig. 16 is an illustration of the oxygen sensitivity calibration plot for aqueous stereocomplexed boron nanoparticles. The relationship between oxygen concentration and fluorescence/phosphorescence intensity ratio at two fixed wavelengths (450 nm & 530 nm) is shown for oxygen concentrations ranging between 0-21%.

[0038] Fig. 17 is an illustration of the UV signal of GPC spectra showing the presence of high-molecular-weight compounds present in BNP samples after several weeks. The arrow denotes the peak shoulder where the aggregated/cross-linked species is observed.

[0039] Fig. 18 is an illustration of the emission spectra of aqueous BNP suspensions showing a decrease in RTP (solid arrow) over several weeks, as well as a blue-shift in fluorescence (dashed arrow). Spectra were obtained after purging BNP solutions with N₂ and normalized against fluorescence λ_em.

[0040] Fig. 19 is an illustration of in vivo ratiometric imaging of tissue oxygenation in mouse window chamber tumor model (breast cancer 4Tl mammary carcinoma xenograft). Data is plotted as the fluorescence (430-480 nm) to phosphorescence (530-600 nm) intensity ratio. Dark streak on left = highly oxygenated blood vessels. Light area (middle) and upper right = hypoxic tumor region. [0041] Fig. 20 is an illustration of emission spectra of the amine (i.e. piperidyl) dyes illustrated in Table 7.

[0042] Fig. 21 is an illustration of a dye blended with PLA in vial w/wo H⁺ in Nitrogen.

Detailed Description

[0043] Boron substituted compounds (e.g., boron difluoride) can be bound to functionalized diketones (e.g., dibenzoylmethane) and used as initiators for polymerization of lactide and other monomers. Luminescent dye groups are introduced in the polymeric material (e.g., biodegradable and biocompatible polylactide) on specific sites in the polymer architecture with control. Block copolymers capable of nanoscale self assembly are possible. The disclosed compounds and compositions of the invention are useful as imaging agents, probes, readily processable photosensitizers, sensors (e.g., oxygen, ratiometric, both intensity and lifetime based; temperature; moisture; pH), laser dyes, optical fibers, waveguides, light emitting materials for displays, biocompatible polymers, solvatochromic materials, lithographic materials, photodegradable materials, photoactivated oxidizing agents, colorants, inks, reactive dyes, and the like. The photosensitizers can be used to produce a beneficial effect in photodynamic therapy for treatment of tumors and other conditions. The disclosed materials are useful even when phosphorescence feature is quenched and only fluorescence is present at higher O₂ conditions. For example, the fluorescence properties alone are useful as cellular imaging agents, interoperative probe in surgery, etc. If the conditions change and the situation becomes hypoxic, the phosphorescence will light up too, adding another useful feature to fluorescence imaging alone. If the oxygen levels drop, that can turn on

phosphorescence, the second optical feature, and activate an oxygen sensing capability too, making the materials dual functional as imaging agents and sensors, i.e. as materials that fluoresce (internal standard, optical imaging) and phosphoresce (optical imaging at a different wavelength and O₂ sensing).

[0044] The dual emissive dye-polymer compounds or compositions (materials) are useful as optical probes and imaging agents with and without the sensing feature. At high oxygen concentrations the fluorescence is still present but the phosphorescence may be quenched. In this case, the fluorescence is useful for imaging, i.e., the location of the material can be followed or viewed (e.g. in a cell, sub-cellular compartment, tissue, organ, in vivo, as a molecular probe, interoperative probe). This aspect is further enhanced if the material is also modified with a targeting group, to direct the material to a given cell, tissue, organ, etc. For agents having specific groups are known for targeting and/or facilitating uptake in cells for identification or diagnosis or treatment of cancer, e.g., colon cancer. The agents can be used to identify and diagnose cancer, for instance, but hypoxia is also a marker that can be used to monitor the effectiveness of therapies (e.g., chemotherapy, radiation, etc). For example, PLA- X or PLA-PEG-X systems where X = conjugates which are known to target colon cancer such as folate (folate receptor) (See e.g., Low P, et al. Curr Opin Chem Biol. 2009;13(3):256-62 or Tsai H, et al. Biomaterials. 2010;31(8):2293-301.), galactose (galectins) (See e.g., Minko T., Adv Drug Deliv Rev. 2004;56(4):491-509, Nagasaki Y, et al, Biomacromolecules.

2001;2(4):1067-70, Kassab R, et al, Bioorg Med Chem. 2002; 10(6): 1767-75 or Otsuka H, et al, Adv Drug Deliv Rev. 2003;55(3):403-19.), indomethacin (COX-2), (See, e.g., Uddin M, et al., Cancer Res. 2010;70(9):3618-27.), or to facilitate colon uptake, lectins such as the wheat germ agglutinin (WGA) (See, e.g., Gao X, et al, Biomaterials. 2006;27(l 8):3482-90.) or Biotin-PEG-PLA analogues open up many possibilities, via biotin-streptavidin

technologies (See e.g., Pulkkinen M, et al, Eur J Pharm Biopharm. 2008;70(l):66-74.).

[0045] The targeting groups may be combined with the dye-polymer compounds and dye polymer compositions in a number of different ways. First, it may be chemically attached to the dye-polymer compound through a bond most commonly to the polymer (e.g. end group, side chain) though it could also be attached through some aspect of the dye or one of its substituents. For the dye polymer composition or blend, it could be attached to the polymer (end group, side chain) that is blended with the dye, or to some component of the dye that is blended with the polymer or support. This chemical attachment (covalent or non-covalent) could occur before or after fabrication of the dye-polymer systems as particles, fibers, films, bulk materials, etc. Alternatively, the targeting group could be covalently attached or in some way associated, non-covalently, to another separate polymer or carrier (e.g. lipid) for blending with the dye-polymer systems in the fabrication step to make particles, films, fibers, bulk materials, etc. It could also be applied as a surface coating on the dye-polymer material. [0046] In the case of materials having ionizable groups, if the state of ionization changes (e.g. protonation or deprotonation, metal bound or not), the emission spectra will respond accordingly. Fluorescence (Fl) will usually shift to a different wavelength (F2) upon analyte binding regardless of environment oxygen levels but the phosphorescence spectra response may be more complicated at low oxygen levels. Specifically, when the sensor molecule is not bound to an analyte, the phosphorescence (Pl) may or may not be present depending on the aryl groups of the dye. IfPl is absent, a phosphorescence band (P2) may appear, e.g. when the sensor dye binds to the proton or ion analyte. That is, it will be turned on, in addition to the fluorescence change (from Fl to F2). If Pl is present, and when the sensor dye binds to the analyte, the phosphorescence usually shifts to a different wavelength (Pl to P2) in addition to the fluorescence change (from Fl to F2). In this case, both peaks, fluorescence and phosphorescence can shift and report on binding or dissociation events (e.g., proton or metal ion) and phosphorescence can also report on oxygen levels too.

[0047] In these respects the materials are multi-functional. They are probes/imaging agents reporting on the location of the luminescent material and its concentration in a given location, and they are sensors, in that they can respond to analytes or physical conditions, and these features may be present separately or together. For example, in the case of turn on sensor materials, there is no or low fluorescence imaging until sensing is activated, i.e.

BF₂dbm(I)PLA materials with low polymer molecular weights. It may be hard to see the material in vivo at first, since fluorescence is so weak, until the oxygen levels drop and the tissue becomes hypoxic and low in oxygen, then the phosphorescence is visible (lights up). In this case, sensing and imaging (i.e. on the phosphorescence channel) are activated at once, because phosphorescence serves as an optical imaging feature too, but at a different more red- shifted wavelength than fluorescence typically. Balanced invariant fluorescence and oxygen quenchable phosphorescence may be achieved by using intermediate polymer molecular weights, and in this case, nanosensors fabricated from the materials are ideal for ratiometric sensing. In this case, fluorescence is present to image and help locate the material and quantify it in a given place (i.e. as a standard), and the phosphorescence intensity varies depending on the level of oxygen that is present. The phosphorescent peak intensity is high for low O₂ levels, and it is low or quenched for high O₂ levels. Furthermore, if pH or the ion sensing feature is added to molecular weight tuning, proton or ion binding may also alter the fluorescence to phosphorescence ratio at low oxygen conditions. For example, strong dye-dye interactions can enhance phosphorescence for lower molecular weight samples (high dye loadings in dye/polymer blends), upon protonation, the interactions are diminished and this may decrease relative phosphorescence intensity.

[0048] The disclosed compounds and compositions (materials) are sensors for oxygen (particularly, at low oxygen levels; that is, you can detect the presence of oxygen, because the phosphorescence is quenched, alternatively, you can detect the absence of O₂, because the material will light up, phosphorescence increases). The disclosed materials are suitable as sensors and imaging agents for conditions/situations that produce low oxygen conditions (hypoxia, anoxia). To illustrate this point, when nitrogen gas is passed over a film (e.g., a coating on glass surface) of a polymer, the gas flow patterns light up and may be imaged. For example, "one could write with a nitrogen gas pen". Thus, the disclosed compounds and compositions can "image nitrogen". In addition, other non-oxygenated gasses, e.g., natural gas, can be "imaged". The action, of contacting the disclosed materials with O₂ free gases eliminate O₂ from the immediate environment and can cause the phosphorescence to increase.

[0049] The disclosed compounds and compositions (materials) are also suitable as molecular probes, e.g., for fluorescence emission. These materials are useful in cells in vitro, in tissue ex vivo, or for in vivo contexts. Non- limiting examples of probes include

interoperative probes, such as, imaging agents used in the operating room, during surgery, and the like. The materials may be conjugated (bonded) or associated by assembly or aggregation with active targeting groups to form bioconjugates which can be used to target specific cells or organs. The bioconjugates can be used to deliver the materials to particular cells and tissues and organs for e.g., surface attachment via a receptor or chemical or ionic association, or for cellular uptake. Examples of targeting groups include but are not limited to materials such as folate, streptavidin, RGD analogues, aptamers, peptides selected by phage display, proteins, biotin, antibodies, galactose and other carbohydrates, tumor antigens, wheat germ aglutinin, and other such groups for more selective delivery to particular cells, tissues, and organs.

[0050] When the oxygen levels drop in a cell, tissue, or in vivo context, then the

phosphorescence will light up. Thus, the materials function as a fluorescent probe, and indicate another aspect of the environment as well. The disclosed materials (e.g. fabricated as nanoparticles) provide combined spatial and temporal resolution for hypoxia imaging and generating oxygen maps of tissues by this optical method. Thus, the materials can be used for optical imaging and detection.

[0051] The nanoparticles can have diameters of from about 20 nm to about 1000 nm.

Preferably, nanoparticles can have diameters of from about 50 nm to about 500 nm. More preferably, nanoparticles can have diameters of from about 50 nm to about 150 nm. The sizes of the nanoparticles were determined using dynamic light scattering (DLS). DLS measurements showed that when the DMF/THF solvent phase was used, the resulting BNPs possessed a hydrodynamic diameter (Dh) of 120 ± 20 nm, with a polydispersity (Pd) of 0.28 ± 0.05. However, when DMF was used as the solvent phase, both Dh and Pd of the BNPs were reduced to 83 ± 7 nm and 0.17 ± 0.03, respectively. These findings correlate well with previous studies, as the use of DMF as the solvent phase has been shown to result in NPs with diameters ranging from 40-80 nm, whereas the use of THF gives rise to larger NPs (-240 nm). Acetone is another solvent commonly used for generating nanoparticles by

nanopreciptation (i.e. solvent displacement).

[0052] The disclosed compounds and compositions (materials) are useful in situations or uses that require the elimination or drastic reduction of oxygen levels and validation that the low oxygen levels or oxygen-free conditions have been maintained. Non-limiting examples of articles that can use the disclosed compounds or compositions include but are not limited to packaging for products that degrade in the presence of oxygen, such as food, drugs, components for the electronics industry and other reagents and materials that are air sensitive. Security applications where it is important to know in an easy read out if a package has been opened or tampered with (e.g. packed under vacuum or inert gas and phosphorescence is present. If opened or compromised, then oxygen is present and the phosphorescence afterglow is quenched and absent.)

[0053] Different dye-polymer compounds of formula I and dye-polymer compositions may be used in combination to generate composite materials with optimized properties. This may involve mixing prior to fabrication, e.g. as nanoparticles, or fabricating each separately, e.g. as nanoparticles, and the mixing the two or more nanoparticle batches after fabrication. This can provide a complete range of properties. For example, one nanoparticle batch may have one dye compound/composition and/or targeting group associated with it and another material may have another dye compound/composition and/or targeting group. If mixed, the two kinds of particles may be administered together, at the same time with the same injection or administration, but then be delivered to different cells or tissues, or subcellular compartments upon cellular uptake for in vivo or in vitro, basic research or analytical applications.

[0054] Nanoparticles prepared from the disclosed luminescent dye polymers and dye compositions can be taken up and internalized by cells, which can be used for research or diagnostic purposes such as imaging. Also, the leaky vasculature of tumors allows for passive targeting via the enhanced permeation and retention (EPR) effect, with the possibility for imaging. Active targeting to tissues is possible by conjugating compounds to the materials that specifically target cells and tissues and organs. The oxygen sensing ability of the luminescent dye polymers and compositions will allow analysis using equipment available for oxygen sensing or imaging. Generation of singlet oxygen (e.g., via phosphorescence quenching) can serve as a photodynamic therapy.

[0055] The disclosed luminescent dye polymers and dye compositions can be used to prepare oxygen sensors that can be placed in food or drug packages and allow measurement of oxygen within the package. The luminescent dye polymers and compositions can be used as a film, on fibers within the packaging, etc.

[0056] Typically the dyes are excited or activated by UV irradiation. They are also 2- photon absorbers, and compatible with multiphoton microscopy as illustrated in cell uptake studies with boron nanoparticles (See Contreras ACS Nano Vol. 4, 2735-2747, 2010). That is, the dye compound/compositions can be activated or excited using a laser at -800 nm, and they will emit fluorescence at about 400 nm. Additionally, they can be activated or excited with radiation of the kind used in cancer radiation therapy.

[0057] The following definitions are used, unless otherwise described: halo includes fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl" embraces only the straight chain radical, a branched chain isomer such as "isopropyl" being specifically referred to. Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms. The heteroatoms include non- peroxide oxygen, sulfur, silane, nitrogen and phosphorous wherein suitable substituents as known in the art can be attached to the hetero atoms, e.g., hydrogen, O, (CrCi₂)alkyl, phenyl or ben2yl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.

[0058] As used herein the term "color tuning" refers to changing the ligand or polymer composition and molecular weight of the dye-polymer conjugate (or dye ligand and the dye loading for dye/polymer blends/compositions) in order to modify the wavelength {e.g., peak position) of the light that is emitted. Additionally, the color that is visible to the eye is the combined effect of the fluorescent and phosphorescent emission at different wavelengths.

[0059] As used herein the term "intensity tuning" refers to the magnitude or height of the fluorescent and phosphorescent peaks at given wavelengths. The fluorescent/phosphorescent tuning for ratiometric sensing and imaging, means tuning the relative heights of fluorescent and phosphorescent peaks in emission spectra. In this respect, intensity tuning is different than color tuning, which refers to the position (i.e. wavelength) of the peaks along the spectrum.

[0060] It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms.

Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

[0061] Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only. They do not exclude other defined values or other values within defined ranges for the radicals and substituents. Specifically, (Ci-Ci₂)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl and the like; (C₃-

Ci₂)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl and the like; (C₃- C₁₂)cycloalkyl(C₁-C₈)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2- cyclohexylethyl and the like; (Ci-C₁₀)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy and the like; (C₂-C₁₂)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2- pentenyl, 3-pentenyl, 4-pentenyl, 1- hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl and the like; (C₂-C i₂)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3- butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1- hexynyl, 2-hexynyl, 3-hexynyl, A- hexynyl, or 5-hexynyl and the like; (Ci-Ci2)alkanoyl can be acetyl, propanoyl or butanoyl and the like; halo(Ci-C₆)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl and the like; hydroxy(d-C₁₂)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1- hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1- hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl and the like;

(C₁-C₁₂)alkoxycarbonyl can be methoxy carbonyl, ethoxy carbonyl, propoxy carbonyl, isopropoxy carbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl and the like; (Ci-C₁₂)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isoburylthio, pentylthio, or hexylthio and the like; (C₂-Ci₂)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy and the like; (C₆- C₂₂)aryl can be phenyl, naphthyl, anthrcyl, phenanthryl, pyryl, naphthacyl, pentacyl, or indenyl and the like; and (C₅-C₁₃)heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, tbiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide) and the like.

[0062] The polymers, Q, that are conjugated to form the formula I compounds or blended with the formula II compounds include any polymeric material that can be conjugated or blended with a boron containing dye compound. In one embodiment, non-toxic

pharmaceutically acceptable, biologically stable (or biodegradable ) polymers are preferred. Non-limiting examples of pharmaceutically acceptable polymers include polylactide (PLA), polyglycolide, lactide-glycolide copolymer, polycaprolactone, or polyethylene glycol polylactide polymers, polyhydroxybutyrate (PHB), polyhydroxybutyrate-valerate copolymer (PHBV), polybutylene succinate (PBS), polybutylene adipate-co-terephthalate (PBAT), sugar based polymers (e.g., cellulose or starch and the like), peptides, or mixtures thereof. Other exemplary polymers include polyurethanes, polyamides, polyesters, and vinylic polymers. Non-limiting examples of vinylic polymers include acrylates such as polymethyl methacrulate (PMMA), acrylonitrile butadiene styrene (ABS), styrene acrylonitrile (SAN), polystyrenes (PS), polyethylene (PE), polyethylenechlorinates (PEC), polybutadiene (PBD),

polydicyclopentadiene (PDCP), polypropylene (PP) Polymethylpentene (PMP), and the like. Other exemplary polymers include silicon-based organic polymers such as

polydimethylsiloxane (PDMS), polyesters such as polyethylene terephthalate (PET), glycolized polyester (PETG), polycarbonate (PC) and the like.

[0063] Additional exemplary polymers or matrices that can be prepared as Q groups or blended with the light emitting compounds include silica, sol gels, aerogels, xerogels cellulosic polymers, e.g., hydroxypropylmethylcellulose, hydroxyl propyl cellulose, ethyl cellulose and the like; epoxy containing polymers, Ethylene vinyl alcohol, (E/VAL), fluoroplastics, e.g., polytetrafluoroethylene (PTFE), liquid crystal polymers, (LCP), melamine formaldehyde, (MF), phenol-formaldehyde plastic (PF), polyacetal, polyacrylates, polymethacrylates, polyacrylonitrile, (PAN), polyamide, (PA), e.g., nylon, polyamide-imide (PAI), polyaryletherketone (PAEK), polyetheretherketone (PEEK), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PTA), Polysulfone (PSU), polyurethane (PU), polyurea, polyvinylchloride (PVC), polyvinylidene Chloride (PVDC), polyvinylidenedifluoride (PVDF) silicone polymers, poly(ethylene glycol) (PEG), poly(ethylene terephthalate) (PET), polysiloxanes, silicones.

[0064] In one embodiment, the composition includes pharmaceutically acceptable polymers, FDA approved polymers or a mixture thereof. In another embodiment, the compositions include polymers prepared from vinyl monomers known in the art. In another embodiment, he invention also provides pharmaceutical compositions comprising a compound of luminescent dye having formula II, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable diluent or carrier.

[0065] The compounds having formula I can be conjugated to a polymer through any suitable functional linking group. For example the conjugate can include dye-X, dye-X-Y, dye-X-R-Y, where X and Y can be initiators, terminators or coupling partner with a complementary reactive group on a polymer (e.g., at the chain end, main chain, side group, etc.). Alternatively, the diketone and the polymer may be linked to generate a "macroligand" (e.g., dbmPLA or dbmPMMA), which is subsequently reacted with a B (e.g. Y¹) precursor such as BF₃ to generate the luminescent material (e.g., BF₂dbmPLA or BF₂dbmPlVIMA). A reactive group (initiator group) can be placed in a formula II compound using any means known in the art. The initiator groups can react with monomers, polymers or oligomers to form at least one polymer chain. In some cases the initiator can be part of the R¹, R², R⁵, X¹ or X² and used for direct coupling, (e.g., initiation). Examples of initiator groups include primary alcohol linking group (e.g., a group having the formula -(CH₂)_Z-OH, where z is an integer from 1 to about 25; or -O-R⁶-O-H where R⁶ is alkylene, or alkenylene having at least two carbon atoms). Exemplary alcohol containing groups include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, -0-CH₂CH₂-OH or ArOH, and the like.

Polymer groups prepared from vinyl groups can use a radical forming linking group (e.g., a diazo or peroxy group). Other exemplary initiator groups include thiocarbonylthio compounds, such as dithioesters, dithiocarbamates, trithiocarbonates, xanthates, and the like.

[0066] Examples of linking groups for attaching the dye compounds to the polymer include groups having the formula -O-R⁶-O-, -NR²¹-R⁶-O, or -NR²¹-R⁶-NR²¹- -S-R⁶-O-, -S-R⁶-S-, -O-R⁶-S-, or -NR²¹-R⁶-S- -S-R⁶-NR²¹- where R⁶ is alkylene, alkenylene having at least two carbon atoms and R²¹ is hydrogen, alkyl, alkenyl, (C₃-C ₁₂)cycloalkyl, aryl, aralkyl or haloalkyl.

[0067] The polymer chains can be formed using any compatible polymer synthesis method known in the art such as; 1) Nitroxide-mediated polymerization (NMP); 2) Reversible addition-fragmentation chain transfer (RAFT) polymerization using compounds having thiocarbonylthio initiator groups, such as dithioesters, dithiocarbamates, trithiocarbonates, and xanthates; 3) ATRP: using compounds having activated RCH₂X initiator groups where X is a halogen (e.g., chlorine or bromine), α-haloesters such as α-bromobutyrolactone, allyl chloroacetate, vinyl chloroacetate, hydroxyethyl 2-bromobutyrate, t-butyl 2-bromobutyrate, glycidol 2-bromopropionate, and the like or α-haloamides such as 2-chloroacetamide and the like. Ring opening polymerization methods can use also compounds having alcohols or metal alkoxide, and carboxylic acid or metal carboxylate as initiator groups. Cationic

polymerization methods can uses compounds having alkyl halide, tosylate, Lewis acid or alcohol initiator groups. Anionic polymerization reactions can use compounds having alcoholic or nucleophilic base initiator groups, such initiators for anionic polymerization are known in the art.

[0068] The molecular weight of the polymer group, Q, or polymeric compound can be modified to have number average molecular weight from about 150 Da to about 1,000,000 Da for PLA. Preferably, the polymer group, Q, or polymeric compound (e.g. PLA) can be modified to have a number average molecular weight from about 500 Da to about 100,000 Da. More preferably, the polymer group, Q, or polymeric compound (e.g. PLA) can be modified to have a number average molecular weight of from about 1000 Da to about 15,000 Da. [0069] The invention includes dimers, e.g., compounds where R³ and R⁴ taken together with the Y¹ atom form a chelate ring having the formula:

e.g., R³ and R⁴ form a ring together with the atom to which they are attached to provide a compound having formula III

III

where each R¹, R², X¹, X² and R⁵ are independently selected from the definitions above. Additionally, R³ and R⁴ taken together form a bidentate chelate, such as deprotonated acid or diacid group, HOC(=O)CH₂C(=O)OH (malonic acid) or HOC(=O)CH₂C(=O)OH (oxalic acid), silica group, or chelating group such as acid-alcohol, acid-ether, with two donor groups, or R³ and R⁴ taken together with the boron atom form a ring having the formula:

[0070] Preferred R² groups include phenyl, naphthyl, anthracyl or have the formula

where each X⁴ is independently O, S, NR^a, or PR^a, where R^a is hydrogen, alkyl or aryl.

[0071] More preferred R¹ and R² groups include phenyl, naphthyl, anthracyl or have the formula:

[0072] Even more preferred R² groups include phenyl, or naphthyl.

[0073] Specific substituents include halo, hydroxy(Ci-Cι₂)alkyl, halo(Ci-C]₂)alkyl, R¹⁵O(C₁-C₁₂)alkyl,

-OR¹⁵, -CO₂R¹⁵, -OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, - N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, or -SO₂R²⁰.

[0074] Additional substituents include a Lewis acid, or a Lewis base functional group.

[0075] Additional functional groups include a carboxylic acid, or an amine.

[0076] Preferred R³ and R⁴ substituents are halo, hydroxy(C₁-C₁₂)alkyl, phenyl, halo(Ci- Ci₂)alkyl, R¹⁵O(C,-C₁₂)alkyl, R¹⁵O(C,-Ci₂)alkyl-O-, -OR¹⁵, -CO₂R¹⁵, -OC(O)R¹⁶, -C(O)R¹⁶, - NR¹³R¹⁴, -N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, or -SO₂R²⁰.

[0077] Additional preferred R³ and R⁴ substituents include electron- withdrawing groups such as halo, e.g., fluorine.

[0078] Additional preferred R³ and R⁴ substituents include electron-donating groups such as OR¹⁵.

[0079] The polymeric luminescent dye compounds and compositions can be processed into various forms such as a powder, film, particle, fiber, coating, gel, network, assembly, suspension or composite. Non-limiting examples include a film, nanoparticle, nanofiber or nanoscale assembly. The nanoparticles, films, coatings, fibers or nanofibers can be used in articles such as medical devices or oxygen sensors. The sensors can be used e.g., to detect low levels of oxygen in, e.g., blocked vasculature, hypoxic tumors. Typical sensors include, an oxygen sensor, a pH sensor, or a temperature sensor.

[0080] The invention provides a method for detecting the presence or absence of oxygen in gas, liquid, or solid or mixtures thereof comprising contacting the gas or liquid or solid, or mixtures thereof, with the disclosed compounds or a compositions and viewing the presence or absence of delayed emission phosphorescence or delayed emission (e.g. phosphorescence and/or delayed fluorescence) from the compound or composition. [0081] The materials can be used for ratiometric sensing or imaging in a diagnostic method for oxygen sensing or imaging where a test substance is contacted with a disclosed dye compound or composition and detecting the pθ₂ levels. In these techniques the fluorescence can be used as a standard and the phosphorescence can be used to determine the pθ₂ level. This can be important, e.g., in in vivo oxygen sensing or imaging in a mammal. The disclosed dye materials can be used for oxygen sensing or imaging of tumors, vasculature, wounds, brain imaging, high altitude drug testing, monitor drugs that delivery oxygen to tissues, organ transplantation or tissue transplantation, or cell transplantation, tissue engineering, cells, e.g., stem cells, or other tissues. The diagnostic imaging can provide an oxygen concentration map of tissues examined.

[0082] A preferred method for using the compound or composition is for ratiometric sensing or imaging.

[0083] Diketone synthesis is modular. Different R¹ and R² groups may be added to either side of the diketone to modulate optical properties (e.g., luminophores). Additionally, the R¹ and R² can contain a linker group (e.g. initiator, terminating agent, coupling partner, targeting group for particular cell or tissue types) or a group that can be readily converted using standard chemical techniques to an initiator site. Various commercially available starting compounds that have initiator sites may be readily used or modified to form compounds having formula I. The compounds having formula II can be modified to tune polymer architecture, materials and optical properties. Block copolymers can be also prepared, by sequential monomer addition or by modifying dibenzoylmethane and related diketones with two different kinds of initiator sites. These can self assemble to generate nanostructured films, bulk materials, solution assemblies, particles, etc. Other polymer compositions can be also prepared, e.g., by varying the initiator group. A preferred initiator group for lactide or caprolactone ring opening polymerizations is a primary alcohol. Alpha bromoesters are good initiator groups for ATRP, affording PMMA and other polymers. The diketone ligand molecules can be readily prepared using standard methods known to a person skilled in organic synthesis. In addition many ligands are commercially available and have groups that can be used or transformed into initiator groups. Exemplary ligand molecules include molecules having a heavy atom or ionizable group and general formulas below:

[0084] Synthesis of the boron polymer, e.g., BF₂dbm(I)PLA, begins with l-{4-[2- (tetrahydropyran-2-yloxy) -ethoxy]-phenyl}-ethanone and methyl p-iodobenzoate. The reagents are added to sodium hydride in THF and heated at reflux for four hours. The reaction was cooled and quenched by addition of a saturated aqueous solution of sodium bicarbonate and then acidified with 3M HCl. THF was then removed in vacuo and the residue was extracted with ethyl acetate. The product was dissolved in THF (50 mL) and water (15 mL) in the presence of 15 mg TsOH as catalyst and was heated at 55 ⁰C for 18 h. The reaction mixture was concentrated in vacuo at 30 ⁰C to remove THF, the white solid in aqueous layer was then collected by filtrated and thoroughly washed by water. [0085] The boron polymer, BF₂dbm(I)PLA, 2, is generated from BF₂dbm(I)OH and DL- lactide using tin octoate, Sn(oct)2, as the ROP catalyst under solvent-free conditions

(l:lactide:Sn(oct)₂ = 1:200:1/50) with heating at 130 °C under nitrogen. The reaction is stopped after 1.5 hours (-50% monomer conversion) to avoid broader molecular weight distributions (i.e. higher PDIs) noted for longer reaction times, suggestive of

transesterifϊcation and thermal depolymerization. After purification by precipitation from CH₂Cl₂/cold MeOH and CH₂Cl₂/hexanes, a pale greenish yellow polymer is obtained (75% yield, corrected for monomer consumption). The synthesis is illustrated in Fig. 1.

[0086] Iodide-substituted difluoroboron dibenzoylmethane-poly(L-lactide)

(BF₂dbm(I)PLL A, 2) and methoxy-terminated polyethylene glycol)-δ-poly(D-lactide)

(mPEG-PDLA, 3) (Fig. 9) were synthesized by ring opening polymerization using the catalyst Sn(oct)₂ as described above. Nanoparticle fabrication was performed using the

nanoprecipitation method where both 2 and 3 were dissolved in a water-miscible solvent or solvent mixture (e.g., DMF, THF, acetone), followed by drop-wise addition of the polymer solution to water, leading to NP formation, as shown in Fig. 9. For these studies, DMF and DMF/THF (10:1 v/v) were chosen as the solvent phases. DMF is commonly chosen as the solvent phase due to its propensity to create NPs with small (< 100 nm) diameters; however, the DMF/THF binary solvent mixture was also tested due to the poor solubility of PEG in DMF. The organic phase was subsequently removed from the aqueous medium by means of dialysis. Following fabrication, the size, optical properties, and dye/polymer stability of the stereocomplexed BNPs were studied.

Examples

[0087] Materials. 3,6-Dimethyl-l,4-dioxane-2,5-dione (D,L-lactide, Aldrich) was recrystallized twice from ethyl acetate and stored under nitrogen. (3S)-cis-3,6-Dimethyl-l,4- dioxane-2,5-dione (L-lactide, Aldrich) was recrystallized twice from ethyl acetate and stored under nitrogen. D-lactide (99.5%) was a generous gift provided by Purac. Polyethylene glycol 2000 monomethyl ether (mPEG, Fluka) was azeotropically distilled from toluene, freeze-dried from benzene, and stored under nitrogen. Solvents, CH₂Cl₂ and THF, were dried and purified by passage through alumina columns. Tin(II) 2-ethylhexanoate (Sn(oct)₂, Spectrum), boron trifluoride diethyl etherate (Aldrich, purified, redistilled) and all other reagents and solvents were used as received without further purification. Syringe filters (13 mm, disposable filter device, 0.2 μm nylon filter membrane) were obtained from Whatman. All other reagents and solvents were used as received without further purification.

[0088] Methods. ¹H NMR (300 MHz) spectra were recorded on a Unitylnova 300/51 instrument in CDCl₃ unless indicated otherwise. ¹H NMR spectra were referenced to the signal for residual protio chloroform at 7.26 ppm and coupling constants are given in hertz. UV/vis spectra were recorded on a Hewlett-Packard 8452A diode-array spectrophotometer. Fluorescence spectra were measured on a Spex Fluorolog 2+2 spectrofluorometer. Molecular weights were determined by GPC (THF, 20 °C, 1.0 niL/min) vs polystyrene standards with RI and UV/vis detection (λ = 396 nm), and a correction factor of 0.58 was applied to all data. Reported yields are corrected for monomer consumption. Polymer Labs 5μm mixed-C columns along with Hewlett-Packard instrumentation (Series 1100 HPLC) and Viscotek software (TriSEC GPC Version 3.0, Viscotek Corp) were used in the GPC/RI or GPC/UV analysis. Elemental analysis was performed by Atlantic Microlab, Inc., Norcross, GA.

[0089] Molecular weights were determined by gel permeation chromatography (GPC) (THF, 20 °C, 1.0 mL/min) versus polystyrene standards on a Hewlett-Packard instrument (series 1100 HPLC) equipped with Polymer Laboratories 5 μm mixed-C columns and connected to UV- vis and RI (Viscotek LR 40) detectors. A 0.58 correction factor was applied. Data were processed with the OmniSEC software (version 4.2, Viscotek Corp). UV-vis spectra were recorded on a Hewlett-Packard 8453 diode array spectrophotometer.

Photographs were taken in the dark using a Canon PowerShot SD600 Digital Elph camera with the automatic setting (no flash).

[0090] Powders were analyzed as precipitated. A Laurell Technologies WS-650S spin- coater was used to cast polymer films for luminescence measurements with the default setup (30 s at a constant speed of 4000 rpm). Boron polymer films were spin-cast from CH₂Cl₂ solutions (-2.5% w/w) onto Fischer Scientific glass cover slides (22 x 22 mm) at the spin speed of 4000 rpm. The coated slides were cut into ~3 mm x 22 mm strips, placed in a transparent glass vial under a N₂ atmosphere, and were sealed with Teflon caps for measurements. (Note: Both borosilicate glass cover slides and glass vials are optically inactive using excitation wavelength > 368 ran.)

[0091] Steady-state fluorescence and phosphorescence emission spectra were recorded on a Horiba Fluorolog-3 Model FL3-22 spectrofluorimeter (double-grating excitation and double- grating emission monochromators). Prior to recording phosphorescence emission spectra, the aqueous nanoparticle solution (~3 mL) was purged with N₂ for 20 min in a quartz fluorimeter cell equipped with a septum screw top. Room-temperature phosphorescence (RTP) spectra were recorded under the same conditions as phosphorescence spectra except that a pulsed xenon lamp (X_6x = 369 nm; duration < 1 ms) was used and spectra were collected with a 1 ms delay after excitation. Time-correlated single-photon counting fluorescence lifetime measurements were carried out with a NanoLED-370 (369 nm) excitation source and a DataStation Hub as the single-photon counting controller. Phosphorescence lifetimes were measured with a 500 ns multichannel scalar card excited with a pulsed xenon lamp (λ_eX = 369 nm; duration < 1 ms). Lifetime data were analyzed with DataStation v2.4 software from Horiba Jobin Yvon.

[0092] Fluorescence quantum yields, Φ_F, for BF₂dbm(I)OH (1) and BF₂dbm(I)PLA (P1-P3) in CH₂Cl₂ were calculated versus anthracene in EtOH as a standard, as previously described by Crosby et al. J. Phys. Chem. 75, 991-1024 (1971), using the following values: Φ_F anthracene = 0.27, n_O ²⁰ EtOH = 1.360, n_O ²⁰ CH₂Cl₂ = 1.424. Optically dilute CH₂Cl₂ solutions of BF₂dbm(I)OH and BF₂dbm(I)PLA, and EtOH solutions of the anthracene standard were prepared in 1 cm path length quartz cuvettes, and absorbances (A < 0.1) were recorded and steady state emission spectra were obtained (λe_X = 350 nm; emission integration range: 365-700 nm).

[0093] For oxygen sensitivity and intensity-based Stern- Volmer plot measurements, gas mixtures of O₂ and N₂ of various concentrations were prepared via two Cole-Parmer 65-mm flow meters. For each concentration, the pre-mixed gas was passed through a 1 cm path quartz fluorometer cell equipped with septum screw top containing the spin-cast Pl film for 15 min and then the spectrum was recorded. The oxygen sensitivity for the P2 nanoparticle aqueous suspension was calibrated similarly except that the pre-mixed gas was first evenly dispensed into a distilled- water chamber before passing through a quartz cell containing 1 mL of the optically dilute (abs < 0.1) sample.

[0094] A window chamber was implanted using previously described techniques (See e.g., Sorg, B. S., et ah, J. Biomed. Opt. 10, 44004 (2005).) Briefly, the mouse was anesthetized using i.p. injection of 100 mg/kg ketamine, 10 mg/kg xylazine. The skin on the back of the animal was stretched into a metal frame to allow insertion of a titanium window frame, having a 12 mm diameter window. The front fact of the skin fold was excised, and approximately 20000 4Tl murine mammary carcinoma cells were injected into the underlying fascia. A cover glass was placed over the open face of the window, and the tumor was allowed to incubate for 10 days prior to imaging.

[0095] Confocal Microscopy Imaging Setup. Fluorescence and transmission images were acquired using a Leica SP5 confocal microscope. This system is equipped with an acousto- optical tunable filter for both the excitation and emission, enabling multispectral imaging with tunable wavelength ranges, as well as multiple PMT detectors for simultaneous acquisition of multiple channels. A 10x dry objective was used along with a 405 nm laser diode as the excitation source. Three separate collection channels were used: 1) bandpass emission from 420nm - 495nm (BNP fluorescence), 2) bandpass emission from 496nm - 623nm, (BNP phosphorescence) and 3) a transmission image. The scanning speed was set to 10 Hz.

[0096] Confocal Imaging Experiments. Aqueous BNPs (100 μL) were injected via the tail vein. Images were acquired prior to injection and semi-continuously monitored for -80 min post- injection.

[0097] Confocal Imaging Data Analysis. Vessels were segmented using a regional intensity threshold by selecting pixels with a lower than mean value based on a 15 pixel radius moving window. Morphological opening using linear structural elements at angle increments of 15 degrees was then done to isolate linear segments. The accumulation could then be tracked as a function of time for the vascular and surrounding tissue BNP accumulation.

[0098] An approximately 100 μL suspension of P2 nanoparticles (~1 mg/mL) was injected into the space between the window chamber and cover glass. Approximately 5 minutes passed between the injection and imaging, which allowed for stabilization of the signal. The animal was placed on an upright fluorescence microscope equipped with a dapi excitation filter, and a liquid crystal tunable emission filter (VariSpec, Cambridge Research and Instrumentation, Inc.). This allowed acquisition of an emission spectrum, which was acquired in 10 run intervals from 430-600 nm. Gas was administered by nose cone at a rate of 5 L/min, except for room air, during which the gas was shut off. The carbogen breathing was begun at the beginning of the experiment, with approximately 5 min passing prior to the initial acquisition. The gas was then switched to room air for a period of 3 min, and finally nitrogen for 30 s. The images shown in Figure 3 were taken at the end of each respective period of gas breathing. Data were processed by taking the ratio of the fluorescence to phosphorescence signals, which were defined as the average signal acquired from 430-480 nm and 530-600 nm, respectively.

[0099] Dbm(I)OH A suspension of sodium hydride (36 mg, 1.5 mmol) and THF (-40 mL) was prepared in a 100 mL, dried Schlenk flask. The NaH suspension was transferred to a 100 mL 2-neck round-bottom flask containing l-{4-[2-(tetrahydropyran-2-yloxy) -ethoxy]- phenyl}-ethanone (See Bender, J. L. et al. J. Am. Chem. Soc. 124, 8526-8527 (2002).) (200 mg, 0.76 mmol) and methyl p-iodobenzoate (200 mg, 0.76 mmol) via cannula. The resulting reaction mixture was heated at reflux for 4 h, cooled to room temperature, and then further cooled in an ice bath. The reaction was quenched by the dropwise addition of a saturated aqueous solution of sodium bicarbonate (5 mL) and then acidified (pH~3) by the addition of 3M HCl. THF was then removed in vacuo and the remaining aqueous layer was extracted with ethyl acetate (2 x 100 mL). The combined organic layer was dried over sodium sulfate, and concentrated in vacuo to give a brown, oily residue. The residue was dissolved in THF (50 mL) and water (15 mL) in the presence of 15 mg TsOH as catalyst and was heated at 55 ⁰C for 18 h. The reaction mixture was concentrated in vacuo at 30 ⁰C to remove THF, the white solid in aqueous layer was then collected by filtrated and thoroughly washed by water. The crude material was purified by column chromatography (1:1 EtO Ac/Hex) to provide the desired product as white/silvery flaky crystals: 142.3 g (46 %) ¹H NMR δ 16.90 (s, IH, enol OH), 7.98 (d, J= 9.0, 2Η, 2',6'-ArH), 7.84 (d, J= 8.8, 2Η, 2",6"-ArH), 7.68 (d, J= 8.8, 1Η, 3', 5'-ArH), 7.01 (d, J= 8.9, 1Η, 3", 5"-ArH), 6.75 (s, 1Η, COCHCO), 4.17 (t, J= 4.4, 2Η, HOCH₂CH₂OAr), 4.02 (m, 2Η, HOCH₂CH₂OAr), 1.96 (t, J= 6.3, HOCH₂CH₂OAr), Anal. Calcd for C₁₇H₁₅IO₄: C, 49.78; H, 3.69. Found: C, 49.98; H, 3.60. UV/vis (CH₂Cl₂): X_013x = 360 nm, ε_max = 35,000 M^'W¹.

[00100] BF₂dbm(I)OH (1) Dbm(I)0H (125.0 mg, 0.305 mmol) was added to a flame-dried 2-neck round bottom flask under nitrogen, and dissolved in a mixture solvent of THF/CH₂C1₂ (5/15 mL) to give a colorless solution. Boron trifluoride diethyl etherate (72 μL, 0.710 mmol) was added via syringe and the solution turned bright yellow. The flask was equipped with a reflux condenser and heated in an oil bath at 60 ⁰C (15 h). The solution was then cooled to room temperature, and then the solvent was removed on vacuo, resulting in a yellow solid. The crude material was purified by column chromatography (1:1 EtO Ac/Hex) followed by recrystallization in acetone/hexanes to give BF₂dbm(I)OH as dark yellow fine crystals: 106.3 mg (73%). m.p. 262-264 ⁰C. ¹H NMR (CDCl₃) δ 8.17 (d, 2H, J= 8.9, 2',6'-AiH), 7.92 (d, 2H, J= 8.6, 2",6"-AiH), 7.82 (d, 2H, J= 8.8, 3", 5"-AxH), 7.07 (d, J= 8.8, 2H, 3',5'-AxH), 7.06 (s, IH, COCHCO), 4.23 (t, J= 4.7, 2H, HOCH₂CH₂OAr), 4.04 (m, 2Η,

HOCH₂CH₂OAr), 1.94 (t, J= 6.3, HOCH₂CH₂OAr). HRMS Calcd for C₁₇Hi₄BO₄F₂Cl (M+Cl): 492.9692; Found: 492.9707; UV/vis (CH₂Cl₂): Xm_3x = 407 nm, ε_max = 57,900 M^-1Cm^"1.

[00101] BF₂(IbHi(I)PLA. A representative preparation for Pl is as follows: BF₂dbm(I)OH (1) (22.8 mg, 0.05 mmol), lactide (0.360 g, 2.5 mmol) and Sn(oct)₂ (0.4 mg, 1.0 μmol) (loading: 1:50:1/50) in hexanes were combined in a sealed Kontes flask under N₂. The entire bulb of the flask was submerged in a 130 °C oil bath for 30 min. Crude polymer was purified by precipitation from CH₂Cl₂/cold MeOH. The polymer was collected by centrifugation, the filtrate was decanted, and the rubbery solid was washed with additional cold MeOH (2^χ). The resulting solid was reprecipitated from CH₂Cl₂/hexanes, collected by centrifugation, washed with hexanes, and dried in vacuo to give a yellow foam: 206 mg (78%, corrected for 64% monomer conversion). M_n (GPC/RI) = 2,700, PDI = 1.11; M_n (NMR) = 4,000. ¹H NMR (CDCl₃) δ ¹H NMR (CDCl₃) 8.16 (d, 2H, J= 8.9, 2',6'-ArH), 7.92 (d, 2Η, J= 8.8, 2",6"-ArH), 7.82 (d, 2Η, J= 8.6, 3", 5"-ArH), 7.08 (s, 1Η, COCHCO), 7.04 (d, J= 8.9, 2Η, 3',5'-ArH), 5.12-5.30 (m, broad, 50 Η, PLA CH), 4.55 (m, 2Η, CH₂CH₂OAr), 4.32 (m, 2H,

CH₂CH₂OAr), 2.69 (q, 1Η, PLA OH), 1.54-1.60 (m, broad, 150 Η, PLA CH₃). UV/vis (CH₂Cl₂): λmax = 406 nm, ε = 33,400 M^-1Cm^"1, λ = 320 nm, ε = 6280 M^-1Cm^"1. [00102] In Fig. Ia, the Ring-opening polymerization of BF₂dbm(I)PLA (P1-P3) is illustrated. In Figs, lb-e, the steady-state emission spectra of polymers P1-P3 as powders (b and c) and spin-cast films (d and e) under air (b and d) and under N₂ (c and e) are illustrated. In Figs, lf-g, simple-cast P1-P3 (right to left) films in vials under air (If) and N₂ (Ig) (UV excitation: λ_eX = 365 nm) are illustrated. Reaction and molecular weight data for P1-P3 are shown in Table 1. The P1-P3 polymers are characterization data are shown in Table 2. The RTP Lifetime Data for Polymers P1-P3 are shown in Table 3.

[00103] A simple demonstration of the effects of MW is presented in Fig. 1, which shows changes in color and brightness for P1-P3 films under air (Fig. If) versus nitrogen (Fig. Ig). Thus, with a very simple MW adjustment, these boron materials are easily tailored for different sensor platforms and imaging and detection schemes.

Table 1. Polymerization and Molecular Weight Data for P1-P3

Loading³ Time" M₁(GPCyPDI M₁(NMR) Conversion⁰ Yield"

P3 1:300 3 17,600/1.17 21,700 55 66

a. Molar ratio of initiator to monomer

b. Polymerization was stopped after this time

c. Percent monomer consumption

d. Polymer product yield corrected for unreacted monomer

Table 2. Characterization of polymers P1-P3 in solution.

M_n (Da)^a PDI^b λ_abs(nm)^c E(M-¹Cm^"1)⁰ λ_em(nm)^d Φ_F ^e TF(DS)*

1 458 1 407 58,000 441 0.55 1.03

Pl 2700 1.11 406 33,000 435 0.41 0.95

P2 7300 1.15 406 40,000 436 0.44 0.96

P3 17600 1.17 406 40,700 435 0.43 0.95 a. In THF vs polystyrene (PS) standards

b. Polydispersity index (PDI) = MJM_n

nm

d. Steady-state fluorescence spectra excited at 369 nm

e. Fluorescence quantum yields in CH₂Cl₂ relative to anthracene in EtOH

f. In CH₂Cl₂, single exponential decay Table 3. RTP Lifetime Data for Polymer Powders Under Air^a

Pl 2,300 0.42

P2 7,300 0.55

P3 17,600 0.57

a. 21 % oxygen content

b. Pre-exponential weighted lifetime.

[00104] The absorption spectrum of BF₂dbm(I)PLA (1) in CH₂Cl₂ is illustrated in Fig. 4. The emission spectra of BF₂dbm(I)OH (1) and BF₂bm(I)PLA (P1-P3) in CH₂Cl₂ are illustrated in Fig. 5. Under these conditions, all three polymers have nearly identical absorption (λa_bs = 406 nm) and emission spectra (XF = 441 nm, Φ_F ~ 0.4,x _F ~ 0.95 ns). (Note: Spectra for P1-P3 overlap.) As expected, iodide substitution results in enhanced intersystem crossing, lower ΦF and shorter τ_F. Phosphorescence is absent for P1-P3 in solution; RTP is a solid-state effect for these boron biomaterials.

[00105] For BF₂dbm(I)PLA powders, RTP and MW dependent fluorescence emission color tuning are observed. Correspondingly, two distinct emission bands were observed in spectra for P2 (466 nm, 527 nm) and P3 (456 nm, 525 nm); whereas, for low MW Pl, a single emission peak at 535 nm was evident with only a small shoulder at -480 nm (Fig. Ib, Table 2). The higher energy band is attributed to fluorescence and the other much stronger, red- shifted band is RTP emission. Under nitrogen, the emission colors of P1-P3 are greenish yellow, green, and cyan respectively (Fig. If). As the energy gap, ΔE, decreases (i.e.

BF₂dbm(I)PLA MW decreases), the singlet-triplet coupling becomes stronger (i.e. larger δ), intersystem crossing is favored and RTP intensity increases.

[00106] Luminescence lifetime data were also collected for P1-P3 as powders (Table 2d). All lifetimes fit to triple-exponential decay due to the heterogeneity of the polymer matrix and possibly F-F interactions too. The fluorescence lifetimes become shorter (0.43-0.37 ns) as the polymer chain decreases (17.6-2.3 kDa) presumably due to enhanced intersystem crossing (Table 2). Shorter RTP and delayed fluorescence (DF) lifetimes (P1-P3: 4.50-4.06 ms) with decreasing MW suggest smaller singlet-triplet energy splitting where the thermal repopulation from triplet to singlet states is more probable. Surprisingly, when the samples were exposed to air, long-lived RTP was still detectable (0.42, 0.55, 0.57 ms for P1-P3, respectively).

Consistent with the spectral data in Fig. Ib where RTP bands remain visible, these results indicate a much faster triplet decay rate (Table 3) compared to BF₂dbmPLA, which is entirely quenched in air. The steady-state emission spectra under air and nitrogen are given in Fig. Id and Fig. Ie respectively, where it is noted that the fluorescence contributions are clearly larger than for the bulk polymer samples. Table 4. Luminescence characterization of P1-P3 in the

solid state at 24 ⁰C under nitrogen.

Powder Film

λ_F ^a _ d

τ_F ^b λRTP ^RTP λ_F ^a τ_F ^b λRTP tRTP

(nm) (ns) (nm) (ms) (nm) (ns) (nm) (ms)

Pl 485 0.37 535 4.06 458 0.48 532 4.25

P2 470 0.42 527 4.39 445 0.54 526 4.37

P3 456 0.43 525 4.50 438 0.64 523 4.41 a. Steady-state fluorescence spectra excited at 369 nm

b. Excitation source: 369 nm LED; fluorescence lifetime fit to triple-exponential decay

c. Excitation source: xenon flash lamp at 405 nm

d. Pre-exponential weighted lifetime

[00107] The oxygen sensitivity calibration for the Pl film. Relationship between the oxygen level (0-21%) and the F/P emission intensity ratio at two fixed wavelengths (450 nm and 525 nm) is illustrated in Fig. 6.

[00108] Film fluorescence lifetimes were longer than their powder counterparts. The steady- state emission spectra of the Pl film were recorded under different O₂ concentrations (Fig. 2a). The ratio of the invariant fluorescence (λ_F= 458 nm) to the oxygen-dependent RTP (λ_P= 532 nm) steadily increases with increasing oxygen levels (Fig. 2c). LJp to 1% O₂, a linear dependency is observed (R² = 0.996). Beyond this point, the F/P plot continues to rise up to ambient levels (i.e. 21% O₂) but with curvature (Fig. 6). Fig. 2b shows the oxygen distribution on the surface of a Pl film under UV excitation (λe_X = 365 nm), where the bright yellow streaks provide dramatic visualization of nitrogen gas flow dynamics.

[00109] BF₂dbm(I)PLA Nanoparticles. P2 nanoparticles were fabricated as previously reported. See Pfister, et al, ACS Nano 2, 1252-1258 (2008). Briefly, BF₂dbm(I)PLA (P2, 25 mg) was dissolved in DMF (2.5 niL). The solution was added dropwise to distilled H₂O (25 mL) with stirring. Samples were dialyzed against distilled H₂O with replacement of fresh water every hour for six hours before dialyzing overnight. On the following day, distilled H₂O was replaced again and samples were dialyzed for an additional hour. The suspension was subsequently passed through filter paper (VWR Filter Paper Qualitative Grade: 413), and transferred to vials for storage. Nanoparticle sizes were determined by dynamic light scattering (DLS) (90° angle) on the Photocor Complex (Photocor Instruments Inc., USA) equipped with a He-Ne laser (Coherent, USA, Model 31-2082, 632.8 nm, 10 mW). Size and polydispersity analysis were performed using DynaLS software (Alango, Israel). Data for P2 nanoparticles are provided in Table 5.

Table 5. Characterization of P2 nanoparticles.

M_n ^a PDI ^b Diameter⁰ _{pD c},_d λ_em F ^e τ/ λ_em P ^g τ_P ^h

(Da) (nm) (nm) (ns) (nm) (ms)

7,300 1.15 98 0.04 450 0.45 528 4.82 a. Determined by GPC in THF vs polysytrene standards

b. GPC PDI = polydispersity index

c. Determined by DLS

d. PD = polydispersity

e. Fluorescence emission maximum for aqueous nanoparticle suspension. (K_n^x,

absorption = 406 nm)

f. Fluorescence lifetime

g. Phosphorescence emission maximum for aqueous nanoparticle suspension purged

h. Phosphorescence lifetime under N₂

[00110] The emission spectra of the P2 boron nanoparticles in aqueous suspension under increasing oxygen levels (0-21%, indicated by the arrow) normalized to the fluorescence band are illustrated in Fig. 7.

[00111] The oxygen sensitivity calibration for the P2 nanoparticles are illustrated in Fig. 8. Relationship between the oxygen level and the F/P emission intensity ratio at two fixed wavelengths (450 nm and 528 nm). Different oxygen concentration ranges shown for comparison: a, linear between 0-1%; b, reasonably linear up to 3%; c, more significant curvature approaching ambient level (21%).

[00112] The polymer P2, with balanced fluorescence and phosphorescence emission, was also fabricated into boron nanoparticles (BNPs) (98 nm) via nanoprecipitation. The emission spectra (λ_F = 450 nm, λ_RTp = 528 nm) (Fig. T), lifetimes (τ_F ~ 0.45 ns, τt_RTp ~ 4.82 ms) and oxygen calibration (Fig. 8) are comparable to P2 films. The linear range for this nanosensor (-0-3%) corresponds with hypoxia in biological contexts. For example, tumor hypoxia (pθ₂ <1%) is associated with increased invasiveness and resistance to radiation and chemotherapy. Despite its importance for cancer treatment and tumor biology, hypoxia is difficult to image with good spatial and temporal resolution, particularly in combination. Due to their small size, biocompatibility, photostability, dual-emissive features, and high oxygen sensitivity, BNPs have the potential to address some of these challenges.

[00113] To test their potential as ratiometric tumor hypoxia imaging agents, P2 nanoparticles were used in combination with a mouse dorsal window chamber breast cancer 4Tl mammary carcinoma model for hyperspectral imaging. Tissue oxygen maps of the window region presented as F/P ratios during carbogen, air, and brief nitrogen breathing (95, 21 and 0% O₂ respectively), show excellent contrast between the microvasculature (red) and the tumor tissue (blue), which remained hypoxic regardless of the breathing gas (Fig. 3). Emission intensity was averaged from 430-480 nm (F) and 530-600 nm (P). Several blood vessels run vertically on the left side of the images (dark lines in brightfield image; more oxygenated yellow-red regions in F/P images), with the tumor comprising the region to the right of the vessels (less oxygenated blue regions in F/P images). The signal was relatively stable -1-2 minutes after changing the breathing gas, which may be a function of the biology of tissue oxygen delivery and consumption more so than the inherent response of the nanosensors. The BNP tumor oxygenation maps are complementary to existing optical methods such as hemoglobin saturation imaging which provides vascular oxygenation.

[00114] Nanoparticle Fabrication. A representative preparation is as follows: an equimolar mixture of 1 (20 mg, M_n = 5700 Da) and 2 (24 mg, M_n = 6600 Da) was dissolved in either 10:1 (v/v) DMF/THF or DMF only (4.4 mL). In the case of DMF, the solution was briefly heated and sonicated to insure complete dissolution. The resulting solution was then cooled to room temperature and added dropwise via syringe to H₂O (44 mL) at a rate of 0.5 mL min^' K During addition of the solution, the H₂O phase was stirred at a fast rate, and the resulting homogenous suspension was stirred an additional 30 min. The suspension was then dialyzed using dialysis tubing (SpectraPor, 12-14 kDa MWCO, Fisher Scientific) against distilled H₂O under slow stirring conditions according to the literature for complete removal of DMF or DMF/THF. Following dialysis the aqueous nanoparticle suspension was passed through filter paper (Whatman, Qualitative Grade 2) and stored in vials. The suspension is characterized as is or freeze-dried for further characterization.

[00115] The optical properties of the aqueous BNP suspensions were studied using UV- vis and fluorescence spectroscopy. As shown in Fig. 10, the UV- vis spectrum of the freshly prepared, freeze-dried BNPs in CH₂Cl₂ exhibit an absorption maximum at 405 nm with a smaller peak at 392 nm, identical to the spectrum for the starting material 2. In contrast, the spectrum of the aqueous BNP suspension shows a shift in these two peaks, with an absorption maximum at 391 nm and a minor shoulder peak at 406 nm. When the fluorescence spectra were measured in CH₂Cl₂, solutions of freeze-dried BNPs displayed a maximum emission seen at λem = 438 nm. In contrast, BNPs in aqueous suspensions displayed a red-shifted fluorescence at λe_m = 454 nm. This red-shift in fluorescence has been observed previously and may indicate dye-dye interactions within individual BNPs.

[00116] A UV-vis absorption spectra for 2 (-3.7 μM) and freeze-dried nanoparticles (BNP, ~3.4 μM) dissolved in CH₂Cl2 (top) and for the stereocomplexed nanoparticles in aqueous suspension (-0.13 mg/mL; bottom) is illustrated in Fig. 10.

[00117] Long-lived room- temperature phosphorescence (RTP) and high sensitivity to oxygen quenching is also observed for the stereocomplexed BNPs. Under atmospheric conditions aqueous BNP suspensions exhibit a blue emission when excited at 365 nm. However, when purged with nitrogen for 20 min, the BNP suspensions exhibited a strong, green-yellow RTP at λe_m = 530 nm, with a delayed fluorescence seen at X_«m ~ 450 nm (Fig. 11). In order to determine the sensitivity of the BNPs to oxygen quenching, the aqueous suspensions were purged with various O₂/N₂ gas mixtures with O₂ concentrations ranging from 0% (pure N₂) to 21% (ambient conditions), and the fluorescence/phosphorescence intensity ratios (F/P) were recorded. The emission spectra, normalized against fluorescence maxima, λ_F, show how RTP decreases as a function of increasing O₂ concentration (Fig. 12). As shown in Fig. 13 a plot of F/P vs. O₂ concentration exhibits a linear trend between 0% and -3% O₂, similar to that for BF₂dbm(I)PLA NPs. The extended linear trend makes the system especially well-suited for hypoxic tissue imaging in biological systems, where O₂ concentrations typically range from 0.1 to 1%.

[00118] In vivo experiments using mouse models were performed to examine tumor accumulation of the pegylated BNPs. Fig. 15a shows the fluorescence image using 420-495 nm bandpass emission prior to injection, and at 10 minutes post injection (Fig. 15b), which was found to be the approximate time of peak uptake. Punctate regions of bright fluorescence appear to be clustered proximal to the blood vessels, which can be seen in the overlay with the transmission image (Fig. 15c). The time course is plotted for the vascular and surrounding tissues which shows rapid uptake, peaking around 10 minutes, with a slow falloff from there (Fig. 15d). The phosphorescence channel (496-623 nm; not shown) did not show a significant increase over baseline, possibly due to high tissue autofluorescence or the long lifetime of the BF₂dbm(I) dye relative to the scanning speed of the present detection system. The confocal microscopy system used here to investigate BNP vascular localization is different than the wide-field fluorescence microscope previously used to detect

phosphorescence for the purposes of ratiometric sensing using non-pegylated BNPs injected directly into the tissue.

[00119] Synthesis of β-Diketones. Method A. The β-diketone ligands la-Id were prepared by Claisen condensation in the presence of NaH as previously described. (See Zhang, G., et al. Macromolecules 2009, 42, 3162-3169, or Zhang, G. et al., Macromolecules, 2009, 42, 3092-3097.) A representative synthesis is as follows. Acetophenone (400 mg, 3.30 mmol), methyl 6-bromo-2-naphthoate (1.16 g, 4.28 mmol) and THF (20 rnL) were added sequentially to a 50 mL round bottom flask. After stirring the mixture for 10 min, a suspension containing NaH (125 mg, 4.95 mmol) in THF (10 mL) was added dropwise at room temperature under N₂. The mixture was stirred for 20 h before saturated aqueous NaHCO₃ (1 niL) was added to quench the reaction. THF was removed in vacuo before 1 M HCl (20 niL) was added. The aqueous phase was extracted with CH₂Cl₂ (3 x 20 niL). The combined organic layers were washed with distilled water (2x10 rnL) and brine (10 mL), and dried over Na₂SO₄ before concentration in vacuo. The residue was purified by column chromatography on silica gel eluting with hexanes/ethyl acetate (6:1) to give 6'-bromonaphthoyl benzoyl methane Ib (755 mg, 65%) as a grey solid. ¹H NMR (300 MHz, CDCl₃) δ 16.92 (s, IH, COCHCOH), 8.50 (s, IH, 1'-ArH), 8.06 - 8.02 (m, 4Η, 3', 4', 5', 8'-ArH), 7.84 (dd, J= 2.7, J= 8.7, 2Η, 2", 6"- ArH)), 7.57 - 7.49 (m, 4Η, 6', 3", 4", 5"-ArH), 6.98 (s, 1Η, COCHCO); MS (MALDI): m/z calcd for C₁₉Hi₄BrO₂ [M+H]⁺ 353.02, found 352.99.

[00120] Method B. Ligands Ie and If were prepared using LDA instead of NaH as the base. A representative synthesis is as follows. 4-Piperidinoacetophenone (836 mg, 4.00mmol), methyl 2-naphthoate (891 m g, 4.78 mmol) and THF (20 mL) were added sequentially to a 50 mL round bottom flask. After stirring for 10 min, a solution of LDA (1.73 M in hexanes, 3.00 mL, 6.59 mmol) was added dropwise at -78 °C under N₂. The mixture was maintained at -78 ⁰C for 4 h, before it was allowed to warm to room temperature and stirred for an additional 4 h. The reaction was then quenched with saturated aqueous NH₄Cl (10 mL). The aqueous phase was extracted with CH₂CI₂ (3 x 20 mL). The combined organic layers were washed with water twice (2x10 mL) and brine (10 mL), and dried over Na₂SO₄ before concentration in vacuo. The residue was purified by column chromatography on silica gel with

hexanes/ethyl acetate (8:1) to give 4-piperidinobenzoyl naphthoyl methane Ie (770 mg, 54%) as a brown solid. ¹H NMR (300 MHz, CDCl₃) δ 17.28 (s, IH, COCHCOH), 8.56 (s, 1Η, 1'- ArH), 8.06 - 7.90 (m, 6Η, 3', 4', 5', 6', 8'-ArH), 7.64 - 7.54 (m, 2Η, 2", 6"-ArH), 6.99 - 6.95 (m, 3Η, 3", 5"-ArH, COCHCO), 3.42 (m, 4Η, 2"', 6"'-piperidino H), 1.72 (s, 6Η, 3"', 4'", 5"'- piperidino H); MS (MALDI): m/z calcd for C₂₄H₂₃NO₂ [M]⁺ 357.17, found 357.14.

[00121] 2-naphthoyl benzoyl methane Ia. Same method as for Ib, but with acetophenone (500 mg, 4.12 mmol), methyl 2-naphthoate (1.03 g, 5.36 mmol) and sodium hydride (167 mg, 6.61 mmol) in THF (40 mL). Column chromatography on silica gel eluting with hexanes/ethyl acetate (6:1) to give Ia (700 mg, 62%) as a pale yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 16.98 (s, IH, ArCOH), 8.62 (s, IH, V-AiH), 8.08 - 7.87 (m, 6H, 3', 4', 5', 8'-ArH. 2", 6"- ArH), 7.62 - 7.49 (m, 5Η, 6', T, 3", 4", 5"-ArH), 7.01 (s, 1Η, COCHCO); MS (MALDI): m/z calcd for C₁₉Hi₅O₂ [M+H]⁺ 275.11, found 275.13.

[00122] 2-naphthoyl 6-bromobenzoyl methane Ic. Same method as for Ib, but with 4- bromo acetophenone (1.00 g, 5.00 mmol), methyl 2-naphthoate (1.16 g, 6.03 mmol) and sodium hydride (200 mg, 7.92 mmol) in THF (40 mL). Ic was obtained after recrystallized from acetone (968 mg, 55%) as a pale yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 16.91 (s, IH, ArCOH), 8.54 (s, 1Η, 1'-ArH), 8.03 - 7.88 (m, 6Η, 3', 4', 5', 8'-ArH. 2", 6"-ArH), 7.66 - 7.53 (m, 4Η, 6', T, 3", 5"-ArH), 6.96 (s, 1Η, COCHCO); MS (MALDI): m/z calcd for C₁₉H₁₄BrO₂ [M+H]⁺ 353.02, found 353.00.

[00123] 6-bromo-2-naphthoyI 4-bromobenzoyl methane Id. Same method as for Ib, but with 4-bromo acetophenone (626 g, 3.08 mmol), methyl 2-naphthoate (1.00 g, 3.70 mmol) and sodium hydride (117 mg, 4.62 mmol) in THF (40 mL). Id was obtained after

recrystallized (2X) from dichloromethane (675 mg, 51%) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 16.91 (s, IH, ArCOH), 8.50 (s, 1Η, 1'-ArH), 8.07 (s, 1Η, 5'-ArH), 8.03 (dd, J = 1.8 , J = 8.7, 1Η, 4'-ArH), 7.91 - 7.84 (m, 4Η, 5', 8'-ArH. 2", 6"-ArH), 7.67 - 7.63 (m, 4Η, T, 3", 5"-ArH), 6.94 (s, 1Η, COCHCO); MS (MALDI): m/z calcd for Ci₉H_nBr₂O₂ [M+H]⁺ 432.91, found 432.93.

6- bromo-2-naphthoyl 4-piperidinobenzoyI methane If. Same method as for Ie, but with 4- piperidonoacetophenone (646 mg, 3.08 mmol), methyl 6-bromo-2-naphthoate (1.00 g, 3.70 mmol) and LDA (1.30 M in heptanes/tetrahydrofuran/ethylbenzene, 2.85 ml, 3.70 mmol) in THF (40 mL). Crude If was obtained after recrystallized from acetone (968 mg, 55%) and used for the next step without further purification. The pure If was obtained from the elimination of difluoroboron from 2f in methanol. (2f (25.1 mg, 0.050 mmol) was dissolved in 10 mL methanol. The mixture was refluxed for 12 h under N₂ before it was concentrated under vacuo. The residue was purified by column chromatography on silica gel with hexanes/ethyl acetate (8:1) to provide If (19.1 mg, 88%) as a yellow solid.) ¹H NMR (300 MHz, CDCl₃) δ 17.21 (s, IH, ArCOH), 8.47 (s, 1Η, 1'-ArH), 8.05 (s, 1Η, 5'-ArH 8.03 - 8.00 (dd, J= 1.8, J= 8.7, 1Η, 8'-ArH), 7.96 - 7.93 (d, J = 9.0, 2Η, 2", 6"-ArH), 7.87 - 7.81 (m, 2H₅ 3', 7'-ArH), 7.63 - 7.60 (dd, J= 1.8, J- 8.7, 1Η, 4'-ArH), 6.93 - 6.90 (d, J= 9.0, 2Η, 3", 5"-ArH), 6.89 (s, 1Η, COCHCO); MS (MALDI): m/z calcd for C₂₄H₂₂BrNO₂ [M]⁺ 435.08, found 435.06.

[00124] Difluoroboron β-Diketonate Complex Synthesis. A representative synthesis is as follows. Difluoroboron 6-bromo-2-naphthoyl benzoyl methane (2b). To a solution of Ib (208 mg, 0.59 mmol) in 30 mL CH₂Cl₂, boron trifluoride diethyl etherate (74 μL, 0.59 mmol) was added at room temperature under N₂. The mixture was refluxed for 2 h. The precipitate was filtered and recrystalized over acetone to give difluoroboron 6-bromonaphthoyl benzoyl methane (183 mg, 78%) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 8.76 (s, IH, 1'- ArH), 8.22 (s, 1Η, 5'-ArH), 8.20 - 8.19 (m, 1Η, 4'-ArH), 8.14 - 8.11 (m, 2Η, 3', 8'-ArH), 7.91 - 7.88 (d, 2Η, 2", 6"-ArH), 7.76 - 7.69 (m, 2Η, 3", 5"- ArH), 7.62 - 7.59 (m, 2Η, 3', 7"-ArH); MS (MALDI): m/z calcd for Ci₉H₁₁BBrF₂O₂Na [M+Na]⁺ 422.99, found 423.03.

[00125] Difluoroboron 2-naphthoyI benzoyl methane 2a. Same method as for 2b, but with Ia (191 mg, 0.70 mmol) and BF₃-OEt₂ (88 μL, 0.70 mmol) in CH₂Cl₂ (20 mL). 2a was obtained as a yellow powder (147 mg, 65%) after recrystallization in acetone. ¹H NMR (300 MHz, CDCl₃) δ 8.80 (s, IH, 1'-ArH), 8.22 (d, J= 8.1, 2Η, 2", 6"-ARH), 8.10 - 7.92 (m, 4Η, 3', 4', 5', 8'-ArH), 7.73 - 7.59 (m, 5Η, 6', 7', 3", 4", 5"-ArH), 7.35 (s, 1Η, COCHCO); MS (MALDI): m/z calcd for Ci₉H₁₂BF₂O₂Na [M+Na]⁺ 345.09, found 345.00

[00126] Difluoroboron 2-naphthoyl 4-bromobenzoyI methane 2c. Same method as for 2b, but with Ic (191 mg, 0.54 mmol) and BF₃-OEt₂ (68 μL, 0.54 mmol) in CH₂CI₂ (20 mL). 2c was obtained as a yellow powder (64 mg, 30%) after recrystallization (2X) from acetone. ¹K NMR (300 MHz, CDCl₃) δ 8.80 (s, IH, 1'-ArH), 8.12 - 7.92 (m, 6Η, 3', 4', 5', 6', 7', 8'- ArH), 7.75 - 7.61 (m, 4Η, 2", 3", 5", 6"-ArH), 7.30 (s, 1Η, COCHCO); MS (MALDI): m/z calcd for Ci₉H_nBBrF₂O₂Na [M+Na]⁺ 422.99, found 422.96.

[00127] Difluoroboron 6-bromo-2-naphthoyl 4-bromobenzoyI methane 2d. Same method as for 2b, but with Id (405 mg, 0.94 mmol) and BF₃-OEt₂ (178 μL, 1.41 mmol) in CH₂Cl₂ (50 mL). 2d was obtained as a grey powder (272 mg, 61%) after recrystallization (3X) from dichloromethane. ¹H NMR (300 MHz, CDCl₃) δ 8.75 (s, IH, 1'-ArH), 8.18 - 8.04 (m, 4Η, 3', 4', 5', 8'-ArH), 7.91 - 7.88 (d, J= 8.4, 2H, 2", 6"-ArH), 7.75 - 7.72 (d, J= 8.4, 2Η, 3", 5"- ArH), 7.28 (s, 1Η, COCHCO); C₁₉Hi₀BBr₂F₂O₂Na [M+Na]⁺ 502.91, found 502.90.

[00128] Difluoroboron 2-naphthoyl 4-piperidinobenzoyl methane D. Same method as for 2b, but with Ie (134 mg, 0.38 mmol) and BF₃-OEt₂ (118 μL, 0.98 mmol) in CH₂Cl₂ (30 mL). The crude product was purified with column chromatography (silica gel, hexanes - EtOAc, 2:1) to afford 2e as an orange solid (132 mg, 88%). ¹R NMR (300 MHz, CDCl₃) δ 8.71 (s, IH, 1'-ArH), 8.10 - 7.88 (m, 5Η, 3', 4', 5', 6', 8'-ArH), 7.65 - 7.54 (m, 3Η, 6'-ArH, 2", 6"- ArH), 7.13 (s, 1Η, COCHCO), 6.88 (d, J= 9.3, 2Η, 3", 5"-ArH), 3.53 (m, 4H, 2"', 6"'- piperidino H), 1.72 (s, 6H, 3'", 4"', 5"'-piperidino H); MS (MALDI): m/z calcd for

C₂₄H₂₂BF₂NO₂Na [MH-Na]⁺ 428.15, found 428.10.

[00129] Difluoroboron 6-bromo-2-naphthoyl 4-piperidinobenzoyl methane E. Same method as for 2b, but with If (106 mg, 0.24 mmol) and BF₃-OEt₂ (31 μL, 0.24 mmol) in CH₂Cl₂ (50 mL). E was obtained as an orange solid (65 mg, 56%) after recrystallization in acetone. ¹U NMR (300 MHz, CDCl₃) δ 8.67 (s, IH, 1'-ArH), 8.10 - 8.04 (m, 4Η, 3', 4', 5', 8'- ArH), 7.85 (dd, J= 6.3, J= 8.7, 2Η, 2", 6"-ArH), 7.65 (dd, J= 1.8, J= 9.2, 1Η, 6'-ArH), 7.10 (s, 1Η, COCHCO), 6.88 (d, J= 9.3, 2Η, 3", 5"-ArH), 3.54 (m, 4H, 2"\ 6"'-piperidino H), 1.73 (s, 6Η, 3'", 4"', 5"'-piperidino H); MS (MALDI): m/z calcd for C₂₄H₂₁BBrF₂NO₂Na [M+Na]⁺ 506.06, found 506.10.

Table 6. Absorption and Luminescence Emission Data for Boron-Diketonate Compounds in Dichloromethane Solution and the Solid State PLA Matrices at Room

Temperature.

Boron- ■Λmax/in ■Λmax/FL in •Λmax/delayed diketonates solution film FL in film

N [nm] [nm]

A 506 544 517

B 487 517 560

C 436 562 563

D 561 558 NA

E 553 559 NA

"Reference: quinine sulfate in 0.1M sulfuric acid aqueous

solution.

*Excitation source: 369nm light emitting diode.

'Excitation source: 369 nm xenon lamp; dyes are 1.3 wt% in

polylactic acid.

[00130] The emission spectra of the salts in Table 6 are illustrated in Fig. 21.

[00131] A dye blended with PLA in vial w/wo H+ in Nitrogen is illustrated in Fig. 22. The dye was blended with PLA. This shows that the solvent used to process the film makes a difference. In THF, the emission spectra showed a considerable blue shift under nitrogen after the dye was exposed to acid. This illustrates emission modulation in dye/polymer

compositions with an ionizable group.

Boron-diketonates ■Λmax/in ^•max/in •^max of neutral dyes •"■max of acidified dyes in various solvents neutral solution acidic solution FL in PLA film FL in PLA film

[nm] [nm] [nm] [nm]

DCM 458/557 504 577 577

THF 424/557 520 577 441/538 [00132] All patents, patent applications and literature cited in the specification are hereby incorporated by reference in their entirety. In the case of any inconsistencies, the present disclosure, including any definitions therein will prevail. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the invention. [00133] Having thus described the preferred embodiments of the present invention, those of skill in the art will readily appreciate that the teachings found herein may be applied to yet other embodiments within the scope of the attached claims.

Claims

What is claimed is:

1. A luminescent dye compound having formula I:

wherein R¹ is (C₆-C₂₂)aryl or (C₅-C₂₁)heteroaryl; R¹ is substituted with at least one substituent which is a heavy atom or an ionizable group and optionally substituted with additional substituent groups;

R² is (C₆-C₂₂)aryl, (C₅-C₂₁)heteroaryl, or (Ci-Ci₈)alkyl; R² is optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substituent groups;

where the substituent groups on R¹ or R² are halo, (Ci-C₁₂)alkyl, hydroxy(Ci- Ci₂)alkyl, halo(Ci-Ci₂)alkyl, R¹⁵O(C_rCi₂)alkyl, R¹⁵O(C,-Ci₂)alkyl-O-, (C₂-C i₂)alkenyl,

(C₇-C₂₆)aralkyl, (C₅-C₁₃)heteroaryl, -OR 1¹5³, oxo (>C=O), -CN, -NO₂, -CO₂R » 1"5, - OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, -N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, -SO₂R²⁰; -

OSi(R²⁵)₃, -Si(R²⁵)₃ -Si(R >2°5x)i(OR >2°5)Nj, -P(OR >2^/5³)x₃, -P(R >2^5)v₃, isocyanate, isothiocyanate, urea, or thiourea; or two substituent groups can form a ring together with the atom to which they are attached optionally having from 3 to 8 ring atoms and optionally having 1, 2, or 3 heteroatoms; each R²⁵ is independently hydrogen, alkyl or aryl; and i and j are independently integers from 1-3 wherein the sum of i and j is 3;

Y¹ is B; X¹ and X² are independently a bond, alkyl, alkenyl, alkynyl or aryl, optionally substituted with 1, 2, 3, 4, 5, or 6 substituent groups; where the substituent groups are halo,

hydroxy(C₁-C₁₂)alkyl, halo(C_rC)₂)alkyl, R¹⁵O(C₁- Cj₂)alkyl, R¹⁵O(C₁-Ci₂)alkyl-O-, (C₂-C₁₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅-C ₁₃)heteroaryl, -OR¹⁵, oxo (>C=0), -CN, -NO₂, -CO₂ R¹⁵, -OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴,

-N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, -SO₂R²⁰, -OSi(R²⁵)₃, -Si(R²⁵)₃, -Si(R²⁵)i(OR²⁵)_j; - P(OR²⁵)₃, -P(R²⁵)₃, isocyanate, isothiocyanate, urea, thiourea or two substituent groups can form a ring together with the atom to which they are attached optionally having from 3 to 8 ring atoms and optionally having 1, 2, or 3 heteroatoms; R³ and R⁴ are independently, halo, hydroxy, R¹⁵O(Ci-Ci₂)alkyl, R¹⁵O(Ci-Ci₂)alkyl, (C₆-C₂₂)aryl, or -OR¹⁵; or R³ and R⁴ taken together form a bidentate chelate, such as deprotonated acid or diacid group, HOC(=O)CH₂C(=O)OH (malonic acid) or

HOC(=O)CH₂C(=O)OH (oxalic acid), or chelating group such as acid-alcohol, acid- ether, or silica (SiO) group, with two donor groups, or R³ and R⁴ taken together with the boron atom form a ring having the formula:

R⁵ is hydrogen, halo, (Q-C^alkyL hydroxy(d-C₁₂)alkyl, halo(d-C₁₂)alkyl, (C₂- C₁₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅-C₁₃)heteroaryl, -OR¹⁵, -CN, -NO₂, -CO₂ R¹⁵, - OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, -N(R²³)C(O)R²⁴, -C(O)NR¹⁷ R¹⁸, -SR¹⁹, -SO₂R²⁰ or - SO₃H; or wherein R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²³ and R²⁴ are independently hydrogen, alkyl, alkenyl, (C₃-C₁₂)cycloalkyl, aryl, aralkyl or haloalkyl; each Q is a polymer chain where each chain is conjugated directly to the compound through one of R¹, R², R⁵, X¹, X² or to a substituent attached to R¹, R², R⁵, X¹, or X²; wherein the polymer chain optionally comprises a targeting group; and n is 1, 2, 3, 4, 5, 6, 7 ,8, 9, 11, 12, 13, 14, or 15; or a pharmaceutically acceptable salt thereof.

2. The luminescent dye compound of claim 1, wherein the heavy atom is iodine, bromine, chlorine, sulfur or a metal-containing group.

3. The luminescent dye compound of claim 1, wherein the heavy atom is iodine or bromine.

4. The compound of claim 3, wherein R¹ is iodo-phenyl and R² is phenyl, naphthyl, or

anthrycyl.

5. The compound of claim 1, wherein the ionizable group is a Lewis acid or a Lewis base.

6. The compound of any of claims 1-5, wherein R² is phenyl.

7. The compound of any of claims 1-5, wherein R² is naphthyl.

8. The compound of any of claims 1-5, wherein R² is heteroaryl.

9. The compound of any of claims 1-5, wherein R² has the formula

where X⁴ is O, S, or NR^a and each X⁴ is independently S, or NR^a, where R^a is hydrogen, alkyl or aryl.

10. The compound of claim 9, wherein R² has the formula:

11. The compound of any of claims 1-10, wherein the R² substituents are independently halo, hydroxy(C₁-Ci₂)alkyl, halo(d-C₁₂)alkyl, R¹⁵O(Ci-Ci₂)alkyl, R¹⁵O(Ci-C₁₂)alkyl-O-, - OR¹⁵, -CO₂R¹⁵, -OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, -N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, or -SO₂R²⁰.

12. The compound of claim 11, wherein the R² substituents are independently OR¹⁵, or

R¹⁵O(Ci-Ci2)alkyl-O-.

13. The compound of claim 12, wherein the R² substituents are independently -Oalkyl or OH.

14. The compound of any of claims 1-13, wherein the R³ and R⁴ substituents are

independently halo, hydroxy(C₁-C₁₂)alkyl, phenyl, halo(Ci-C₁₂)alkyl, R¹⁵O(C₁-Ci₂)alkyl, R¹⁵O(C!-Ci₂)alkyl-O-, -OR¹⁵, -CO₂R¹⁵, -OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, - N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, or -SO₂R²⁰.

15. The compound of claim 14, wherein the R³ and R⁴ substituents are independently halo.

16. The compound of claim 15, wherein the R³ and R⁴ substituents are fluorine.

17. The compound of any of claims 1-12, wherein the R³ and R⁴ substituents are taken

together to form a bidentate chelate.

18. The compound of claim 17 wherein the chelate is a diacid, acid-alcohol, or acid-ether or silica group.

19. The compound of claim 18 wherein the diacid group is HOC(=O)CH₂C(=O)OH or HOC(=O)CH₂C(=O)OH.

20. The compound of claim 17 wherein R³ and R⁴ taken together with the boron atom form a ring having the formula:

21. The compound of any of claims 1-20, wherein the polymer is attached through a group having the formula -O-R⁶-O-, -NR²¹-R⁶-O-, or -NR²¹-R⁶-NR²¹- -S-R⁶-O, -S-R⁶-S-, - O-R⁶-S-, or -NR²¹-R⁶-S- -S-R⁶-NR²¹- where R⁶ is alkylene, alkenylene having at least two carbon atoms and R²¹ is hydrogen, alkyl, alkenyl, (C₃-C₁₂)cycloalkyl, aryl, aralkyl or haloalkyl.

22. The compound of any of claims 1-21, wherein each Q is independently a polylactide, polyglycolide, lactide-glycolide copolymer, polyethylene glycol polylactide,

polycaprolactone, polycaprolactone-polylactide block copolymers, poly(methyl methacrylate), 2-hydroxyethyl methacrylate, polystyrene, polyurethane,

poly(fluoroalkanes), hydroxyl propyl cellulose, polyhydroxybutyrate, polyhydroxy- butyrate-valerate copolymer, polybutylene succinate, polybutylene adipate-co- terephthalate, polyethylene glycol, polyurethanes, proteins, nucleic acids, poly(silanes), polysiloxanes, polyphosphazenes, silica, alumina, polycarbonates, a sugar based polymer or a mixture thereof.

23. The compound of claim 22, wherein each Q is independently a polylactide,

polyglycolide, poly(ethylene glycol), polycaprolactone, lactide-glycolide copolymer, poly(ethylene glycol)-polylactide, polycaprolactone-polylactide, poly(ethylene glycol)- polycaprolactone poly(ethylene glycol)-polylactide-co-glycolide block copolymers, or a mixture thereof.

24. A luminescent dye composition comprising a polymer and a compound having formula II:

II

wherein R¹ (C₆-C₂₂)aryl or (C₅-C₂₁)heteroaryl, or (C₁-C₁₈)alkyl; R¹ is substituted with at least one substituent which is a heavy atom or an ionizable group and optionally substituted with additional substituent groups;

R² is (C₆-C₂₂)aryl or (C₅-C₂₁)heteroaryl; R² is optionally independently substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substituent groups;

where the substituent groups on R¹ or R² are halo, (d-C₁₂)alkyl, hydroxy(Ci- C_]2)alkyl, halo(Ci-C₁₂)alkyl, R¹⁵O(d-Ci₂)alkyl, R¹⁵O(d-C₁₂)alkyl-O-, (C₂-C₁₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅-C₁₃)heteroaryl, -OR¹⁵, oxo (>C=O), -CN, -NO₂, -CO₂R¹⁵, - OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, -N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, -SO₂R²⁰; - OSi(R²⁵)₃, -Si(R²⁵)₃ -Si(R²⁵);(OR²⁵)j, -P(OR²⁵)₃, -P(R²⁵)₃, isocyanate, isothiocyanate, urea, or thiourea; or two substituent groups can form a ring together with the atom to which they are attached optionally having from 3 to 8 ring atoms and optionally having 1 , 2, or 3 heteroatoms; each R²⁵ is independently hydrogen, alkyl or aryl; and i and j are independently integers from 1-3 wherein the sum of i and j is 3;

Y¹ is B; X¹ and X² are independently a bond, alkyl, alkenyl, alkynyl or aryl, optionally substituted with 1, 2, 3, 4, 5, or 6 substituent groups; where the substituent groups are halo, (Ci-C₁₂)alkyl, hydroxy(Ci-C₁₂)alkyl, halo(C]-C₁₂)alkyl, R¹⁵O(C₁- Ci₂)alkyl, R¹⁵O(d-C₁₂)alkyl-O-, (C₂-C ₁₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅-C ₁₃)heteroaryl, -OR¹⁵, oxo (>C=0), -CN, -NO₂, -CO₂ R¹⁵, -OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴,

-N(R²³)C(O)R²⁴, -C(O)NR¹⁷R¹⁸, -SR¹⁹, -SO₂R²⁰, -OSi(R²⁵)₃, -Si(R²⁵)₃, -Si(R²⁵)i(OR²⁵)_j, - P(OR²⁵)₃, -P(R²⁵)₃, isocyanate, isothiocyanate, urea, thiourea or two substituent groups can form a ring together with the atom to which they are attached optionally having from 3 to 8 ring atoms and optionally having 1, 2, or 3 heteroatoms;

R³ and R⁴ are independently, halo, hydroxy, R¹⁵O(C_rC₁₂)alkyl, R¹⁵O(d-Ci₂)alkyl, (C₆-C₂₂)aryl, or -OR¹⁵; or R³ and R⁴ taken together form a bidentate chelate, such as deprotonated acid or diacid group, HOC(=O)CH₂C(=O)OH (malonic acid) or HOC(=O)CH₂C(=O)OH (oxalic acid), or chelating group such as acid-alcohol, acid- ether, or silica (SiO), with two donor groups, or R³ and R⁴ taken together with the boron atom form a ring having the formula:

R⁵ is hydrogen, halo, (Ci-C₁₂)alkyl, hydroxy(C]-Ci2)alkyl, halo(Ci-C_]2)alkyl, (C₂- C₁₂)alkenyl, (C₇-C₂₆)aralkyl, (C₅-Ci₃)heteroaryl, -OR¹⁵, -CN, -NO₂, -CO₂ R¹⁵, - OC(O)R¹⁶, -C(O)R¹⁶, -NR¹³R¹⁴, -N(R²³)C(O)R²⁴, -C(O)NR¹⁷ R¹⁸, -SR¹⁹, -SO₂R²⁰ or - SO₃H; or wherein R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²³ and R²⁴ are independently hydrogen, alkyl, alkenyl, (C₃-C ₁₂)cycloalkyl, aryl, aralkyl or haloalkyl; or a

pharmaceutically acceptable salt thereof.

25. The luminescent dye composition of claim 24, wherein the heavy atom is iodine,

bromine, chlorine, sulfur or a metal-containing group.

26. The luminescent dye composition of claim 25, wherein the heavy atom is iodine or bromine.

27. The luminescent dye composition of claim 26, wherein R¹ is iodo-phenyl and R² is phenyl, naphthyl or anthrycyl.

28. The luminescent dye composition of claim 24, wherein the ionizable group is a Lewis acid, or a Lewis base.

29. A composition comprising a compound of claim I₅ or a composition of claim 24,

optionally in combination with a carrier.

30. The composition of claim 29, wherein the composition is in the form of particles,

nanoparticles, films, coatings, fibers or nanofibers, powders, foams, gels, network, assembly, suspension or composite, or bulk material.

31. An analytical or diagnostic method for detecting the presence or absence of oxygen in gas, liquid, solid or mixtures thereof comprising contacting the gas or liquid, solid, or mixtures thereof, with a compound of claim 1 or a composition of claim 24 and viewing the presence or absence of delayed light emission from the compound or composition.

32. The analytical or diagnostic method of claim 31 where the light emission is from

phosphorescence or fluorescence.

33. The method of claim 31 or 32, where the compound or composition is a used for

ratiometric sensing or imaging.

34. The analytical or diagnostic method for oxygen sensing or imaging comprising

contacting a test material with an effective amount of a compound of claim 1 or a composition of claim 24 and detecting the pθ₂ levels.

35. The analytical or diagnostic method of claim 32, wherein the fluorescence is used as a standard and the phosphorescence is used to determine the pθ₂ level.

36. The analytical or diagnostic method of any of claims 31-35, wherein in vivo measurement of oxygen in a mammal is important.

37. The analytical or diagnostic method of any of claims 31-36, wherein the oxygen sensing or imaging is for tumors, vasculature, wounds, brain, high altitude drug testing, monitoring drugs that deliver oxygen to tissues, organ transplantation or tissue transplantation, or cell transplantation, tissue engineering, cells, or other tissues, in vivo, ex vivo, or in vitro.

38. The analytical or diagnostic method of claim 37, wherein the cells are stem cells.

39. The analytical or diagnostic method of any of claims 31-38, wherein the imaging

provides an oxygen concentration map of tissue examined.

40. A method for tuning the relative phosphorescence and fluorescence intensities of

the luminescent dye compound of claim 1, comprising modifying the molecular weight of the polymer chain; or

the luminescent dye composition of claim 24 by varying the dye loading to provide a suitable balance between the phosphorescence and fluorescence intensities.

41. A method for tuning the phosphorescence and fluorescence of a luminescent dye compound of claim 1 or luminescent dye composition of claim 24 comprising;

modifying the compound of formula I or formula II, by addition of a functional group which can be protonated, deprotonated, ionized or deionized; or

altering the molecular weight of the polymeric component to modulate

phosphorescence and fluorescence emission wavelengths and intensities.

42. The method of claim 40 or 41, wherein the functional group is a Lewis acid, or a Lewis base.

43. The method of claim 42, wherein the functional group is a carboxylic acid, or an amine.

44. The method of claim 43, wherein the functional group is a cyclic amine.

45. An article prepared using a luminescent dye polymeric compound of claim lor a

composition of claim 24.

46. The article of claim 45 which is in the form of particles, nanoparticles, a film, coating, fibers, nanofibers, powders, foams, gels, networks, assemblies, suspensions or composites, or bulk materials.

47. The article of claim 45 or 46, which is a sensor or imaging agent.

48. The article of claim 47 wherein the sensor is an oxygen sensor, pH sensor, ion sensor, or a temperature sensor.

49. The article of claim 48, which is a sensor.

50. The article of claim 49, wherein the sensor is an oxygen sensor, pH sensor, ion sensor, or a temperature sensor.

51. The luminescent dye compound of any of claims 1-23, wherein the polymer component comprises includes a targeting group.

52. The luminescent dye compound of claim 51 , wherein the polymer targeting group has the formula PLA-X or PLA-PEG-X where X is a folate, galactose, indomethacin, lectins, or Biotin.

3. The luminescent dye compound of claim 52, wherein the lectins is wheat germ agglutinin (WGA).