Brush-arm star polymer imaging agents and uses thereof

Abstract

Disclosed are methods, compositions, reagents, systems, and kits to prepare nitroxide-functionalized brush-arm star polymer organic radical contrast agent (BASP-ORCA) as well as compositions and uses thereof. Various embodiments show that BASP-ORCA display unprecedented per-nitroxide and per-molecule transverse relaxivities for organic radical contrast agents, exceptional stability, high water solubility, low in vitro and in vivo toxicity, and long blood compartment half-life. These materials have the potential to be adopted for tumor imaging using clinical high-field .sup.1H MRI techniques.

Claims

1. A brush-arm star polymer comprising one or more repeating units of Formula (I) and one or more repeating units of Formula (II): ##STR00023## or a salt thereof, wherein: the combined number of repeating units of Formula (I) and repeating units of Formula (II) is at least 100; each A is independently C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 heteroalkylene, wherein each of the alkylene and heteroalkylene is independently optionally substituted with 1-2; each A.sup.1 is C.sub.1-C.sub.12 heteroalkylene, wherein each of the heteroalkylene is independently optionally substituted with 1-2 oxo; each B is C.sub.1-C.sub.12 alkylene, wherein each of the alkylene is independently optionally substituted with one oxo; each X is an imaging agent, wherein at least one imaging agent is an organic nitroxide radical-containing imaging agent; each P is a polyethylene glycol; each instance of L is independently selected from the group consisting of C.sub.1-C.sub.12 alkylene, C.sub.1-C.sub.12 heteroalkylene, (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 heteroalkylene), and a combination of any two thereof; ##STR00024##  wherein: each of the alkylene, heteroalkylene, arylene, and heteroarylene is independently optionally substituted with 1-24 independently selected R.sup.1 selected from the group consisting of alkyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, and —S(O).sub.mR.sup.A; each of the heteroarylene is independently monocyclic, 5- or 6-membered heteroarylene; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; each m is independently 1 or 2; and each of p and o are independently an integer between 0 and 20, inclusive; each of a and b is 1; when “1”, “2”, “3”, “4”, “5”, and/or “6” shown in Formula (I) or Formula (II) are terminal groups, the terminal groups are independently selected from the group consisting of optionally substituted alkenyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl; and each y is independently an integer between 1 and 100, inclusive.

2. The brush-arm star polymer of claim 1, wherein P is a polyethylene glycol with a number average molecular weight about 2000, about 2500, about 3000, about 3500, or about 4000, g/mol.

3. The brush-arm star polymer of claim 1, wherein: each B is C.sub.1-C.sub.12 alkylene, wherein each of the alkylene is substituted with one.

4. The brush-arm star polymer of claim 1, wherein each L of Formula (I) is independently selected from ##STR00025##

5. The brush-arm star polymer of claim 1, wherein when the brush-arm star polymer comprises two or more imaging agents, at least one imaging agent is a chelated metal, inorganic compound, or organic compound, or a salt thereof.

6. The brush-arm star polymer of claim 1, wherein at least one organic nitroxide radical-containing imaging agent is of the formula: ##STR00026##

7. The brush-arm star polymer of claim 1, wherein: each instance of L of Formula (I) is independently C.sub.1-C.sub.12 alkylene, C.sub.1-C.sub.12 heteroalkylene, (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 heteroalkylene), or a combination of any two thereof, wherein each of the alkylene, heteroalkylene, arylene, and heteroarylene is independently optionally substituted with 1-24 independently selected R.sup.1 selected from the group consisting of alkyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, and —S(O).sub.mR.sup.A.

8. The brush-arm star polymer of claim 1, wherein at least one repeating unit of Formula (I) is of formula: ##STR00027##

9. The brush-arm star polymer of claim 1, wherein: each instance of L of Formula (I) is independently C.sub.1-C.sub.12 alkylene, C.sub.1-C.sub.12 heteroalkylene, (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 heteroalkylene), or a combination of any two thereof, wherein each of the alkylene, heteroalkylene, arylene, and heteroarylene is independently optionally substituted with 1-24 independently selected R.sup.1 selected from the group consisting of alkyl, heteroalkyl, halo, cyano, oxo, nitro, and —OR.sup.A.

10. The brush-arm star polymer of claim 1, wherein the repeating unit of Formula (II) is of formula: ##STR00028##

11. The brush-arm star polymer of claim 1, wherein the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is between about 1:20 to about 20:1, respectively.

12. A method of producing a brush-arm star polymer of claim 1 comprising: (a) reacting one or more macromonomers of Formula (III): ##STR00029## or a salt thereof, with a metathesis catalyst to form a living polymer; and (b) mixing a crosslinker of Formula (IV): ##STR00030## or a salt thereof, with the living polymer.

13. A method of imaging a subject, the method comprising steps of: administering to a subject an effective amount of the polymer of claim 1; and acquiring an image.

14. A method of performing magnetic resonance imaging of a subject, the method comprising steps of: administering to a subject an effective amount of the polymer of claim 1; and acquiring a magnetic resonance image; wherein at least one imaging agent is useful for performing magnetic resonance imaging.

15. A method of performing near-infrared fluorescence imaging of a subject, the method comprising steps of: administering to a subject an effective amount of the polymer of claim 1; and acquiring a near-infrared fluorescence image; wherein at least one imaging agent is useful for performing near-infrared fluorescence imaging.

16. The brush-arm star polymer of claim 1, wherein each P is polyethylene glycol with a number average molecular weight ranging from about 200 g/mol to about 6000 g/mol, inclusive.

17. The brush-arm star polymer of claim 1, wherein each L of Formula (II) is of the formula: ##STR00031## wherein each q is independently an integer between 0 and 20, inclusive.

18. The brush-arm star polymer of claim 1, wherein each A is C.sub.1-C.sub.12 heteroalkylene, wherein each of the heteroalkylene is optionally substituted with 1-2.

19. The brush-arm star polymer of claim 1, wherein each y is 1.

20. The brush-arm star polymer of claim 5, wherein at least one imaging agent is a near-infrared fluorescence imaging agent.

21. The brush-arm star polymer of claim 5, wherein at least one imaging agent is of the formula: ##STR00032##

22. The brush-arm star polymer of claim 5, wherein at least one repeating unit of Formula (I) is of formula: ##STR00033##

23. The brush-arm star polymer of claim 1, wherein the brush-arm star polymers form a particle of a diameter between about 10 nm and about 1000 nm, inclusive.

24. The brush-arm star polymer of claim 1, wherein each A.sup.1 is C.sub.1-C.sub.12 heteroalkylene, wherein each of the heteroalkylene is substituted with 1-2.

25. The brush-arm star polymer of claim 1, wherein each L of Formula (II) is independently (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), or a combination of any two thereof, wherein each of the alkylene, heteroalkylene, and arylene is independently substituted with 1-24 independently selected R.sup.1 selected from the group consisting of alkyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, and —S(O).sub.mR.sup.A.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings, which constitute a part of this specification, illustrate several exemplary embodiments of the invention and together with the description, serve to explain certain principles of the invention. The embodiments disclosed in the drawings are exemplary and do not limit the scope of this disclosure.

(2) FIGS. 1A-1B show chemical structures of BASP components (FIG. 1A) and the general brush-first ROMP procedure (FIG. 1B). Branched MMs chex-MM and Cy-MM are combined in the ratio j:0.01j. This combination of MMs is exposed to 1.0 equivalents of Grubbs III initiator to produce a living bottlebrush with an average degree of polymerization (DP)=j+0.01j=m. N equivalents of Acetal-XL is then added (in aliquots of 5 eq. Acetal-XL every 5 minutes) to provide the final BASP-ORCA. The properties of the BASP-ORCAs are defined by their m and N values (see FIG. 15).

(3) FIGS. 2A-2C show the transmission electron microscopy image of BASP-ORCA1 (D.sub.TEM=37±7) after being negatively stained with uranyl acetate (FIG. 2A), electron paramagnetic resonance (EPR) spectra for BASP-ORCA1 and chex-MM (FIG. 2B), and T.sub.1 and T.sub.2-weighted MRI phantoms for BASP-ORCA1, chex-MM, PBS buffer, chex-bottlebrush, and a PEG-BASP lacking chex (FIG. 2C). In FIG. 2C the concentration of chex-containing samples (BASP-ORCA1, chex-MM, and chex-bottlebrush) ranges from 1 mM to 4 mM chex. The concentration of PEG-BASP lacking chex ranges from 6 mg/mL to 21 mg/mL, which is equivalent to the mass per volume concentration range of BASP-ORCA1.

(4) FIGS. 3A-3C are EPR spectra for BASP-ORCA1 at 1 minute, 40 minutes, and 180 minutes following exposure to 20 equivalents of sodium ascorbate (Asc) per nitroxide moeity (FIG. 3A), ascorbate reduction kinetics for BASP-ORCA1, chex-bottlebrush, and chex-MM (FIG. 3B), and Cy5.5 emission at 700 nm in response to Asc and glutathione (GSH) (FIG. 3C).

(5) FIGS. 4A-4B show in vivo NIRF images of NCR nude mouse before and 20 hours after injection of BASP-ORCA1 (FIG. 4A) and ex vivo NIRF images of selected organs (FIG. 4B). Units of radiant efficiency:

(6) $\frac{p\text{/}\sec \text{/}{\mathrm{cm}}^2\text{/}\mathrm{sr}}{\mu W\text{/}{\mathrm{cm}}^2}.$

(7) FIGS. 5A-5D show in vivo MRI imaging with BASP-ORCA. FIG. 5A shows T.sub.2-weighted MR images of tumor bearing NCR nude mouse before (top row) and 20 hours after (bottom row) injection of 0.16 mmol chex/kg (“low dose”) of BASP-ORCA1. Each series of images corresponds to progressive slices in the z-axis through the tumor of the same mouse. FIG. 5B shows T.sub.2-weighted MR images of tumor bearing NCR nude mouse before (top row) and 20 hours after (bottom row) injection of 0.23 mmol chex/kg (“high dose”) of BASP-ORCA1. Each series of images corresponds to progressive slices in the z-axis through the tumor of the same mouse. FIG. 5C shows T.sub.2-weighted coronal MR images before (top) and 20 hours after (bottom) injection of 0.23 mmol chex/kg (“high dose”) of BASP-ORCA1. FIG. 5D shows the percent MRI contrast change at various times following BASP-ORCA1 injection compared to pre-injection.

(8) FIGS. 6A-6B show EPR spectra obtained for homogenized tissue samples collected for the same mice imaged in FIGS. 5A-5D 22 hours following BASP-ORCA1 injection (FIG. 6A) and fluorescence radiant efficiencies of the homogenized tissue samples (FIG. 6B). Right-hand bars: Muscle-normalized concentration of chex per milligram of protein as obtained from EPR integration of tissue homogenates. Left-hand bars: Muscle-normalized concentration of Cy5.5 in tissue homogenates as obtained from NIRF imaging.

(9) FIGS. 7A-7B show GPC traces. FIG. 7A shows GPC traces of BASP-ORCAs with different brush length (m) and cross-linker equivalents (N). *indicates negligible residual MM; **denotes uncoupled bottlebrush. In all cases, the MM-to-bottlebrush conversions were almost quantitative, while the bottlebrush-to-BASP conversions were ≥85%. FIG. 7B shows GPC traces of chex-bottlebrush and PEG-BASP used for phantom MRI comparison with BASP-ORCA 1.

(10) FIG. 8 shows the EPR spectra of BASP-ORCAs of varying composition.

(11) FIGS. 9A-9D show computational analysis of EPR spectra obtained during reduction kinetics experiments. t1=1 minutes, t16=40 minutes, t29=180 minutes following addition of Asc solution.

(12) FIG. 10 shows the excitation and emission spectra of BASP-ORCA1.

(13) FIG. 11 shows the cell viability assay for BASP-ORCA1 in the toxin-sensitive HUVEC and cancerous HeLa cell lines as measured by CellTiter Glo. No toxicity was observed until high concentrations were reached (up to 0.3 mg/mL and 5 mg/mL for HUVEC and HeLa, respectively).

(14) FIG. 12 shows in vivo gross toxicity of BASP-ORCA1 following intravenous injections in BALB/c mice.

(15) FIGS. 13A-13C show pharmacokinetics (PK) (FIG. 13A), biodistribution (BD) (FIG. 13B), and excrements collected 24 hours after administration of BASP-ORCA1 in BALB/c mice as imaged by NIRF (λ.sub.ex/λ.sub.em=640/700 nm) (FIG. 13C). PK data were fit into a two-component model using standard procedures (R.sup.2=0.95)..sup.101,102

(16) FIG. 14 shows ex vivo BD as assessed by NIRF imaging (λ.sub.ex/λ.sub.em=640/700 nm) of subcutaneous tumor-bearing NCR-NU mice following injection of BASP-ORCA1.

(17) FIG. 15 shows characterization data for BASP-ORCAs and control compounds.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

(18) The present disclosure provides methods, compounds, particles, nanoparticles, compositions, systems, and kits focused on the synthesis and uses of brush-arm star polymers containing at least one imaging agent. In certain embodiments, the polymers are brush-arm star polymer organic radical contrast agents (BASP-ORCAs). In certain embodiments, the brush-arm star polymer organic radical contrast agents are comprised of brush-arm polymers covalently linked to a polymer core via crosslinkers. In certain embodiments, BASP-ORCAs contain a high concentration of reduction-resistant nitroxide groups bound between a poly(ethylene glycol) (PEG) shell and a polyacetal core.

(19) These polymers are shown to be effective for medical imaging (e.g., brain, heart, lung, liver, kidney, spleen, muscle, tissue, and tumor). In certain embodiments, the imaging modality is magnetic resonance imaging. In certain embodiments, the imaging modality is near-infrared fluorescence imaging.

(20) Brush-Arm Star Polymers

(21) One aspect of the present disclosure relates to brush-arm star polymers comprising at least 100 repeating units selected from Formula (I) and Formula (II):

(22) ##STR00008##
or a salt thereof, wherein: each of A, A.sup.1, and B is independently C.sub.1-C.sub.12 alkylene, C.sub.2-C.sub.12 alkenylene, C.sub.2-C.sub.12 alkynylene, or C.sub.1-C.sub.12 heteroalkylene, C.sub.2-C.sub.12 heteroalkenylene, C.sub.2-C.sub.12 heteroalkynylene, wherein each alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene is optionally substituted with 1-24 independently selected R.sup.1; X is an imaging agent; P is alkylene, heteroalkylene, or polymer; L is a bond, —O—, —S—, —S—S—, C.sub.1-C.sub.12 alkylene, C.sub.2-C.sub.12 alkenylene, C.sub.2-C.sub.12 alkynylene, C.sub.1-C.sub.12 heteroalkylene, (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heterocyclylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heterocyclylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-aryl-(C.sub.0-C.sub.12 heteroalkylene), or (C.sub.0-C.sub.12 heteroalkylene)-heterocyclylene-(C.sub.0-C.sub.12 heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R.sup.1, and combinations thereof; each R.sup.1 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, or —S(O).sub.mR.sup.A; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; each of a and b are independently an integer between 1 and 10000, inclusive; each of “1”, “2”, “3”, “4”, “5”, and “6” is independently a terminal group selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, optionally substituted hydroxyl, optionally substituted amino, and optionally substituted thio; or represents a bond to a structure of Formula (I) or Formula (II); y is an integer between 1 and 100, inclusive; and m is 1 or 2.

(23) In certain embodiments, P is a polyether, polyester, polyacrylamide, polycarbonate, polysiloxane, polyfluorocarbon, polysulfone, or polystyrene. In certain embodiments, P is a polyether selected from the group consisting of polyethylene glycol (PEG), polyoxymethylene (POM), polypropylene glycol (PPG), polytetramethylene glycol (PTMG), poly(ethyl ethylene) phosphate (PEEP), and poly(oxazoline). In certain embodiments, P is a polyester selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyhydroxyalkanoate (PHA), polyhydroxybutryate (PHB), polyethylene adipate (PEA), polybutylene succinate (PBS), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). In certain embodiments, P is a poly(N-alkylacrylamide). In certain embodiments, P is a polycarbonate selected from the group consisting of poly(Bisphenol A carbonate), poly[Bisphenol A carbonate-co-4,4-(3,3,5-trimethylcyclohexylidene)diphenol carbonate], or poly(propylene carbonate). In certain embodiments, P is a polysiloxane. In certain embodiments, P is polydimethylsiloxane (PDMS). In certain embodiments, P is a polyfluorocarbon selected from the group consisting of poly(chlorotrifluoroethylene), poly(ethylene-co-tetrafluoroethylene), poly(tetrafluoroethylene), poly(tetrafluoroethylene-co-perfluoro(propylvinyl ether)), poly(vinylidene fluoride), and poly(vinylidene fluoride-co-hexafluoropropylene). In certain embodiments, P is a polysulfone selected from the group consisting of poly[1-[4-(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanediyl, sodium salt], poly(1-hexadecene-sulfone), poly(oxy-1,4-phenylenesulfonyl-1,4-phenylene), poly(oxy-1,4-phenylenesulfonyl-1,4-phenylene), and polyphenylsulfone.

(24) In certain embodiments, P is poly(ethylene glycol) with a molecular weight ranging from about 200 g/mol to about 6000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 200 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 200 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 1000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 1500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 2000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 2500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 3000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 3500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 4000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 4500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 5000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 5500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 6000 g/mol.

(25) In certain embodiments, B is C.sub.1-C.sub.12 alkylene, optionally substituted with 1-24 independently selected R.sup.1; R.sup.1 is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, or —S(O).sub.mR.sup.A; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; and m is 1 or 2.

(26) In certain embodiments, A is C.sub.1-C.sub.12 alkylene, optionally substituted with 1-24 independently selected R.sup.1; R.sup.1 is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, or —S(O).sub.mR.sup.A; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; and m is 1 or 2.

(27) In certain embodiments, A.sup.1 is C.sub.1-C.sub.12 alkylene, optionally substituted with 1-24 independently selected R.sup.1; R.sup.1 is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, or —S(O).sub.mR.sup.A; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; and m is 1 or 2.

(28) In certain embodiments, L is selected from a group consisting of

(29) ##STR00009##
wherein: q, p, and o are independently an integer between 0 and 20, inclusive.

(30) In certain embodiments, L is independently selected from

(31) ##STR00010##

(32) In certain embodiments, X is a chelated metal, inorganic compound, organometallic compound, organic compound, or salt thereof. In certain embodiments, the imaging agent contains a metal selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, rutherfordium, dubnium, seaborgium, bohrium, hassium, meitnerium, gadolinium, gallium, thallium, and barium. In certain embodiments, X is and inorganic compound. In certain embodiments, X is an organic compound. In certain embodiments, X is metal-free.

(33) In certain embodiments, the imaging agent is an magnetic resonance imaging (MRI) agent. In certain embodiments, the MRI agent is a chelated gadolinium. In certain embodiments, the MRI agent is a nitroxide radical-containing compound.

(34) In certain embodiments, the imaging agent is a nuclear medicine imaging agent. In certain embodiments, the nuclear medicine imaging agent is selected from the group consisting of .sup.64Cu diacetyl-bis(N.sup.4-methylthiosemicarbazone) (.sup.64Cu-ASTM), .sup.18F-fluorodeoxyglucose (FDG), .sup.18F-fluoride, 3′-deoxy-3′-[.sup.18F]fluorothymidine (FLT), and .sup.18F-fluoromisonidazole (FMISO), chelated gallium, chelated technetium-99m, and chelated thallium.

(35) In certain embodiments, the imaging agent is radiographic imaging agent. In certain embodiments, the radiographic imaging agent is selected from the group consisting of chelated barium, gastrografin, metrizoic acid, iotalamic acid, ioxaglate, iopamidol, iohexol, ioxilan, iopromide, iodixanol, and ioversol.

(36) In certain embodiments, the imaging agent X is a radical-containing compound. In certain embodiments, the imaging agent is a nitroxide radical-containing compound. In certain embodiments, the imaging agent X is of the formula:

(37) ##STR00011##

(38) In certain embodiments, the imaging agent X is an organic compound. In certain embodiments, the imaging agent is a salt of an organic compound. In certain embodiments, the imaging agent X is of the formula:

(39) ##STR00012##

(40) In certain embodiments, the repeating unit of Formula (I) is of formula:

(41) ##STR00013##
or a salt thereof, wherein: each of A, A.sup.1, and B is independently C.sub.1-C.sub.12 alkylene, C.sub.2-C.sub.12 alkenylene, C.sub.2-C.sub.12 alkynylene, or C.sub.1-C.sub.12 heteroalkylene, C.sub.2-C.sub.12 heteroalkenylene, C.sub.2-C.sub.12 heteroalkynylene, wherein each alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene is optionally substituted with 1-24 independently selected R.sup.1; X is an imaging agent; P is alkylene, heteroalkylene, or polymer; L is a bond, —O—, —S—, —S—S—, C.sub.1-C.sub.12 alkylene, C.sub.2-C.sub.12 alkenylene, C.sub.2-C.sub.12 alkynylene, C.sub.1-C.sub.12 heteroalkylene, (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heterocyclylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heterocyclylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-aryl-(C.sub.0-C.sub.12 heteroalkylene), or (C.sub.0-C.sub.12 heteroalkylene)-heterocyclylene-(C.sub.0-C.sub.12 heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R.sup.1, and combinations thereof, each R.sup.1 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, or —S(O).sub.mR.sup.A; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; y is an integer between 1 and 100, inclusive; and m is 1 or 2.

(42) In certain embodiments, the repeating unit is of formula:

(43) ##STR00014##

(44) In certain embodiments, the repeating unit is of the formula:

(45) ##STR00015##

(46) In certain embodiments, the repeating unit of Formula (II) is of formula:

(47) ##STR00016##
or a salt thereof, wherein: L is a bond, —O—, —S—, —S—S—, C.sub.1-C.sub.12 alkylene, C.sub.2-C.sub.12 alkenylene, C.sub.2-C.sub.12 alkynylene, C.sub.1-C.sub.12 heteroalkylene, (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heterocyclylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heterocyclylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-aryl-(C.sub.0-C.sub.12 heteroalkylene), or (C.sub.0-C.sub.12 heteroalkylene)-heterocyclylene-(C.sub.0-C.sub.12 heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R.sup.1, and combinations thereof; each R.sup.1 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, or —S(O).sub.mR.sup.A; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; and m is 1 or 2.

(48) In certain embodiments, the repeating unit is of formula:

(49) ##STR00017##

(50) In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is between about 1:20 to about 20:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (H) is about 1:20, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (11) is about 1:19, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:18, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:17, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:16, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:15, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:14, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:13, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (11) is about 1:12, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:11, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:10, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:9, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:8, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:7, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:6, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:5, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:4, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:3, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:2, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 2:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 3:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 4:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 5:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 6:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (11) is about 7:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 8:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 9:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 10:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 11:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 12:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 13:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 14:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 15:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 16:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 17:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 18:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 19:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 20:1, respectively.

(51) In certain embodiments, the polymer forms a particle or nanoparticle of a diameter between about 10 nm and about 1000 nm. In certain embodiments, the polymer forms a particle of a diameter between about 10 nm and about 100 nm. In certain embodiments, the polymer forms a particle of a diameter between about 100 nm and about 200 nm. In certain embodiments, the polymer forms a particle of a diameter between about 200 nm and about 300 nm. In certain embodiments, the polymer forms a particle of a diameter between about 300 nm and about 400 nm. In certain embodiments, the polymer forms a particle of a diameter between about 400 nm and about 500 nm. In certain embodiments, the polymer forms a particle of a diameter between about 500 nm and about 600 nm. In certain embodiments, the polymer forms a particle of a diameter between about 600 nm and about 700 nm. In certain embodiments, the polymer forms a particle of a diameter between about 700 nm and about 800 nm. In certain embodiments, the polymer forms a particle of a diameter between about 800 nm and about 900 nm. In certain embodiments, the polymer forms a particle of a diameter between about 900 nm and about 1000 nm. In certain embodiments, the polymer forms a particle of a diameter between about 28 nm and about 49 nm. In certain embodiments, the polymer forms a particle of a diameter between about 25 nm and about 40 nm.

(52) Methods for Preparing Brush-Arm Star Polymers

(53) In another aspect of the present disclosure, a method of producing a brush-arm star polymer comprising an imaging agent is described herein, the method comprises the steps of: reacting one or more macromonomers containing an imaging agent with a metathesis catalyst to form a living polymer; and mixing a crosslinker with the living polymer. In certain embodiments, at least two different macromonomers each containing a different imaging agent are reacted.

(54) In certain embodiments, the brush-arm star polymer is prepared by reacting macromonomer

(55) ##STR00018##
macromonomer

(56) ##STR00019##
a and ring-opening metathesis catalyst in a solvent to form a living polymer in the first step. In the second step the living polymer is then mixed with crosslinker

(57) ##STR00020##
to form the brush-arm star polymer. In certain embodiments the ring-opening methathesis catalyst is Grubbs 3.sup.rd generation bispyridyl catalyst (Grubbs III).

(58) In certain embodiments, the reaction time of the first step is between about 10 minutes and about 60 minutes. In certain embodiments, the reaction time of the first step is about 30 minutes. In certain embodiments, the reaction time of the second step is between about 1 hour and about 24 hours. In certain embodiments, the reaction time of the second step is about 6 hours.

(59) In certain embodiments, the solvent used to prepare the brush-arm star polymer can be polar or non-polar, protic or aprotic. Common organic solvents useful in the methods described herein include, but are not limited to, acetone, acetonitrile, benzene, benzonitrile, 1-butanol, 2-butanone, butyl acetate, tert-butyl methyl ether, carbon disulfide carbon tetrachloride, chlorobenzene, 1-chlorobutane, chloroform, cyclohexane, cyclopentane, 1,2-dichlorobenzene, 1,2-dichloroethane, dichloromethane (DCM), N,N-di methylacetamide N,N-dimethylformamide (DMF), 1,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone (DMPU), 1,4-dioxane, 1,3-dioxane, diethylether, 2-ethoxyethyl ether, ethyl acetate, ethyl alcohol, ethylene glycol, dimethyl ether, heptane, n-hexane, hexanes, hexamethylphosphoramide (HMPA), 2-methoxyethanol, 2-methoxyethyl acetate, methyl alcohol, 2-methylbutane, 4-methyl-2-pentanone, 2-methyl-1-propanol, 2-methyl-2-propanol, 1-methyl-2-pyrrolidinone, dimethylsulfoxide (DMSO), nitromethane, 1-octanol, pentane, 3-pentanone, 1-propanol, 2-propanol, pyridine, tetrachloroethylene, tetrahyrdofuran (THF), 2-methyltetrahydrofuran, toluene, trichlorobenzene, 1,1,2-trichlorotrifluoroethane, 2,2,4-trimethylpentane, trimethylamine, triethylamine, N,N-diisopropylethylamine, diisopropylamine, water, o-xylene, p-xylene. In certain embodiments, the solvent used to prepare the brush-arm star polymer is tetrahyrdofuran (THF).

(60) In certain embodiments, the molar ratio of chex-MM and Cy-MM is between about 1:1 and about 1000:1. In certain embodiments, the molar ratio of chex-MM and Cy-MM is about 100:1.

(61) In certain embodiments, the molar ratio of (chex-MM+Cy-MM) and ring-opening metathesis catalyst is between about 1:1 and about 100:1. In certain embodiments, the molar ratio of (chex-MM+Cy-MM) and Grubbs (III) is about 5.05:1. In certain embodiments, the molar ratio of (chex-MM+Cy-MM) and Grubbs (III) is about 7.07:1. In certain embodiments, the molar ratio of (chex-MM+Cy-MM) and Grubbs (III) is about 9.99:1.

(62) In certain embodiments, the molar equivalents of Acetal-XL with respect to Grubbs III is between about 1 equivalent and about 100 equivalents. In certain embodiments, the molar equivalents of Acetal-XL with respect to Grubbs III is about 15 equivalents. In certain embodiments, the molar equivalents of Acetal-XL with respect to Grubbs III is about 20 equivalents. In certain embodiments, the molar equivalents of Acetal-XL with respect to Grubbs III is about 30 equivalents.

(63) In certain embodiments, the macromonomer is of Formula (III):

(64) ##STR00021##
or a salt thereof, wherein: each of A, A.sup.1, and B is independently C.sub.1-C.sub.12 alkylene, C.sub.2-C.sub.12 alkenylene, C.sub.2-C.sub.12 alkynylene, or C.sub.1-C.sub.12 heteroalkylene, C.sub.2-C.sub.12 heteroalkenylene, C.sub.2-C.sub.12 heteroalkynylene, wherein each alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene is optionally substituted with 1-24 independently selected R.sup.1; X is an imaging agent; P is alkylene, heteroalkylene, or polymer; L is a bond, —O—, —S—, —S—S—, C.sub.1-C.sub.12 alkylene, C.sub.2-C.sub.12 alkenylene, C.sub.2-C.sub.12 alkynylene, C.sub.1-C.sub.12 heteroalkylene, (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 alkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 heteroalkylene)-arylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heteroarylene-(C.sub.0-C.sub.12 heteroalkylene), (C.sub.0-C.sub.12 alkylene)-heterocyclylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-heterocyclylene-(C.sub.0-C.sub.12 alkylene), (C.sub.0-C.sub.12 heteroalkylene)-aryl-(C.sub.0-C.sub.12 heteroalkylene), or (C.sub.0-C.sub.12 heteroalkylene)-heterocyclylene-(C.sub.0-C.sub.12 heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R.sup.1, and combinations thereof; each R.sup.1 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, or —S(O).sub.mR.sup.A; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; y is an integer between 1 and 100, inclusive; and m is 1 or 2.

(65) In certain embodiments, P is poly(ethylene glycol) with a molecular weight ranging from about 200 g/mol to about 6000 g/mol.

(66) In certain embodiments, B is C.sub.1-C.sub.12 alkylene, optionally substituted with 1-24 independently selected R.sup.1; R.sup.1 is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, or —S(O).sub.mR.sup.A; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; and m is 1 or 2.

(67) In certain embodiments, A is C.sub.1-C.sub.12 alkylene, optionally substituted with 1-24 independently selected R.sup.1; R.sup.1 is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, or —S(O).sub.mR.sup.A; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; and m is 1 or 2.

(68) In certain embodiments, A.sup.1 is C.sub.1-C.sub.12 alkylene, optionally substituted with 1-24 independently selected R.sup.1; R.sup.1 is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR.sup.A, —N(R.sup.A).sub.2, —NR.sup.AC(O)R.sup.A, —NR.sup.AC(O)OR.sup.A, —NR.sup.AC(O)N(R.sup.A).sub.2, —C(O)N(R.sup.A).sub.2, —C(O)R.sup.A, —C(O)OR.sup.A, —OC(O)R.sup.A, —OC(O)OR.sup.A, —OC(O)N(R.sup.A).sub.2, —SR.sup.A, or —S(O).sub.mR.sup.A; each R.sup.A is independently hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 heteroalkyl, or C.sub.1-C.sub.6 haloalkyl; and m is 1 or 2.

(69) In certain embodiments, the metathesis catalyst is a ring-opening metathesis polymerization (ROMP) catalyst. In certain embodiments, the metathesis catalyst is a transition metal complex. In certain embodiments, the metal is selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, rutherfordium, dubnium, seaborgium, bohrium, hassium, and meitnerium. In certain embodiments, the metathesis catalyst is a ruthenium complex. In certain embodiments, the metathesis catalyst is a molybdenum complex. In certain embodiments, the metathesis catalyst is a zirconium complex. In certain embodiments, the metathesis catalyst is selected from the group consisting of RuC13/alcohol mixture, bis(cyclopentadienyl)dimethylzirconium(IV), dichloro[1,3-bis(2,6-isopropylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine)ruthenium(II), dichloro[1,3-Bis(2-methylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine) ruthenium(II), dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][3-(2-pyridinyl)propylidene]ruthenium(II), dichloro(3-methyl-2-butenylidene)bis (tricyclopentylphosphine)ruthenium(II), dichloro[1,3-bis(2-methylphenyl)-2-imidazolidinylidene](2-isopropoxyphenylmethylene)ruthenium(II) (Grubbs C571), dichloro(benzylidene)bis(tricyclohexylphosphine)ruthenium(II) (Grubbs I), dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine) ruthenium(II) (Grubbs II), and dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)bis(3-bromopyridine)ruthenium(II) (Grubbs III). In certain embodiment, the metathesis catalyst is of the formula:

(70) ##STR00022##
Compositions and Kits

(71) In one aspect of the present disclosure, compositions and kits are described herein. In certain embodiments, a composition is comprised of a polymer described herein and a pharmaceutically acceptable excipient. In certain embodiments, a composition is comprised of an effective amount of a polymer described herein.

(72) Compositions described herein can be prepared by any method known in the art. In general, such preparatory methods include bringing the polymer described herein into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

(73) Compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

(74) Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

(75) Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

(76) Although the descriptions of compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

(77) The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.

(78) The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound or polymer described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell.

(79) Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

(80) The compound or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

(81) Also encompassed by the disclosure are kits. The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.

(82) In certain embodiments, the kits are comprised are comprised of a polymer described herein and instructions for use. In certain embodiments, the kits are comprised of a composition described herein and instructions for use.

(83) In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.

(84) Methods of Treatment

(85) In one aspect of the present disclosure, methods of imaging a subject or a portion of a subject are described herein, the method comprising steps of: administering to a subject a polymer described herein, or a composition described herein; and acquiring an image. In certain embodiments, the imaging modality is selected from the group consisting of radiography, magnetic resonance imaging (MRI), nuclear medicine, ultrasound, elastography, tactile imaging, photoacoustic imaging, tomography, echocardiography, near-infrared fluorescence (NIRF) imaging, and magnetic particle imaging. In certain embodiments, the imaging modality is magnetic resonance imaging (MRI). In certain embodiments, the imaging modality is near-infrared fluorescence (NIRF) imaging.

(86) In certain embodiments, the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In some embodiments, the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs).

(87) In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 minute and about 100 hours. In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 hour and about 100 hours. In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 hour and about 50 hours. In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 hour and about 20 hours. In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 hour and about 10 hours. In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 hour and about 5 hours.

EXAMPLES

(88) In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods, compositions, and systems provided herein and are not to be construed in any way as limiting their scope.

(89) BASP-ORCA Design and Synthesis

(90) One of the most common ways to increase the relaxivity of MRI contrast agents (including nitroxides) involves attaching them to a rigid macromolecular scaffold. For example, Rajca et al., appended a spirocyclohexyl nitroxide derivative (“chex”).sup.69 to the surface of dendrimers to produce chex-dendrimer ORCAs where the per-chex r.sub.1 was 0.42 mM.sup.−1s.sup.−1 compared to r.sub.1=0.14 mM.sup.−1s.sup.−1 for the model nitroxide 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-CP). In another study, chex was appended to the core of PEGylated branched-bottlebrush polymers..sup.70 The resulting polymers had a per-chex r.sub.1 of 0.32 mM.sup.−1s.sup.−1, which was approximately 50% greater than the chex-macromonomer used to synthesize these polymers (chex-MM, FIG. 1A). In this system, r.sub.2 also increased from 0.30 mM.sup.−1s.sup.−1 for chex-MM to 0.82 mM.sup.1s.sup.1 for the chex-bottlebrush polymer, thus demonstrating that increasing the macromolecular size and chex density leads to increases in both r.sub.1 and r.sub.2, with a greater increase in r.sub.2. In an effort to further increase these relaxivity values, the aim was to incorporate chex into the BASP macromolecules. Moreover, it was hypothesized that BASPs could provide enhanced nitroxide stability potentially making tumor imaging in vivo possible. The control and robustness of BASP synthesis would enable the scalable production of BASP-ORCAs with optimal sizes for tumor accumulation.

(91) BASP-ORCAs were synthesized following the brush-first ring-opening metathesis polymerization (ROMP) strategy (FIGS. 1A-1B)..sup.71′72 Norbornene-based branched macromonomers (MMs, FIG. 1A) featuring 3 kDa (PEG) and either chex (chex-MM) or Cy5.5 dye (Cy-MM, FIG. 1A) were copolymerized by exposure to Grubbs 3.sup.rd generation bis-pyridine initiator.sup.73 (Grubbs III, FIG. 1A; reaction stoichiometry: j equivalents chex-MM: 0.01j Cy-MM: 1.0 Grubbs III) for 30 minutes (FIG. 1B). The resulting living bottlebrush polymers with an average degree of polymerization (DP) of: ˜j+0.01j=m were crosslinked via slow addition of N equivalents of bis-norbornene acetal crosslinker (Acetal-XL, FIG. 1A) directly to the reaction mixture to generate the desired BASP-ORCA (FIG. 1B). With this method, the BASP-ORCA size is determined by the MM:Grubbs III:Acetal-XL ratios (i.e., m and N values). Much less Cy-MM (0.01j) relative to chex-MM (j) was used to bridge the difference in concentration requirements between MRI (mM to μM) and NIRF (nM to pM).

(92) To identify optimal conditions for the synthesis of BASP-ORCAs with narrow size distributions within the range of ˜25-40 nm, as well as high water solubility and relaxivity, m and N values from 5-10 and 15-30, respectively were screened (see FIG. 15). Gel permeation chromatography (GPC) revealed nearly quantitative MM-to-bottlebrush conversion as well as ≥85% bottlebrush-to-BASP conversion for all m and N values (FIGS. 7A-7B). The NP diameters as determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM) ranged from ˜28 to ˜49 nm (see FIG. 15). A representative TEM image for the m=7.07 and N=20 BASP-ORCA (referred to as BASP-ORCA1) is provided in FIG. 2A. The hydrodynamic diameter (D.sub.h) of this particle was 31±4 nm, which is suitable for extended in vivo circulation and tumor accumulation.

(93) Characterization of BASP-ORCA magnetic properties

(94) Electron paramagnetic resonance spectroscopy (EPR) was used to confirm the presence of chex in BASP-ORCAs, as well as to study the chex environment in BASP-ORCA1. The spin concentrations were ≥85% for all BASP-ORCAs. The height-normalized EPR spectra for BASP-ORCA1 and chex-MM are shown in FIG. 2B. The spectrum for BASP-ORCA1 is significantly broader than chex-MM, which is consistent with the larger and more rigid BASP nanostructure where the chex molecules are bound at the dense interface between the acetal crosslinker core and the PEG shell (FIGS. 1B and 2B). The BASP-ORCA1 spectrum was simulated using the procedure developed by Budil and Freed.sup.74, which allows for characterization of the chex mobility in terms of the correlation time for rotational diffusion (τ). The spectrum was best fitted by superimposing two computed components (FIGS. 9A-9D): 22% corresponded to a relatively fast-moving nitroxide with τ=0.2 ns while 78% corresponded to a very slow-moving nitroxide with τ=10.0 ns. The faster-moving component likely corresponds to nitroxides that are furthest from the BASP-ORCA1 acetal core (FIG. 1B), while the slow-moving component corresponds to nitroxides that are close to and/or entangled within the rigid acetal core. Notably, the τ of 10.0 ns measured for the slow component in BASP-ORCA1 is very large, which suggests that the majority of chex molecules are in a rigid environment. For comparison, in the previously reported chex-dendrimer ORCAs, TEMPO-labeled bottlebrush polymers, and BASPs, the largest τ measured was ˜1 ns.

(95) Next, the longitudinal (r.sub.1) and transverse (r.sub.2) relaxivities of these BASP-ORCAs were evaluated using a Bruker 7 T MRI scanner. The per-chex r.sub.1 values as a function of m and N (FIG. 15) ranged from 0.27-0.53 mM.sup.−1s.sup.−1; they were not significantly increased compared to Rajca's chex-dendrimer and the chex-bottlebrush polymers. However, the per-chex r.sub.2 values ranged from 2.90-7.40 mM.sup.−1s.sup.−1, which is ˜3.5-˜9.0-fold greater than the per-chex r.sub.2 in the chex-bottlebrush polymers and ˜17-˜44-fold greater than 3-CP (FIG. 15). BASP-ORCA1 displayed a per-chex r.sub.2 value of 4.67 mM.sup.−1s.sup.−1. Though this value was not the highest that was measured, BASP-ORCA1 was selected because it offered the best balance of high relaxivity, solubility (approximately 50 mg/mL, FIG. 15), and size for translation to biological studies. Given the number-average molar mass of BASP-ORCA1 as determined by gel permeation chromatography and static light scattering (M.sub.n=4.75×10.sup.5 g/mol, D=1.32), it can be estimated that each BASP-ORCA1 particle contains an average of 92 chex groups. Based on this number, the estimated average molecular r.sub.1 and r.sub.2 values for BASP-ORCA1 are 37.6 mM.sup.−1s.sup.−1 and 428.8 mM.sup.−1s.sup.−1, respectively, which are greater than those for the commonly used FDA-approved Gd-based contrast agent Magnevist (r.sub.1=3.1 mM.sup.−1s.sup.−1 and r.sub.2=5.4 mM.sup.−1s.sup.−1 at 7 T) and iron-based NPs such as Feraheme (r.sub.1=3.1 mM.sup.−1s.sup.−1 and r.sub.2=68 mM.sup.4s.sup.−1 at 7 T)..sup.75,76,77,78 The r.sub.2/r.sub.1 ratio for BASP-ORCA1 is approximately one-half that of Feraheme. Thus, BASP-ORCA1 should provide effective T.sub.2-weighted MRI contrast enhancement.

(96) MR phantom images of phosphate-buffered saline (PBS) solutions of BASP-ORCA1, chex-MM, and the previously reported chex-bottlebrush polymer at various chex concentrations (from 1 mM-4 mM chex) as well as a PEG-based BASP that lacks chex (at equivalent concentrations by mass as BASP-ORCA1) are provided in (FIG. 2C), along with images for “blank” PBS buffer. The T.sub.1-weighted images for BASP-ORCA1, chex-MM, and chex-bottlebrush polymer are not obviously different while the T.sub.2-weighted images clearly show a large decrease in signal for BASP-ORCA1 as concentration increases. The PEG-BASP with no chex shows no change in contrast as a function of concentration, which confirms that chex is required to observe any changes in image contrast.

(97) The data presented above demonstrate that the high nitroxide density of BASP-ORCA1, which is a consequence of its unique crosslinked multi-layer nanostructure, affords an increased magnetization capability for r.sub.2 enhancement. This finding is consistent with reports where nitroxides are utilized as magnetic catalysts for outer-sphere relaxation processes..sup.79,80,81 Most importantly, the exceptionally high r.sub.2 of BASP-ORCA1 overcomes one of the major limitations of nitroxide-based contrast agents: inherently low contrast.

(98) Ascorbate Quenching Kinetics of BASP-ORCAs

(99) As discussed above, nitroxide-based ORCAs typically suffer from rapid reduction to diamagnetic hydroxylamines under biologically relevant conditions. Amongst the many potential biological reducing agents, ascorbate (Asc) is known to play a major role in in vivo nitroxide reduction,.sup.82,83 and Asc-induced reduction can be amplified by glutathione (GSH). It was hypothesized that the rigid chex environment in the BASP-ORCAs could help to lower the rate of chex reduction. To test this hypothesis, EPR spectra for BASP-ORCA1 at various times were collected following exposure to 20 equivalents of Asc and 20 equivalents of GSH per nitroxide (both reagents were present in 10 mM concentrations). EPR spectra collected 1 minute, 40 minutes, and 180 minutes after exposure to these conditions are provided in FIG. 3A; the reduction in peak height as a function of time is indicative of nitroxide reduction. The normalized peak height of the EPR spectra are plotted versus time in FIG. 3B. Reduction kinetics data for the previous chex-bottlebrush polymers and chex-MM are provided for comparison. In contrast to the chex-bottlebrush and chex-MM samples, which both display an initial rapid chex reduction phase in the first hour, the reduction of chex in BASP-ORCA1 was significantly retarded with nearly 85% remaining after 1 hour, and 70% remaining after 3 hours (compared to 65% and 57%, respectively, for the chex-bottlebrush). Based on the integrated peak heights as a function of time, the second-order rate constants for BASP-ORCA1 reduction in the initial (first 10 minutes) and late (>1 hour) stages of the reduction process were calculated: k.sub.early=0.0376 M.sup.−1s.sup.−1 and k.sub.late 0.00672 M.sup.−1s.sup.−1) (Table 2). Simulation revealed that the EPR spectra collected during the reduction process still consisted of a “fast” and a “slow” component (FIGS. 9A-9D). Interestingly, T for the “fast” component remained constant at 0.2 ns, while T for the “slow” component became increasingly larger with time (11.0 ns at 40 minutes and 13.2 ns at 180 minutes). Therefore, even after 3 hours there persists an extremely reduction resistant and slow moving nitroxide population, which suggests that BASP-ORCAs could be used for tumor MRI over longer timescales than have been possible with previous nitroxide contrast agents (vide infra).

(100) Nitroxide Reduction Kinetics

(101) TABLE-US-00001 TABLE 2 Kinetics of the reduction of nitroxides with 20-fold molar excess of ascorbate (Asc) and 0-25-fold molar excess of glutathione (GSH). Numerical fits to pseudo-first order rate equation (k′) peak height (PH) or integrated peak height (IPH) of the low-field EPR line. Late Initial Ki- Ki- Avg netics Ni- netics k × k × (>1 h) k × trox Asc. GSH (<1 h) k′ × 10.sup.4 10.sup.4 Range k′ × 10.sup.4 Run Run Data Conc. Conc. Conc. Range 10.sup.4 (M.sup.−1 (M.sup.−1 of 10.sup.4 (M.sup.−1 Compd No. Label used (mM) (mM) (mM) of fits (s.sup.−1) R.sup.2 s.sup.−1) s.sup.−1) fit (h) s.sup.−1) R.sup.2 s.sup.−1) BASP- 1 JP1191 IPH .sup.  0.5 10 10 <1000 3.294 0.8795 329.4 366 ± 25  1.2-2.8 0.672 0.9923 67.2 ORCA1.sup.a IPH*.sup.a 3.40 0.9948 339.7 334 ± 55  0.586 0.9994 58.6 PH 0.836 0.8721 83.6 0.297 0.9943 29.7 2 JP1190 IPH .sup.  0.5 10 10 115-595 3.712 0.7664 371.2 IPH*.sup.a 3.408 0.9910 340.8 PH 3.377 0.2923 33.77 JP1189 IPH .sup.  0.5 10 10 113-613 3.828 0.7646 382.8 IPH*.sup.a 3.238 0.9863 323.7 PH 5.07 0.3068 50.7 4 JP1188 IPH .sup.  0.5 10 10 126-603 3.818 0.5387 381.8 IPH*.sup.a 3.311 0.9938 331.1 PH 5.072 0.3366 50.72 1 YW982 IPH .sup.  0.5 10 5.0 177-897 3.27 0.9633 327.0 306.sup.b chex- PH 3.42 0.9702 342.0 308.sup.b bottle- brush 2 YW983 IPH .sup.  0.5 10 5.0  396-1019 2.85 0.9520 285.0 1.1-2.8 0.416 0.9216 41.6 PH 2.73 0.9895 273.0 0.386 0.9938 38.6 1 YW981 IPH .sup.  0.5 10 0.0 251-851 3.05 0.9439 305.0 296.sup.b chex- PH 2.41 0.9808 241.0 254.sup.b bottle- brush 2 YW985 IPH .sup.  0.5 10 0.0 278-878 2.86 0.9145 286.0 1.3-2.8 0.243 0.8838 24.3 PH 2.68 0.9775 268.0 0.196 0.9735 19.6 chex- 1 JP609  IPH .sup.  0.5 10 0.0  90-390 6.20 0.6609 620.0 603 ± 123 0.8-2.8 0.301 0.6847 30.1 den- drimer PH 6.17 0.9718 617.0  579 ± 59.6 0.354 0.9663 35.4 2 JP610  IPH .sup.  0.5 10 0.0 115-415 7.18 0.6743 718.0 PH 6.09 0.9336 609.0 3 JP611  IPH .sup.  0.5 10 0.0 126-426 4.72 0.7984 472.0 PH 5.10 0.9915 510.0 3-CP 1 JP899  IPH .sup.  0.2 4.0 5.0 <600 2.435 0.9997 608.8 608.0 ± 4.2  PH 2.361 0.9990 590.3 602.6 ± 25   2 JP8100 IPH .sup.  0.2 4.0 5.0 <600 2.438 0.9997 609.6 PH 2.410 0.9996 602.4 3 JP1101 IPH .sup.  0.2 4.0 5.0 <600 2.423 0.9998 605.6 PH 2.461 0.9996 615.2 3-CP 1 JP460  IPH .sup.  0.2 4.0 0.0 <3600 2.547 0.9996 636.8 625 ± 22  PH 2.504 0.9949 636.0 611 ± 44  2 JP461  IPH .sup.  0.2 4.0 0.0 <3600 2.498 0.9975 624.5 PH 2.396 0.9949 599.0 3 JP462  IPH .sup.  0.2 4.0 0.0 <3600 2.459 0.9999 614.8 PH 2.389 0.9961 597.3 >1 1.18  0.9952 295 .sup.aFor BASP-ORCA1, double integration of entire EPR spectra gave initial rate constant k = 449 ± 23 M.sup.−1s.sup.−1, which is somewhat larger than the integrated peak height (IPH) value, k = 366 ± 25 M.sup.−1s.sup.−1; IPH* is the integrated peak height for the center line of the EPR spectrum. .sup.bFor ORCA-Fluor, initial second order rate constants from 4 kinetic runs using 0-10 equivalents of GSH, k = 301 ± 20 and 281 ± 43 M.sup.−1s.sup.−1 for baseline corrected IPH and PH data. Data for chex-bottlebrush,.sup.90 data for chex-dendrimer (baseline corrected) and late kinetics for 3-CP with Asc only, data for 3-CP with 20 equivalents of Asc and 25 equivalents of GSH,.sup.92 and data for 3-CP with Asc only.sup.93 were reported elsewhere.
Fluorescence Properties of BASP-ORCAs

(102) As noted above, Cy5.5 was also incorporated into these BASP-ORCAs (see FIG. 10 for BASP-ORCA1 absorption and emission spectra confirming the presence of Cy5.5) in order to simultaneously use NIRF as an imaging modality for comparison to MRI. Nitroxides are well-known to quench fluorescence via catalysis of non-emissive photophysical processes such as intersystem crossing. This quenching requires close interaction between the nitroxide and the fluorophore; the systems with the greatest quenching typically feature the nitroxide directly linked to the fluorophore via it bonds (i.e., electronic conjugation)..sup.84,85,86 Given the fact that chex and Cy5.5 are incorporated into BASP-ORCAs via two different macromonomers, and noting the limited mobility of chex in these nanoparticles, it was reasoned that Cy5.5 quenching would be minimal; therefore, Cy5.5 emission could be used as a fairly constant descriptor of particle concentration regardless of the extent of chex reduction.

(103) To test this hypothesis, BASP-ORCA1 was exposed to a large excess of Asc (40 to 120 equivalents to chex) in water, and monitored the resulting Cy5.5 emission. In agreement with the expectation, only a 25±2% to 30±2% increase in fluorescence emission was observed (FIG. 3C). Moreover, addition of GSH (60 equivalents) as a co-reductant along with 60 equiv. of Asc gave only a 35±7% increase in fluorescence. Taken together, these data suggest that Cy5.5 fluorescence is only minimally quenched by chex in BASP-ORCA1. For comparison, exposure of the previously reported chex-bottlebrush polymer containing Cy5.5 to excess Asc or Asc+GSH led to 119±5% and 250±5% increases in fluorescence, respectively. Notably, the time required to achieve a fluorescence plateau varied significantly between BASP-ORCA1 (approximately 40 minutes) and the chex-bottlebrush polymer (a few minutes). Collectively, these data suggest that the BASP nanostructure provides greater steric shielding and isolation of chex and Cy5.5 compared to the analogous bottlebrush polymer.

(104) In Vitro Cytotoxicity and In Vivo Gross Toxicity, Pharmacokinetics (PK), and Biodistribution (BD) of BASP-ORCA1 in Non-Tumor Bearing Mice

(105) Motivated by BASP-ORCA1's unprecedented combination of properties, which include nanoscopic size (D.sub.h=31±4 nm) and narrow size distribution, good water solubility, slow reduction kinetics, and exceptionally high r.sub.2 relaxivity for an organic contrast agent, next the performance of this nanomaterial in biological assays was investigated. As discussed above, one potential advantage of ORCAs is their low toxicity. To investigate the toxicity of BASP-ORCA1, first in vitro human umbilical vein endothelial cell (HUVEC) and HeLa cell viability assays were conducted. In these assays, the cells were incubated with varied concentrations of BASP-ORCA1 for 72 h. Cell viability was determined by the CellTiter Glo assay (FIG. 11). The half-maximal inhibitory concentration (IC.sub.50) of BASP-ORCA1, i.e., the concentration that led to 50% cell death, was 1.5 mg/mL (280 μM chex) and 4.5 mg/mL (830 μM chex) in HUVEC and HeLa cells, respectively. These results confirm that BASP-ORCA1 induces negligible in vitro cytotoxicity at practical concentrations. Next, the in vivo gross toxicity of BASP-ORCA1 was assessed. Healthy BALB/c mice were administered increasing doses (from 5 to 30 mg or 0.2 to 1.5 g/kg, respectively) of BASP-ORCA1 via tail vein injection. The animal body masses and behaviors were monitored over the course of 30 days. Loss of ≥10% body mass is generally considered to be a sign of unacceptable toxicity..sup.87,88 As shown in FIG. 12, even the highest dose of BASP-ORCA1 (administered to n=4 animals) induced no significant decrease in body mass, which suggests that these particles are well-tolerated up to their solubility-limiting dose.

(106) The pharmacokinetics (PK) and biodistribution (BD) of BASP-ORCA1 were monitored in healthy, non-tumor bearing BALB/c mice (n=3) using NIRF imaging (IVIS, Cy5.5λ.sub.ex/λ.sub.em=640/700 nm). For PK analysis, blood samples were collected via cardiac puncture at various time points from 1 hour to 48 hours. Percent injected dose was plotted as a function of time (FIG. 13A). As is common for spherical nanoparticles, BASP-ORCA1 exhibited a two-phase clearance behavior, with an early distribution phase of ˜6 hours, followed by a steady elimination phase. Fitting the data presented in FIG. 13A with a standard two-compartment model yielded a blood compartment half-life for BASP-ORCA1 of 10 hours..sup.89 This long half-life is attributed to the nanoscale size of BASP-ORCA1, which limits renal clearance, and its PEGylated corona, which minimizes protein absorption and macrophage uptake. Consistent with these results and a plethora of studies on PEGylated nanoparticles, BD analysis revealed that a majority of BASP-ORCA1 accumulated in the liver, with increasing accumulation over 72 hours (FIG. 13B). Less, but significant, accumulation in the kidney and negligible accumulation in other tissues was observed (Note: fluorescence in extracted lung tissue is attributed to a high concentration of BASP-ORCA1 in the blood). Notably, fluorescence images of fecal samples (FIG. 13C) suggest that BASP-ORCA1 is ultimately cleared from the body via excretion.

(107) BASP-ORCA1 BD in Tumor-Bearing Mice

(108) Given the long circulation of BASP-ORCA1, it was hypothesized that this particle would passively accumulate in subcutaneous tumors following systemic injection. To test this hypothesis, a tumor model was established via subcutaneous injection of a mixture of 2.0×10.sup.6 lung carcinoma cells (A549, ATCC), Matrigel, and PBS buffer into a hind flank of NCR-NU mice (n=4). When the average tumor volume was ˜1 cm, BASP-ORCA1 was administered at a dose of 0.23 mmol chex/kg via tail vein injection. The mice were imaged 20 hours after administration. This choice of imaging time strikes a balance between allowing for sufficient tumor accumulation while limiting the extent of chex reduction in vivo. NIRF images indicated substantial tumor accumulation of BASP-ORCA1, which is consistent with other reports for PEGylated nanoparticles of similar size including the related drug-conjugated BASPs (FIG. 4A). Ex vivo BD data were consistent with the studies on non-tumor bearing BALB/c mice (i.e., liver accumulation and persistence in blood, FIGS. 13A and 13B) with the addition of significant tumor accumulation (FIG. 4C and FIG. 14).

(109) In Vivo MRI and NIRF Imaging with BASP-ORCA1

(110) The low toxicity, long circulation half-life, and tumor accumulation of BASP-ORCA1, along with its exceptional chex stability and relaxivity, suggested that this particle could be suitable for MRI of tumors following systemic injection and accumulation; a feat that has not yet been reported with ORCAs. Two groups of A459 tumor-bearing NCR-NU mice were administered different doses of BASP-ORCA1 via tail-vein injection: the “low dose” group (n=3) received 0.16 s mmol chex/kg (0.8 g BASP-ORCA1/kg) while the “high dose” group (n 4) received 0.23 mmol chex/kg (1.2 g BASP-ORCA1/kg). The mice were anaesthetized and MR images were collected at various time points: 12 hours, 16 hours, and 20 hours post-injection for the low dose group and 20 hours post-injection for the high dose group. The images from each time point were compared to images collected before BASP-ORCA1 injection. FIG. 5A shows T.sub.2-weighted images for a selected mouse from the low dose group imaged before BASP-ORCA1 injection (top row of images) and 20 hours (bottom row of images) after BASP-ORCA1 injection; from left-to-right the images correspond to progressive slices of the same animal in the z-axis with the tumor observed on the bottom right of each image. FIG. 5B shows an analogous set of images for a selected mouse from the high dose group. Contrast differences between the pre-injection and post-injection images can be observed at both dose levels, with greater contrast observed in the high dose animal. Whole animal images similarly revealed a clear difference in tumor contrast (FIG. 5C, arrows).

(111) The percent negative contrast enhancement (i.e., signal reduction) before and after BASP-ORCA1 administration was quantified by image analysis (FIG. 5D). Signal reductions ranging from 14±2% to 16±2% (P≤0.05) were observed for the 12 hour to 20 hour time points in the low dose group (FIG. 5D, low dose bars). In the high dose group, a 24±2% (P≤0.001) signal reduction was observed 20 hours after BASP-ORCA1 administration (FIG. 5D, high dose right bar). The clear BASP-ORCA1 dose-response effect suggests that the observed contrast differences between pre- and post-injection are due to accumulation of BASP-ORCA1 in the tumors. Keeping in mind that MRI phantoms revealed no observable contrast enhancement for PEG-BASPs that lack chex (FIG. 2C), these MRI data imply that 20 hours following injection there is a sufficient concentration of non-reduced chex (i.e., chex radicals) present on the BASP-ORCA1 to impart contrast. To confirm the presence of chex radicals in the tumors, the same mice that were imaged by MRI were sacrificed 21 hours after BASP-ORCA1 administration and their tissue homogenates and blood were analyzed by EPR spectroscopy (FIG. 6A). From these spectra, the radical concentration per g protein in each tissue sample, the latter obtained via a bicinchoninic acid assay (BCA), of the tissue homogenate, was evaluated and normalized by the radical concentration per g protein in muscle tissue (FIG. 6B). In agreement with the MRI data, the concentration of free radicals in the tumor was quite high 22 hours after BASP-ORCA1 injection; the measured value of 0.25 μmol ±0.04 chex/g of protein corresponds to 4.5% of the injected dose of chex radicals (Note: this value does not include any chex radicals that were reduced, and thus the percent injected dose of BASP-ORCA1 in the tumor is likely larger than 4.5%). Moreover, consistent with the in vivo NIRF imaging results (vide supra), relatively high radical concentrations were observed in the liver and kidney, which suggests that the clearance of BASP-ORCA1 proceeded mostly through these organs. Notably, the murine liver contains a high concentration of Asc (millimolar range); the observation of radicals in the liver 22 hours after injection is further evidence of the extremely stable nature of the chex units in βASP-ORCA1 (Note: in the previous chex-bottlebrush polymers, there was very little chex radical in the liver following 30 minutes and none after 24 hours). A high chex concentration was also observed in the heart, which is in accord with a long blood compartment half-life and is consistent with the PK data obtained by NIRF imaging. Finally, NIRF imaging of these homogenates provided fluorescence radiant efficiencies that were in good agreement with the spin concentrations (FIG. 6B), which suggests that the chex radicals and Cy5.5 dyes are still co-localized within the BASP-ORCA1 construct 22 hours after injection. Unlike the previous chex-bottlebrush polymers, which displayed dramatic increases in fluorescence as chex was reduced, the signal uniformity offered by BASP-ORCA1 provides for straightforward multi-modal confirmation of BD.

(112) BASP-ORCA1 is the first nitroxide MRI contrast agent capable of providing significant contrast 20 hours after injection, which is a testament to its unique structural features that combine optimal size for tumor accumulation with a high nitroxide density and stability. To set these results in context, the data outlined herein was compared to recent literature examples of MRI-contrast agents the rely on metals to achieve tumor imaging following systemic administration. For example, Kataoka and coworkers recently reported on a new class of Gd-based nanoparticles (T.sub.1 contrast agents) for MRI of tumors. In their study, a ˜40% contrast enhancement (at 0.05 mmol Gd/kg iv dose) was observed plateauing 4 hours following injection into mice bearing subcutaneous C26 tumors. Notably, commercially available Gd-DTPA, which is a small molecule, exhibited negligible contrast enhancement (at 0.23 mmol Gd/kg iv dose) after 4 hours. This example highlights the importance of a nanoparticle system for extended circulation and tumor imaging, though the impact of Gd nanoparticle accumulation in tissues would need to be addressed prior to clinical translation. In another example, the same group reported novel Fe-based nanoparticles (T.sub.2 contrast agents) for tumor imaging in a similar murine model (subcutaneous C26 tumors). Here, an approximately 25% contrast difference was observed 24 hours following intravenous administration of 0.45 mg Fe/kg. Notably, less than 10% contrast enhancement was observed using commercially available Resovist® (at 0.45 mg Fe/kg intravenous dose).Error Bookmark not defined. It should be noted that the instrument parameters used to obtain T.sub.2-weighted images in this work were similar to those used in the studies described herein; thus, these results for BASP-ORCA1 are on par with recently reported nanoparticle MRI contrast agents that rely on metals to achieve contrast.

Conclusion

(113) In conclusion, a nitroxide-nanoparticle MRI contrast agent -BASP-ORCA1—that enables simultaneous MRI and NIRF imaging in vivo over timescales suitable for tumor imaging following systemic injection was developed herein. BASP-ORCA1 addresses the two major challenges that have historically limited nitroxide-based organic radical contrast agents for MRI: low relaxivity and poor stability. These functions were made possible by the brush-arm star polymer (BASP) nanostructure, which enables the placement of chex nitroxides at the interface between a rigid poly(acetal) core and a hydrophilic PEG shell. Altogether, BASP-ORCA1 displayed unprecedented per-nitroxide and per-molecule transverse relaxivities for organic radical contrast agents, exceptional stability, high water solubility, low in vitro and in vivo toxicity, and a long blood compartment half-life. These features combined to facilitate the imaging of subcutaneous tumors in mice 20 hours after tail-vein injection, providing contrast enhancements on par with commercial and literature examples of metal-based contrast agents. This work suggests that organic radicals can be viable alternatives to metal-based MRI contrast agents, and sets the stage for the development of theranostic systems that combine organic radical contrast agents with therapeutic payloads to achieve simultaneous tumor imaging and drug delivery without concerns over long-term accumulation of metals.

(114) Materials, General Methods, and Instrumentation

(115) All reagents were purchased from commercial suppliers and used without further purification unless stated otherwise. Grubbs 3rd generation bispyridyl catalyst,.sup.73 macromonomers (MMs) chex-MM,.sup.2 Cy-MM,.sup.90 PEG-MM and cross-linker Acetal-XL were prepared according to literature procedures. Size exclusion chromatography (SEC) analyses were performed on an Agilent 1260 Infinity setup with two Shodex KD-806M columns in tandem and a 0.025 M LiBr DMF mobile phase run at 60° C. The differential refractive index (dRI) of each compound was monitored using a Wyatt Optilab T-rEX detector, and the light scattering (LS) signal was acquired with a Wyatt Dawn Heleos-II detector. Column chromatography was carried out on silica gel 60F (EMD Millipore, 0.040-0.063 mm).

(116) Dynamic light scattering (DLS) measurements were performed using a Wyatt Technology Mobius DLS instrument. Samples were prepared at 1.0 mg/mL in either nanopure water (MilliQ), PBS buffer, or 5% glucose solution (in nanopure water). The resulting solutions were passed through a 0.4 μm Nalgene filter (PES membrane) into disposable polystyrene cuvettes, which were pre-cleaned with compressed air. Measurements were made in sets of 10 acquisitions, and the average hydrodynamic diameters were calculated using the DLS correlation function via a regularization fitting method (Dynamics 7.4.0.72 software package from Wyatt Technology).

(117) TEM images were acquired using a FEI Tecnai Multipurpose TEM (G2 Spirit TWIN, 12 kV) at the MIT Center for Materials Science and Engineering. Samples were prepared as follows: 5 μL of a 1.0 mg/mL aqueous solution of BASP-ORCA was pipetted onto a carbon film-coated 200-mesh copper grid (Electron Microscopy Sciences) placed on a piece of parafilm. Next, the solution was carefully absorbed at the base of the droplet using the edge of a Kimwipe, leaving behind the nanoparticles on the TEM grid. The samples were then negatively stained by adding a drop of 2 wt % uranyl acetate (Electronic Microscopy Sciences). After 3 min, the residual uranyl acetate solution was carefully absorbed onto a Kimwipe, and the samples were allowed to dry completely.

(118) Excitation/emission spectra and fluorescence measurements were acquired using a Tecan Infinite® 200 Pro plate reader. Electron Paramagnetic Resonance (EPR) spectra were acquired at the University of Nebraska using a Bruker CW X-band spectrometer equipped with a frequency counter. The spectra were obtained using a dual mode cavity; all spectra were recorded using an oscillating magnetic field perpendicular (TE.sub.102) to the swept magnetic field. DPPH powder (g=2.0037) was used as a g-value reference.

(119) Relaxivity Measurements by MRI

(120) Phantom MRI data were acquired in a 12 cm outer diameter birdcage transceiver for imaging in a 20 cm bore Bruker 7 T Avance III MRI scanner. Samples at varying concentrations (0 up to 5 mM) in PBS buffer were loaded into the wells of a 384-well clear polystyrene plate (Thermo Scientific Nunc), which had been pre-cut in half to optimally fit the coil. Unused wells were filled with PBS buffer. 2 mm slices were imaged through the samples with the field of view of 5×5 cm and the data matrices were 256×256 points. Longitudinal (r.sub.1) and transverse (r.sub.2) relaxivity measurements were acquired using multi-spin multi-echo (MSME) sequences (flip angle=180°). r.sub.1; TE=12 ms, TR=300, 350, 400, 450, 500, 600, 800, 1000, 1200, 1500, 3000, 5000, 10000 ms. r.sub.2; TR=5000 ms, TE=12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 280, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, 324, 336, 348, 360 ms. Custom routines written in Matlab (Mathworks, Natick, Mass.) were used to reconstruct the images and compute relaxation time constants by fitting image intensity data to exponential decay curves.

(121) Kinetics of Nitroxide Quenching by EPR Spectroscopy

(122) A solution was prepared with ascorbic acid (Asc), sodium phosphates (<30 ppm transition metals), sodium hydroxide and diethylenetriaminepentaacetic acid (DTPA, ˜0.1% (mol/mol) to sodium phosphates) at pH 7.4. Reduced L-GSH was then dissolved to provide the Asc/GSH solution. BASP-ORCA solution was prepared in phosphate buffer, which was made from sodium phosphates and DTPA (˜0.1% (mol/mol) to sodium phosphates) at pH 7.4. Equal volumes of the freshly prepared 1 mM (in nitroxide) sample solution and 20 mM Asc/GSH solution were combined and vortexed for 6 seconds, and then added to a 2 mm OD EPR tube. Kinetic studies were performed on 0.5 mM nitroxide solution in the presence of 125 mM sodium phosphates, 10 mM Asc, and 10 mM GSH. The peak height of the low-field line of the triplet was measured as a function of time. Microwave power was kept under 6.5 mW and the temperature was controlled at 295 K with a nitrogen flow system.

(123) Computational Analysis of Nitroxide Quenching by EPR Spectroscopy

(124) The EPR spectra are constituted by a “fast” and a “slow” component. From visual inspection, it was clear that the slow component was changing from one to another sample, while the fast one showed an almost equivalent line shape in the three spectra. Therefore, first a computation (program by Budil&Freed.sup.74) of the fast component to be subtracted from the three spectra to obtain a reliable line shape for the slow components was employed. This succeeded for the fast component shown in FIG. 9A (the subtracted experimental line in black and the computed line is in red). The main parameters used for the computation are shown in the figure and described below. Subtraction of this fast component from the overall spectra produced the three slow components shown in FIGS. 9B, 9C and 9D for 1 min, 40 min, and 180 min, respectively (in FIGS. 9A-9D the spectra are normalized in height). Their computations are shown as well, together with the main parameters used for computation and analysis. The following parameters were calculated.

(125) The g.sub.ii components for the coupling between the electron spin and the magnetic field (accuracy from computation±0.0002). The starting values, which were used in previous studies.sup.91 using nitroxide radicals, are 2.009,2.006,2.003, for g.sub.xx, g.sub.yy, and g.sub.zz, respectively. It was found that these values worked for the computations of the fast component and for the t=1 minute slow component; however, for computing the slow components of t=40 minutes and 180 minutes it was necessary to decrease the g.sub.zz values to 2.0025 and 2.002, respectively. This observation indicated an increased structural anisotropy of the nitroxide labels from 1 minute to 40 minutes to 180 minutes.

(126) The A.sub.ii components for the coupling between the electron spin and the nitroxide-nitrogen nuclear spin (accuracy from computation+0.5 G). These parameters increase with an increase in the environmental polarity of the nitroxide. Mainly, as done in previous studies,.sup.91 the A.sub.xx and A.sub.yy values were maintained constant (6 G) and only A.sub.zz was changed. The polarity was found to be slightly lower for the fast component (A.sub.zz=35 G) than for the slow one (A.sub.zz=36 G); it was constant for the different slow components.

(127) The correlation time for rotational diffusion of the radical, τ (accuracy from computation ±0.05 ns). This parameter increases with an increase in the local viscosity around the nitroxide group and with a decrease in the rotational mobility of the nitroxide. The local viscosity largely increased (the mobility decreased) from the fast component to the slow ones and it also increased (the mobility decreased) from 1 minute (10 ns) to 40 minutes (11 ns) to 180 minutes (13.2 ns). Notably, by performing a subtraction procedure using the double integrals of the components of the spectra, it was found that the fast component was contained in all the three spectra in almost the same relative percentage, that is, about 20% (the accuracy in this percentage is about 1%).

(128) The line width (accuracy from computation ±0.1 G), which measures spin-spin interactions due to a high local concentration of paramagnetic species (like colliding nitroxide groups in fast motion, or nitroxides bound in close proximity in slow motion). The line width was quite high for all samples, indicating a high local concentration of nitroxides, but it was the highest (7.6 G) for the slow component of the t=1 minute sample, and it decreased at 40 minutes (5.5 G) and further decreased at 180 minutes (4.2 G). The latter value is even smaller than the line width of the fast component (4.8 G).

(129) Fluorimetry

(130) Fluorescence analysis was performed using a Tecan Infinite® 200 Pro plate reader. Absorption/emission spectra of BASP-ORCA1 were acquired to determine λ.sub.ex/em, which were 640 nm and 705 nm respectively (as expected for the dye used in these studies: Cyanine5.5). Absorption spectra were acquired using a 1 nm wavelength step size at 9 nm bandwidth; emission spectra were obtained using λ.sub.ex of 640 nm, a 5 nm wavelength step size, and 10 nm bandwidth. To examine the effect of nitroxide-quenching on fluorescence emission intensity, samples were prepared in 96-well plates (Corning, n=3) by mixing 50 μL of 5 mg BASP-ORCA1/mL solution with 50 μL of Asc/GSH solution with one of the following compositions: 120 equivalents (eq, with respect to chex) Asc, 60 eq Asc, 40 eq Asc, and 60 eq Asc+60 eq GSH. Control samples (n=3) were prepared by mixing 50 μL of 5 mg BASP-ORCA1/mL solution with 50 μL of PBS. Fluorescence intensity was monitored continuously for 2 hours; a plateau was typically reached within 40-50 min.

(131) Cell Culture

(132) A549 and HeLa cells (ATCC) were cultured in DMEM media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, VWR) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Human umbilical vein endothelial cells (HUVEC, Lonza) were cultured in EGM.sup.+ media (Lonza) supplemented with 1% penicillin/streptomycin. All cells were housed in 5% CO.sub.2 humidified atmosphere at 37° C.

(133) In Vitro Cell Viability

(134) HUVEC cells were plated at 5,000 cells per well (in 100 μL) in 96-well collagen-coated plates (Corning) and allowed to adhere overnight. The media was then replaced with fresh media containing BASP-ORCA1 at various concentrations. The plate was incubated for 72 hours, and cell viability was then determined using the CellTiter-Glo assay (Promega). HeLa cells were plated in 96-well plates (Corning) and cytotoxicity was studied following the same experimental procedure used for HUVEC cells.

(135) Animal Usage

(136) All experiments involving animals were reviewed and approved by the MIT Committee for Animal Care (CAC). BALB/c mice (female, 8-12 weeks old, Taconic) were used for in vivo toxicity, pharmacokinetic studies, and biodistribution (n=3). NCR-NU nude mice (female, 8-12 weeks old, Taconic) were used for in vivo MRI, NIRF imaging, and biodistribution (n=3). All animals received an alfalfa-free diet (TestDiet) at least 2 weeks prior to the start of the studies to minimize auto-fluorescence.

(137) In Vivo Toxicity

(138) Solutions containing 5.0-30 mg of BASP-ORCA1 in 5% glucose were prepared, passed through sterile 0.2 μm filter (Nalgene, PES membrane), and administered into BALB/c mice via tail vein injection. The mice were monitored over a period of 30 days. Initial injections were performed in one mouse for each dose, all of which appeared to be well-tolerated. The highest dose (30 mg) was then administered to another set of mice 01=3). No adverse physical effects and/or significant weight losses were observed.

(139) In Vivo MR and NIRF Imaging Instrumentation

(140) All imaging experiments were performed at the Koch Institute for Integrative Cancer Research at MIT. In vivo MRI was acquired using a Varian 7T/310/ASR-whole mouse MRI system. Scans were collected with respiratory gating (PC-SAM version 6.26 by SA Instruments Inc.) to avoid confounding noise due to chest movement. The respiratory rate and animal temperature were closely monitored during image collection. Coronal T2WIs were collected using the fast spin echo multiple slices pulse sequence with TR=4000 ms; T.sub.E(eff)=48 ms; ETL=8; FOV=100×50 mm.sup.2; 512×256 matrix and 2 averages over 12 slices of 1 mm thickness and 0 mm gap. Axial T.sub.2WIs were collected using the fast spin echo multiple slices pulse sequence with T.sub.R=4000 ms; T.sub.E(eff)=48 ms; ETL=8; FOV=45×45 mm.sup.2; 256×256 matrix and 2 averages over 10-16 (to capture entire tumor) slices of 1 mm thickness and 0 mm gap.

(141) In vivo NIRF imaging was performed on an IVIS Spectrum-bioluminescent and fluorescent imaging system (Xenogen). Epi-fluorescence imaging was acquired through excitation of the Cy5.5 fluorophore (λ.sub.ex/λ.sub.em=640/700 nm, exposure time 2-10 seconds) present in BASP-ORCA1.

(142) Pharmacokinetics (PK) and Biodistribution (BD) Studies

(143) BASP-ORCA1 doses (5.0 mg in 5% glucose) were prepared, passed through sterile 0.2 μm filters, and injected into BALB/c mice (groups of n=3). Blood samples were taken at 1, 3, 6, 24, and 48 hours via cardiac puncture after euthanization in a CO.sub.2 chamber. The blood samples were subjected to fluorescence imaging (IVIS, Cy5.5λ.sub.ex/λ.sub.em=640/700 nm, Xenogen) for analysis of blood-compartment PK. For BD, organs from these BALB/c mice were harvested and subjected to fluorescence imaging (IVIS, Cy5.5λ.sub.ex/λ.sub.em=640/700 nm, Xenogen).

(144) In Vivo MR and NIRF Imaging in Tumor-Bearing Mice

(145) A549 cells were cultured in DMEM media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO.sub.2 humidified atmosphere (37° C.) to a final concentration of 20%. Cells were then harvested, mixed with Matrigel and sterile pH 7.4 PBS buffer (1:1), filtered through sterile 0.2 μm filters, and injected subcutaneously (2.0×10.sup.6 cells) into the hind flank of NCR-NU mice. Tumor growth was monitored for 2-4 weeks until appropriate cumulative diameters (˜1 cm) were achieved.

(146) MRI and NIRF images were acquired for each animal (n=3-4) before injections. BASP-ORCA1 doses (0.16 mmol chex/kg or 0.23 mmol chex/kg in 5% glucose) were prepared, passed through a sterile 0.2 μm filter, and administered to the tumor-bearing mice via tail vein injection. Tumor imaging was done at pre-determined time points; at the last imaging time point, mice were immediately euthanized in a CO.sub.2 chamber, and organs were collected, imaged by NIRF, and stored in dry ice for EPR analysis.

(147) Ex Vivo EPR Spectroscopy

(148) Harvested organs were shipped on dry ice to the University of Nebraska, where they were stored on dry ice. For EPR sample preparation, each tissue sample, one at a time, was rapidly thawed and transferred to a weighed vial; 900 μL of PBS buffer (0.5 mM, pH 7.2) was then added. The mixture was put into an ice-water bath and homogenized with a rotor stator homogenizer, then pipetted into a 4-mm outer diameter EPR sample tube. The samples were degassed by sonication as needed (for instance, when gas bubbles were visible). The EPR tube was capped, sealed with parafilm, and stored briefly in acetone/dry ice bath before spin concentration measurements.

(149) Spin concentrations of nitroxide radicals in tissues (μmol chex per g protein; Note: see below for details of protein content determination) were measured at −30° C. (243.2 K) to increase signal-to-noise of the aqueous samples. Measurements of tissue samples were alternated with that of the spin concentration reference (see next paragraph) and g-value reference (2,2-diphenyl-1-picrylhydrazyl powder was used as the g-value reference). For tissue samples with low signal-to-noise, the cavity background was recorded with identical parameters, including number of scans and receiver gain. Typical parameters were as follows: microwave attenuation-20 dB, modulation amplitude-5 Gauss, spectral width-300 Gauss, resolution-512 points, conversion-40.96, time constant-10.24, and sweep time-20.97 seconds. These parameters were kept identical for the tissues, references, and cavity backgrounds. The number of scans (8-256) and receiver gain were adjusted as needed for each sample.

(150) The reference for spin concentration was 0.50 mM Proxyl in PBS (pH 7.2). This reference was always stored in dry ice, except during measurements, and occasionally re checked for spin concentration decay.

(151) Protein Content Determination

(152) The protein content of tissue homogenate samples was determined using the BCA Protein Assay Kit (ThermoFisher Scientific). These protein contents were then used as a normalizing parameter to compare nitroxide spin concentration and NIRF signal (FIG. 6B).

(153) Ex Vivo NIRF Imaging

(154) To acquire BD, the collected organs and organ homogenates were subjected to NIRF imaging following the same aforementioned experimental procedure as for in vivo NIRF imaging. Furthermore, tissue homogenate samples were transferred into a 96-well plate and imaged for the correlation of NIRF signal and spin concentration.

(155) In Vivo MRI Data Analysis

(156) Signal intensities pre- and post-injection were compared only using slices where tumors and muscle were clearly visible. Using ImageJ software, a region of interest (ROI) around each component was manually drawn. The average signal intensity and area of the ROI were measured; these data were then normalized against the signal intensity of the muscle tissue. Signal intensity was acquired by multiplying area and normalized signal intensity. This process was repeated for all relevant slices for a given organ; the sum of these signal intensities was then calculated and divided for the total area, affording the volume-averaged signal intensity. Signal enhancement by BASP-ORCA1 was quantified by comparing the volume-averaged signal intensities pre- and post-injection.

(157) Procedure for BASP-ORCA Synthesis

(158) Representative Procedure for BASP-ORCA Synthesis with Brush Length of 7.07 (m) and 20 Equivalents (N) of Cross-Linker (BASP-ORCA1, m=7.07, N=20)

(159) All BASP-ORCA syntheses were performed in a glovebox under N.sub.2 atmosphere; however, similar results are expected under ambient conditions. All ROMP reactions followed the same general procedure, which was modified from literature examples.

(160) To a 4 mL vial, a suspension of Acetal-XL (15.6 mg, 26.8 μmot, 20.0 eq) in THF (268.0 μL, 0.1 M Acetal-XL) was prepared. To a second 4 mL vial containing a stir bar, chex-MM (35.0 mg, 9.4 μmol, 7.0 eq) was added; Cy-MM was then added from a premade 12.5 mg/mL solution in THF (30.6 μL, 0.094 mol, 0.07 eq). To a third vial, a solution of Grubbs 3.sup.rd generation bispyridyl catalyst (Grubbs III, 0.02 M in THF) was freshly prepared. THE (91.8 μL) was then added to the MM vial, followed by the addition of Grubbs III solution (67.0 μL, 1.3 μmol, 1.0 eq) to give the desired MM:Grubbs III ratio of 7.07:1 (1 mol % of the Cy-MM), while achieving a total MM concentration of 0.05 M, affording a dark blue solution. The reaction mixture was allowed to stir for 30 minutes at room temperature before an aliquot (˜5 μL) was taken out and quenched with 1 drop of ethyl vinyl ether for GPC analysis. The Acetal-XL suspension was then added dropwise (in aliquots of 5 eq, or ˜70 μL, every 5 minutes) over the course of 20 minutes into the MM vial, and the polymerizing mixture was allowed to stir for 6 hours at room temperature, affording a dark blue solution. To quench the polymerization, a drop of ethyl vinyl ether was added. The reaction mixture was transferred to an 8 kD molecular weight cutoff dialysis tubing (Spectrum Laboratories) in 10 mL nanopure water, and the solution was dialyzed against water (500 mL×3, solvent exchange every 6 h). The solution of BASP-ORCA was then lyophilized to afford a blue solid.

(161) Other BASP compositions were prepared as follows: MM:Grubbs III ratios of 9.99:1, 7.07:1, or 5.05:1 (m values). Acetal-XL were used in 15, 20, or 30 equivalences (N values). PEG-BASP, which contained no chex-MM, was prepared in an analogous manner to BASP-ORCAs using a PEG-MM lacking chex. Chex-bottlebrush was prepared as previously described..sup.90

Equivalents and Scope

(162) In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

(163) Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

(164) This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

(165) Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

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