CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.

61/764,127, filed February 13, 2013, the entirety of which is incorporated by reference herein.

TECHNICAL FIELD

[0002] The present invention is directed to sulfonic esters of metal oxides and their uses.

BACKGROUND

[0003] Corroles are tetrapyrrolic macrocycles:

Corrole

[0004] Corroles are becoming increasing useful in the field of chemical synthesis as catalysts in, for example, oxidation, hydroxylation, hydroperoxidation, epoxidation, sulfoxidation, reduction, and group transfer reactions. See, e.g., Aviv, I., Gross, Z., Chem. Commun., 2007, 1987-1999. Based on their physico-chemical properties, it is envisioned that corroles could be useful in the sensors field and biomedical field. Id. Corrole-based materials useful in the chemical synthesis, sensor, biomedical, and other fields are needed.

SUMMARY

[0005] The present invention is directed to materials of formula I:

wherein A is a corrolyl or metallated corrolyl; M is a surface comprising Ti0 ₂ , BaTi0 ₃ , Sn0 ₂ , A1 ₂ 0 ₃ , Fe ₂ 0 ₃ , Fe ₃ 0 ₄ , Zr0 ₂ , Ce0 ₂ , CdO, Cr ₂ 0 ₃ , CuO, MnO, Mn ₂ 0 ₃ , Mn0 ₂ , NiO, SnO, Sn0 ₂ , Si0 ₂ , or ZnO; and n is 0 or 1.

[0006] The invention is also directed to materials according to formula II: o

wherein B is -NCO, Ci_i ₀ alkyl, or ; wherein R ¹ is -COOH, -COOC _1-6 alkyl,

Ci_ ₆ alkyl, or aryl optionally substituted with halogen or Ci- ₆ alkyl; M is a surface comprising Ti0 ₂ , BaTi0 ₃ , Sn0 ₂ , A1 ₂ 0 ₃ , Fe ₂ 0 ₃ , Fe ₃ 0 ₄ , Zr0 ₂ , Ce0 ₂ , CdO, Cr ₂ 0 ₃ , CuO, MnO, Mn ₂ 0 ₃ , Mn0 ₂ , NiO, SnO, Sn0 ₂ , Si0 ₂ , or ZnO; and n is 0 or 1.

[0007] Methods of making materials of formulas I and II are described herein. Also described are methods of using the materials of the invention in applications such as optical imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 depicts confocal fluorescence microscopy images of 1-Ti0 ₂ [(a), (b),

(c)], I-AI-T1O2 [(d), (e), (f)], and 1-Ga-Ti0 ₂ [(d), (e), (f)].

[0009] Figure 2 depicts transmission electron microscopic (TEM) images of Ti0 ₂ nanoparticles of the invention before and after dye-functionalization. (a and d) Images of the initial Ti0 ₂ nanoparticles. (b and e) Images of the nanoparticles after peroxide-etching, (c and f) Images of the nanoparticles after dye functionalization. The scale bar is 25 nm for the top row and 100 nm for the bottom row of images.

[0010] Figure 3 depicts electronic absorption spectra for an amphiphilic corrole (H ₃ tpfc(S0 ₂ OH) ) and corrole-Ti0 ₂ nanoconjates of the invention in phosphate buffer saline pH

7.4. [0011] Figure 4 depicts confocal fluorescence microscopic images of U87-Luc cells treated with 0.2 μg/mL of a preferred embodiment of the invention (1-Al-Ti0 ₂ ) after 24 h (a), 48 h (b), and 72 h (c).

[0012] Figure 5 depicts Z-stacked confocal fluorescence micrographic images of individual U87-Luc cells taken at 0.5- ιη slice intervals after (a) 48 h and (b) 72 h of treatment with 0.2 μg/mL of a preferred embodiment of the invention (1-Al-Ti0 ₂ ).

[0013] Figure 6 depicts a cell viability plot of U87-Luc cells treated by of a preferred embodiment of the invention (1-Al-Ti0 ₂ ) at various concentrations (2 ng/mL to 2 mg/mL) using a bioluminescence assay.

[0014] Figure 7 depicts the results of mouse primary hepatocytes (MPH) treated with a preferred embodiment of the invention (1-Al-Ti0 ₂ ) in various concentrations (0.3 ng/mL to 0.3 mg/mL) for 24 and 48 h.

[0015] Figure 8 depicts ATR-IR spectra for Ti0 ₂ nanoparticles and preferred materials of the invention.

[0016] Figure 9 depicts normalized ATR-IR spectra for Ti0 ₂ nanoparticles and preferred materials of the invention.

[0017] Figure 10 depicts X-ray photoelectron spectra for nanoconjugates 1-Ti0 ₂ , 1-Al- Ti0 ₂ , and 1-Ga-Ti0 ₂ exhibiting the F(ls) band.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0018] The present invention is directed to materials, preferably nanoparticulate materials, comprising a metal oxide covalently bonded to a corrole or metallated-corrole through an -S0 ₂ - linkage. The metal oxides for use in making the materials of the invention include those having at least one -OH group. Such metal oxides are known in the art and are described in further detail below.

[0019] One embodiment of the invention is directed to materials according to formula I:

o

wherein A is a corrolyl or metallated corrolyl; M is a surface comprising Ti0 ₂ , BaTi0 ₃ , Sn0 ₂ , A1 ₂ 0 ₃ , Fe ₂ 0 ₃ , Fe ₃ 0 ₄ , Zr0 ₂ , Ce0 ₂ , CdO, Cr ₂ 0 ₃ , CuO, MnO, Mn ₂ 0 ₃ , Mn0 ₂ , NiO, SnO, Sn0 ₂ , Si0 ₂ , or ZnO; and

n is 0 or 1.

[0020] Within the scope of the invention, M is a surface that comprises a metal oxide, for example, a metal oxide that comprises at least one -OH group. The -OH group can be inherently present on the surface. Alternatively, the at least one -OH group can be incorporated by oxidizing the surface with a reagent such as hydrogen peroxide. Preferred surfaces for use in the invention include metal oxides such as Ti0 ₂ , BaTi0 ₃ , Sn0 ₂ , A1 ₂ 0 ₃ , Fe ₂ 0 ₃ , Fe ₃ 0 ₄ , Zr0 ₂ , Ce0 ₂ , CdO, Cr ₂ 0 ₃ , CuO, MnO, Mn ₂ 0 ₃ , Mn0 ₂ , NiO, SnO, Sn0 ₂ , Si0 ₂ , and ZnO. In some embodiments, the surface comprises Ti0 ₂ . In some embodiments, the surface comprises BaTi0 ₃ . In some embodiments, the surface comprises Sn0 ₂ . In some embodiments, the surface comprises A1 ₂ 0 ₃ . In some embodiments, the surface comprises Fe ₂ 0 ₃ . In some embodiments, the surface comprises Fe ₃ 0 ₄ . In some embodiments, the surface comprises Zr0 ₂ . In some embodiments, the surface comprises Ce0 ₂ . In some embodiments, the surface comprises CdO. In some embodiments, the surface comprises Cr ₂ 0 ₃ . In some embodiments, the surface comprises CuO. In some embodiments, the surface comprises MnO. In some embodiments, the surface comprises Mn 0 ₃ . In some embodiments, the surface comprises Mn0 ₂ . In some embodiments, the surface comprises NiO. In some embodiments, the surface comprises SnO. In some embodiments, the surface comprises Sn0 ₂ . In some embodiments, the surface comprises Si0 ₂ . In some embodiments, the surface comprises ZnO.

[0021] In preferred embodiments, the surface is a nanoparticle surface.

[0022] In some embodiments, n is 0. In other embodiments, n is 1.

[0023] Corroles for use in the invention are known in the art and are of the general formula:

[0024] The corroles of the invention described herein can be attached to the M-OS0 ₂ - moiety(ies) of the invention through any available carbon.

[0025] Particularly preferred corroles for use in the invention include those of the following general formula:

wherein Ar is an aryl group, for example, a phenyl or naphthyl group. In some embodiments of the invention, the aryl group is unsubstituted. In other embodiments, the aryl group is substituted. For example, when the aryl group is phenyl, the phenyl can be optionally substituted with halogen, for example, 1 to 5 halogen, that is, one or more of F, CI, Br, or I, with F being a particularly preferred halogen. In exemplary embodiments, the aryl group is pentafluorophenyl. In other embodiments, when the aryl group is naphthyl, the naphthyl can be optionally substituted with 1 to 7 halogen, with F being a particularly preferred halogen.

[0026] Preferred corrolyls for use in the invention are those wherein Ar is

pentafluorophenyl and include

[0027] In addition to being substitued with one or more halogens, the aryl group can be further substituted with -NR ³ R ⁴ , wherein R ³ and R ⁴ are each independently H, Ci_i ₀ alkyl, Ci_ ₁₀ alkenyl, or -alkaryl; or R ³ and R ⁴ , together with the nitrogen atom to which they are attached, form a heterocycloalkyl ring, which may be optionally subsituted with Ci_ ₆ alkyl, for example, methyl or ethyl. Examples of-NR ³ R ⁴ moieties include:

-5-N N—

[0028] Corroles incorporating an -NR ³ R ⁴ substituted aryl group can be accessed using methods known in the art, for example, using nucleophic substitution reactions. See, e.g., Hori, T., Osuka, A. Eur. J. Org. Chem. 2010, 2379-2386. For example, corroles incorporating an -NR ³ R ⁴ substituted aryl group can be accessed using the following synthetic scheme:

Amines that can be used in nucleophilic substitution reactions include, for example, benzylamine, octylamine, sec-butylamine, allylamine, dimethylamine, morphiline, piperidine, and N-methylpiperazine. [0029] Another preferred corrole for use in the invention is of the general formula

wherein each R ² is independently H, Ci_ ₆ alkyl, halogen, or M-0-S0 ₂ -, wherein M is as described above.

[0030] Yet another preferred corrole for use in the invention is of the general formula

wherein Ar and R ² are as previously described.

[0031] Corroles for use in the invention can also be metallated. In metallating a corrole, the nitrogens of the corrole are coordinated to a metal. Metals for use in the metallated corroles of the invention include any metal known in the art to be useful for coordinating to a corrole. Those of skill in the art understand that the function and use of the corrole can be modified by changing the coordinated metal.

[0032] For example, metals for use in metallating the corroles of the invention include Al, Ga, Fe, Mn, Sb, Co, Cr, Rh, Ru, Ro, Ir, V, Re, Cu, Sn, Ge, Ti, and Mo. Particularly preferred metals include Al and Ga. Another preferred metal is Fe. Yet another preferred metal is Mn. Another metal for use in the invention is Sb. Another metal for use in the invention is Co.

Another metal for use in the invention is Cr. Another metal for use in the invention is Rh.

Another metal for use in the invention is Ru. Another metal for use in the invention is Ro.

Another metal for use in the invention is Ir. Another metal for use in the invention is V.

Another metal for use in the invention is Re. Another metal for use in the invention is Cu.

Another metal for use in the invention is Sn. Another metal for use in the invention is Ge.

Another metal for use in the invention is Ti. Another metal for use in the invention is Mo. [0033] The metals for use in metallating the corroles of the invention can be optionally coordinated to one or more ligands. Such ligands are known in the art and include, for example, pyridine, nitrosyl, imido, nitrido, oxo, ether, hydroxyl, chloride, carbonyl, fluoro, bromo, phenyl, iodo, phosphine, arsine, and the like. Those skilled in the art would readily be able to determine a suitable ligand for any particular metal. A particularly preferred ligand for use in the invention is pyridine. Preferred metal-ligand moieties include Al(ligand) ₂ and Ga(ligand), with

Al(pyridine) ₂ and Ga(pyridine) being particularly preferred.

[0034] Exemplary materials of formula I according to the invention include:

wherein Ar is pentafluorophenyl. In particularly preferred embodiments, Ar is

pentafluorophenyl and M is Ti0 ₂ .

[0035] While 2,17 substituted corroles have been set forth herein, these examples are exemplary only and are not meant to limit the invention. It is envisioned that substitution at any position of the corrolyl or metallated corrolyl is within the scope of the invention.

[0036] Materials of formula I can be made according to the following method:

contacting a surface comprising Ti0 ₂ , BaTi0 ₃ , Sn0 ₂ , A1 ₂ 0 , Fe ₂ 0 , Fe 0 ₄ , Zr0 ₂ , Ce0 ₂ , CdO, Cr ₂ 0 ₃ , CuO, MnO, Mn ₂ 0 ₃ , Mn0 ₂ , NiO, SnO, Sn0 ₂ , Si0 ₂ , or ZnO, the surface having at least one -OH group, with a compound of formula III:

(Cl-S0 ₂ ) _m -A (III)

wherein A is corrolyl or metallated corrolyl; and

wherein m is 1 or 2.

Preferably, the synthetic methods of the invention are conducted in an organic solvent such as pyridine, with heat.

[0037] Compounds of formula III can be prepared according to methods known in the art. See, e.g., (a) Mahammed, A.; Goldberg, I.; Gross, Z. Org. Lett. 2001, 3, 3443. (b) Saltsman, I.; Mahammed, A.; Goldberg, I.; Tkachecko, E.; Botoshansky, M.; Gross, Z. J. Am. Chem. Soc. 2002, 124, 7411. See also, Blumenfeld, C. M.; Grubbs, R. H.; Moats, R. A.; Gray, H. B.;

Sorasaenee, K. Inorg. Chem. 2013, 52, All A. One exemplary method of preparing compounds of formula III is shown in Scheme 1.

Scheme 1

[0038] The corrole or metallated corrole used in any of the methods of preparing materials of formula I can be any of the corroles or metallated corroles described herein.

[0039] In preferred methods of the invention, the surface is a nanoparticle surface. In other preferred methods of the invention, the surface comprises Ti0 ₂ .

[0040] Corrole coupling to the metal oxide surfaces of the invention can be performed by mixing metals of the invention bearing hydroxylated surfaces, preferably in nanocrystal form, with solutions of corrole and heating, preferably to reflux. After repeated washing with copious amounts of solvent such as, for example, CH ₂ C1 ₂ , acetone, and water, and drying under vacuum, powders are obtained.

[0041] Preferred corroles for use in the methods of making materials of formula I include

wherein Ar and R ² are as set forth above.

[0042] Preferred metallated corroles for use in the methods of the invention include:

wherein Ar and R ² are as set forth herein above and wherein D is Al, Ga, Fe, Mn, Sb, Co, Cr, Rh, Ru, Ro, Ir, V, Re, Cu, Sn, Ge, Ti, or Mo, each of which is optionally corrdinated to one or more ligands. In preferred embodiments, D is Al(pyridine) ₂ or Ga(pyridine).

[0043] The materials of the invention can be use in synthetic, biomedical, and optical imaging applications. In a preferred embodiment of the invention, the materials of formula I are used in imaging cancer in a patient. For example, a material according to formula I, wherein A is a metallated corrolyl, is administered to a patient. After a period of time sufficient for the material to be taken up by any cancer cells, the cancer cells within the patient are imaged using optical imaging, preferably using fluorescence imaging. Cancers that can be imaged using the methods of the invention will include glioblastoma, melanoma, breast cancer, liver cancer, and colon cancer.

[0044] Preferably, the materials used in the imaging methods of the invention include

o=s=o o=s=o

I I

0 o

1 I

M , and M ;

wherein Ar is pentafluorophenyl and M is preferably Ti0 ₂ .

[0045] The materials of formula I of the invention can also be useful in other fields of endeavor by changing the coordinating metal in the metallated corrole. For example, materials of the invention can be useful in the hydroxylation and hydroperoxidation of alkanes. The materials of the invention are also useful in epoxidation and sulfoxidation reactions. The materials of the invention are also useful in catalysis, for example, reduction and group transfer catalysis.

[0046] In other embodiments, the materials of formula I of the invention are useful in corrole-based sensing applications and dye-sensitized solar cells. In other embodiments, the materials of formula I of the invention will have anticancer activity or will prevent cell death. In other embodiments, the materials of the invention are useful in singlet oxygen sensitization. In other embodiments, the materials of the invention are useful in lipo-protein protection and neuroprotection. [0047] The invention is also directed to materials according to formula II: o

wherein B is -NCO, Ci_ioal kyl, or ; wherein R ¹ is -COOH, -COOCi_ ₆ alkyl,

Ci_ ₆ alkyl, or aryl optionally substituted with halogen or Ci- ₆ alkyl;

[0048] M is a surface comprising Ti0 ₂ , BaTi0 , Sn0 ₂ , A1 ₂ 0 , Fe ₂ 0 , Fe 0 ₄ , Zr0 ₂ , Ce0 ₂ , CdO, Cr ₂ 0 ₃ , CuO, MnO, Mn ₂ 0 ₃ , Mn0 ₂ , NiO, SnO, Sn0 ₂ , Si0 ₂ , or ZnO; and

n is 0 or 1.

[0049] Within the scope of the invention, M is a surface that comprises a metal oxide, for example, a metal oxide that comprises at least one -OH group. The -OH group can be inherently present on the surface. Alternatively, the at least one -OH group can be incorporated by oxidizing the surface with a reagent such as hydrogen peroxide. Preferred surfaces for use in the invention include Ti0 ₂ , BaTi0 ₃ , Sn0 ₂ , A1 ₂ 0 ₃ , Fe ₂ 0 ₃ , Fe ₃ 0 ₄ , Zr0 ₂ , Ce0 ₂ , CdO, Cr ₂ 0 ₃ , CuO, MnO, Mn ₂ 0 , Mn0 ₂ , NiO, SnO, Sn0 ₂ , Si0 ₂ , and ZnO. In some embodiments, the surface comprises Ti0 ₂ . In some embodiments, the surface comprises BaTi0 . In some embodiments, the surface comprises Sn0 ₂ . In some embodiments, the surface comprises A1 ₂ 0 ₃ . In some embodiments, the surface comprises Fe ₂ 0 ₃ . In some embodiments, the surface comprises Fe ₃ 0 ₄ . In some embodiments, the surface comprises Zr0 ₂ . In some embodiments, the surface comprises Ce0 ₂ . In some embodiments, the surface comprises CdO. In some embodiments, the surface comprises Cr ₂ 0 ₃ . In some embodiments, the surface comprises CuO. In some embodiments, the surface comprises MnO. In some embodiments, the surface comprises Mn ₂ 0 ₃ . In some embodiments, the surface comprises Mn0 ₂ . In some embodiments, the surface comprises NiO. In some embodiments, the surface comprises SnO. In some embodiments, the surface comprises Sn0 ₂ . In some embodiments, the surface comprises Si0 ₂ . In some embodiments, the surface comprises ZnO.

[0050] In preferred embodiments, the surface is a nanoparticle surface.

[0051] In some embodiments, n is 0. In other embodiments, n is 1. [0052] In certain embodiments, B is -NCO.

[0053] In other embodiments, B is Ci_ioalkyl, for example methyl, ethyl, propyl, butyl, sec-butyl, tert-butyl, pentyl, and the like.

[0054] In yet other embodiments, B is wherein R ¹ is -COOH, -COOCi_

₆ alkyl; Ci_ ₆ alkyl; or aryl optionally substituted with halogen or Ci- ₆ alkyl.

[0055]

[0056] In other embodiments, B is ^{N /} R ¹ ; wherein R ¹ is -COOCi_ ₆ alkyl, for example, -COOMe, -COOEt, -COOPr, -COOBu, and the like.

[0057] In other embodiments, B is ^{N /} R ¹ ; wherein R ¹ is Ci_ ₆ alkyl,

xample, methyl, ethyl, propyl, butyl, sec-butyl, tert-butyl, pentyl, and the like.

[0058] In yet other embodiments, B is wherein R ¹ is aryl, for example, phenyl or naphthyl. In these embodiments, the aryl can be optionally substituted with one or more substitutents selected from the group consisting of halogen and Cr ₆ alkyl.

[0059] In those embodiments wherein B is preferably

[0060] Methods of making materials of formula II are also within the scope of the invention. According to the invention, materials of formula II can be prepared by contacting a surface comprising Ti0 ₂ , BaTi0 , Sn0 ₂ , A1 ₂ 0 , Fe ₂ 0 , Fe 0 ₄ , Zr0 ₂ , Ce0 ₂ , CdO, Cr ₂ 0 , CuO, MnO, Mn ₂ 0 ₃ , Mn0 ₂ , NiO, SnO, Sn0 ₂ , Si0 ₂ , or ZnO, the surface having at least one -OH group, with a compound of formula IV:

C1-S0 ₂ -R (IV) wherein R is -NCO wherein R ¹ is -C(0)OH, -C(0)OCi_ ₆ alkyl;

Ci_ ₆ alkyl; or aryl optionally substituted with halogen or Ci- ₆ alkyl. [0061] Compounds of formula IV can be prepared using methods known in the art.

[0062] Materials of formula II are useful in the field of chemical synthesis, for example, as catalysts. Materials of formula II are also useful in the field of material science.

[0063] As used herein, the term "halogen" refers to F, CI, Br, or I.

[0064] As used herein, "alkyl" refers to branched or straigh-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, Ci_ioalkyl denotes an alkyl group having 1 to 10 carbon atoms. Preferred alkyl groups include methyl, ethyl, propyl, butyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like.

[0065] As used herein "alkenyl" refers to hydrocarbon chains that include one or more double bonds.

[0066] As used herein, "aryl" refers to phenyl or naphthyl.

[0067] As used herein, "alkaryl" refers to an aryl moiety attached through an alkylene group, for example, benzyl (-CH ₂ -phenyl).

[0068] As used herein, "heterocycloalkyl" refers to a 5 to 7-membered monocyclic or bicyclic saturated ring that includes at least one heteroatom that is N, O, or S. Examples include piperidinyl, piperazinyl, morpholinyl, and pyrrolidinyl.

[0069] As used herein, "corrolyl" refers to a corrole moiety.

[0070] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

M = Al(py) ₂ or Ga(py) M = Al(py) ₂ =1-AI-Ti0 ₂ py = pyridine Ga(py) = 1-Ga-Ti0 ₂ py = pyridine [0071] Materials. 2 M AlMe ₃ in toluene (Aldrich), GaCl ₃ (Aldrich), HS0 ₃ C1 (Aldrich), 21 nm nanopowder Ti0 ₂ (Aldrich), 30% H ₂ 0 ₂ (EMD) were obtained commercially and used as received. The starting material 5, 10,15-tris(pentafluorophenyl) corrole (H ₃ tpfc) was prepared based on the literature method. The solvents pyridine and toluene were dried over a column. Acetone and dichloromethane used were both of reagent and spectroscopic grades depending on the applications. D-Luciferin potassium salt (Promega), Hoechst 34580 (Invitrogen™), Hoechst 33258 (Invitrogen™), Sytox Green (Invitrogen™), and FM® 1-43FX (Invitrogen™) were used as received according to the provider's instruction.

[0072] Chemical Preparation. All preparations were carried out under Ar(g) atmosphere unless otherwise noted.

[0073] 1. Corrole Preparation. Preparation of 2, 17-bischlorosulfonato-5,10, 15- tris(pentafluorophenyl) corrole (H ₃ tpfc(S0 ₂ Cl) ₂ ; 1) was performed according to the literature procedure. The metallocorroles described in this study were prepared in the following manner.

[0074] 1.1. Preparation of 1-Al. To the 20-mL toluene solution of 0.32 g of 1 (0.32 mmol) in a round bottom flask was added 0.8 mL of 2 M AlMe3 (1.6 mmol) in toluene solution at an icebath temperature. The solution was stirred for 10 min followed by the addition of 1 mL anhydrous pyridine. The solution was allowed to stir for another 10 min over ice. The reaction was quenched by an addition of ice chips. The dark green solution was then extracted with CH ₂ C1 ₂ and washed with water. The solvent was removed in vacuo and the dry deep green solid was redissolved in CH2C12 followed by filtration. The filtrate was brought to dryness to afford the dark green solid (0.098 g, 26% yield). ESI-MS (CH ₂ C1 ₂ ): m/z: 1014.87 [M-H]- (Calculated for C ₃₇ H ₆ N ₄ F ₁₅ C1 ₂ S ₂ 0 ₄ A1: 1015.88); Ή-ΝΜΡν (400 MHz, acetone-d ₆ , ppm): δ = 9.76 (s, 1 H), 9.25 (s, 1 H), 8.97 (d, 1 H), 8.85 (d, 1 H), 8.70 (d, 1 H), 8.58 (d, 1 H); 19F-NMR (376 MHz, acetone-d6, ppm): -138.7 (d, 4 F), -140.0 (d, 2 F), -156.9 (t, 1 F), -157.5 (t, 1 F), -158.1 (t, 1 F), -164.9 (m, 2 F), -165.3 (m, 2 F), -167.0 (m, 2 F); UV-Vis (toluene :pyridine, 95 :5): ax (ε M ^"1 cm ^"1 ) = 436 (4.08 10 ⁴ ), 625 (7.66 10 ³ ) nm.

[0075] 1.2. Preparation of 1-Ga. To a heavy-walled Schlenk flask were added 0.20 g of 1 (0.20 mmol) and 0.57 g GaCl (3.3 mmol) under Ar(g). The flask was chilled in N ₂ (l) and evacuated. 15 mL Degassed anhydrous pyridine (15mL) was added to the flask via vacuum transfer. The flask was subsequently sealed and allowed to warm to room temperature. The reaction vessel was heated to 120 °C for 1 h. The pyridine solution was diluted with CH ₂ C1 ₂ and washed with water three times. The solution was then filtered through glass wool and partially concentrated for recrystallization with hexanes overnight. The product was then filtered, dried, and washed with a combination of acetone, CH ₂ C1 ₂ , and toluene. This filtrate collected was brought to dryness in vacuo to afford a dark green solid (0.092 g, 38% yield). ESI-MS

(CH ₂ Cl ₂ :pyridine): m/z: 1056.81 [M-H]- (Calculated for C ₃ 7H ₈ N ₄ F ₁₅ Cl ₂ S ₂ 0 ₄ Ga: 1057.82); ^l H- NMR (500 MHz, CD ₂ C1 ₂ , ppm): δ = 9.99 (s), 8.82 (m), 8.73 (m), 8.57 (m); ¹⁹ F-NMR (376 MHz, acetone-d ₆ , ppm): -138.7 (d), -140.0 (d), -156.9 (t), -157.5 (t), -158.1 (t), -164.9 (m), -165.3 (m), -167.0 (m); UVVis (toluene :pyridine, 95 :5): max (ε M ^"1 cm ^"1 ) = 429 (1.65 10 ⁴ ), 61 1 (5.61 10 ³ ) nm.

[0076] 2. Ti0 ₂ Surface activation. To the solid Ti02 nanoparticle (10 g) in a 2.0-L round bottom flask was added 1.2 L 30% H202 solution. The milky colloidal suspension was stirred under reflux or 5 h. Upon cooling, the off- white solid was isolated from the H ₂ 0 ₂ solution by ultracentrifugation at 4 °C and washed with copious amount of water. The activated Ti0 ₂ nanoparticle (Ti0 ₂ -OH) collected was dried in vacuo for 12 h and stored dry in a vial prior to use.

[0077] 3. Surface Conjugation. The following general procedure was employed for the conjugation of the corroles 1 , 1-Al, and 1-Ga to the activated Ti0 ₂ nanoparticle surface: To the mixed solids containing the activated Ti0 ₂ and corrole in a 25 -mL round bottom flask was charged with anhydrous pyridine. The suspension turned green immediately and was stirred under reflux before the reaction was stopped. The resulting green solid was isolated from the green solution by centrifugation and washed multiple times with dichloromethane, acetone, and deionized water until the centrifuge supernatant became colorless. The solid remained green, was dried in vacuo, and was stored until further use. The detailed preparation procedure for each corrole nanoconjugate is given as follows:

[0078] 3.1. Preparation of 1-Ti0 ₂ . To a 25 mL round bottom flask were added 0.32 g Ti0 ₂ -OH and 0.028 g of 1 (28.1 μιηοΐ), which was subsequently cycled with argon and vacuum. After establishment of the inert atmosphere, 8 mL anhydrous pyridine was added to the flask and the reaction was set to reflux for 2 h. The resulting green solid was collected in a manner following the general centrifugation and washing procedures outlined above.

[0079] 3.2. Preparation of 1-Al-Ti0 ₂ . To a 40 mL vial was added 1.18 g Ti0 ₂ -OH, which was subsequently cycled with argon and vacuum. To this flask, was added 5 mL anhydrous pyridine, followed by sonication to ensure even dispersion. In a second flask, was added 0.03 g of 1-Al (25.5 μιηοΐ) and 7 mL anhydrous pyridine under Ar(g). This solution was stirred and then added to the Ti0 ₂ -OH precursor via syringe. The reaction was sealed and allowed to reflux for 2 h after which, the resulting green solid was collected in a manner following the general centrifugation and washing procedures outlined above. [0080] 3.3. Preparation of 1-Ga-Ti0 ₂ . To a 40 mL vial was added 0.84 g Ti0 ₂ -OH and 0.04 g of 1-Ga (32.8 μηιοΐ), which was subsequently cycled with argon and vacuum. After establishment of the inert atmosphere, 8 mL anhydrous pyridine was added to the flask and the reaction was set to reflux for 2 h. The resulting green solid was collected in a manner following the general centrifugation and washing procedures outlined above.

[0081] Spectroscopies. UV-Vis spectra were either recorded on a Carey 50

spectrophotometer or a Hewlett-Packard 8453 diode-array spectrophotometer at room

temperature from samples in various solvents. IR spectra were recorded with a SensIR

Durascope ATR accessory plate on a Nicolet Magna-IR spectrometer, an uncooled pyroelectric deuterated triglycine sulfate (DTGS) etector, and a KBr beamsplitter. The ^l H and ¹⁹ F NMR spectra were recorded on a Varian Mercury 300 (300 MHz for 1H; 288 MHz for ¹⁹ F)

spectrometer. The NMR spectra were analyzed using MestReNova (v. 6.1.1). ^l H NMR measurements were referenced to internal solvents. Fluorescence spectra were measured with a Jobin-Yvonne/SPEX Fluorolog spectrometer (Model FL3-1 1) equipped with a Hamamatsu R928 PMT. Samples were excited at λεχ = 405-430 nm (the Soret region), 514 nm, and 600-630 nm (Q-band region) with 2-nm band-passes. The fluorescence was observed from λειη = 500-800 nm, depending on the excitation wavelength, at 2-nm intervals with 0.5 s integration times at room temperature.

[0082] Relative Fluorescence Quantum Yield Measurements. The Oem

measurements were performed using degassed toluene solutions of 1 , 1-Al, 1-Ga, and tetraphenylporphyrin (as a standard). Samples were excited at λεχ = 355 nm and the emission was observed from λειη = 500- 800 nm. The standard tetraphenylporphyrin was excited at λεχ = 514 nm and the emission was observed from λειη = 500-800 nm. Oem for tetraphenylporphyrin is 0.1 1.3 All relative fluorescence quantum yields were calculated based on the corresponding fluorescence spectra of the samples and the standard according to the equation:

2

[0083] where Oem(s) and Oem(x) are the relative fluorescence quantum yield of the standard and sample, respectively; As and Ax are the absorbance at the excitation wavelength for the standard and sample, respectively; Fs and Fx are the area under the corrected emission curve for the standard and sample, respectively; and r\s and ηχ are the refractive index of the solvent used for the standard and sample, respectively.

[0084] Mass Spectrometry. Samples were analyzed by direct infusion ESI in the negative ion mode using an LCT Premier XE (Waters) ESI-TOF mass spectrometer operated in the W configuration. The samples were prepared in CH2C12:isopropanol (9: 1 v/v) at ~ 10 μΜ and infused with an external syringe pump at 25 μΕ/ηιίη. Some samples contained 50

pyridine in 1 mL CF^C^iisopropanol mixture.

[0085] Surface characterization. X-ray photoelectron spectroscopy was performed on an M-Probe spectrometer that was interfaced to a computer running the ESCA2005 (Service Physics) software. The monochromatic X-ray source was the 1486.6 eV Al Ka line, directed at 35° to thesample surface. Emitted photoelectrons were collected by a hemispherical analyzer that was mounted at an angle of 35° with respect to the sample surface. Low-resolution survey spectra were acquired between binding energies of 1 and 1100 eV. Higher-resolution detailed scans, with a resolution of 0.8 eV, were collected on the F(ls) XPS line. All binding energies are reported in electronvolts.

[0086] Attenuated total reflectance (ATR) infrared spectra of powdered corrole-Ti0 ₂ nanoconjugate samples were collected using a SensIR Durascope ATR accessory plate on a Nicolet Magna-IR spectrometer, an uncooled pyroelectric deuterated triglycine sulfate (DTGS) detector with a KBr window (400-4000 cm ¹ ), and a KBr beamsplitter. The spectral resolution was 4 cnr ¹ and 64 scans were collected per spectrum. A KBr background spectrum was subtracted from the measured spectrum of the nanoconjugates to provide the desired FTIR characterization data. See Figures 8 and 9.

[0087] Confocal Microscopy. The phantom imaging experiments were performed using a Zeiss LSM 710 Confocal Microscope (Carl Zeiss, Wake Forest, NC). The microscope system consists of a Zeiss 710 confocal scanner, 63 x/ 1.4 Plan-APOCHROMAT oil immersion lens (Zeiss), Axio Observer Zl microscope and diode-pump solid-state lasers. Two visible excitation lines (405 and 561 nm) were used for the experiments. The microscope is equipped with a QUASAR 32 channel spectral detector (two standard PMTs and a 32 channel PMT array) with spectral resolution of 9.7 nm. The software ZEN 2009 was used for hardware control. The laser power used for the experiments is 10% of the total available power (25 mW). ImageJ software was employed to process the resulting data.

[0088] Transmission electron microscopy. The morphologies of the Ti02

nanoparticles before and after surface functionalization were imaged using a FEI Tecnai F30ST transmission electron microscope (TEM) operated at acceleration voltage of 300 kV. Images were recorded using a Gatan CCD camera. For TEM analysis, a small quantity of Ti02 particles was dispersed in IPA by sonication. The dispersions were drop-cast onto C-flatTM holey carbon films on a 200 mesh Cu TEM grid (purchased from Electron Microscopy Sciences).

[0089] Approximation of loading of 1-Al on Ti0 ₂ surface. Calculation of the corrole 1-Al's loading on the surface of Ti0 ₂ was based on the absorbance values obtained from the integrated sphere electronic absorption measurements described as follows.

[0090] Absorption spectroscopy. Thin film transflectance measurements were used to calculate the dye loading on the Ti0 ₂ nanoparticles. Both peroxide-etched and dye- functionalized nanoparticles were dispersed in a polydimethylsiloxane (PDMS) polymer matrix. The weights of the Ti0 ₂ nanoparticles, PDMS base (Sylgard® 184 silicone elastomer base from Dow Corning), and curing agent (Sylgard® 184 silicone elastomer curing agent from Dow Corning) are provided in Table 1 below. The nanoparticles were first dispersed in a minimal amount of isopropanol (IPA) by sonication. The dispersion of Ti0 ₂ nanoparticles in IPA was then mixed with the PDMS base and curing agent using a Vortex mixer. The mixtures were cast into films onto quartz substrates and allowed to cure in air for 12 hours followed by curing in a drying oven at 60° for 2 hours. See Figure 3.

Table 1 Weights of Ti0 ₂ nanoparticles, weights of the PDMS base and curing agnet used to case PDMS films, weight% Ti0 ₂ in the films, film weight, and mass of Ti0 ₂ per volume of PDMS.

a Separate measurements showed that 98% of the IPA evaporated during curing of the PDMS film.

b A value of 0.965 g/cm ³ was used for the density of PDMS.

[0091] Transflectance spectra of the etched and dye-functionalized Ti0 ₂ nanoparticle films were measured using a Cary 5000 UV-Vis-NIR spectrometer from Agilent Technologies equipped with an integrating sphere (External DRA 1800), a PMT detector, a quartz-iodine lamp for the visible region (350-800 nm), and a deuterium lamp for the ultraviolet region (300-350 nm). Because the Ti0 ₂ nanoparticles cause diffuse scattering of the incident illumination, the PDMS films were placed in the center of the integrating sphere such that both the transmitted, T, and the reflected, R, (including the spectrally reflected and diffusely scattered light) light were collected by the PMT detector. The transflectance measurements allow for the absorbance, A, of the films to be determined by A = - log(T+R). The concentration, C, of the dye within the PDMS films was then calculated using the Beer- Lambert law, A = εθ, where ε is the extinction coefficient of the dye and 1 is the film thickness (determined by profilometry, see below). The absorbance values at 426 and 595 nm (corresponding to the Soret and Q bands of the dye, respectively) for the PDMS film containing the dye-functionalized Ti0 ₂ nanoparticles, the estimated extinction coefficients of the dye at these wavelengths, and the film thicknesses are provided in Table 2 below. The absorbance values for the PDMS film containing the

unfunctionalized, peroxide-etched Ti0 ₂ nanoparticles at these wavelengths are also provided, which were subtracted from the absorbance values of the dye-functionalized Ti0 ₂ nanoparticles. The dyeloading was determined to be between 2.3 and 3.5 μιηοΐε of dye per grams of Ti0 ₂ (based on whether the Soret or Q band was used to determine the dye concentration).

Table 2. Absorption values at 426 and 595 nm and thicknesses for PDMS films containing dyefunctionalized and peroxide-etched Ti0 ₂ nanoparticles, and estimated dye loading of the Ti0 ₂ particles based on absorption measurements.

Extinction coefficients measured in toluene :pyridine (95:5) mixture.

[0092] Profilometry. Thickness profiles of the PDMS films were measured using a Bruker DektakXT stylus surface profilometer. The diameter of the diamond-tipped stylus was 2 μπι and a weight of 1 mg was applied to the film, respectively. The stylus was scanned at a rate of 250 um/s. The thickness profiles were used measure the average path length through the PDMS films during the transflectance measurements.

[0093] Cell culture and cell viability assay. Pathogen-free U87-LUC cell line (TSRI Small Animal Imaging and Research Laboratory) was grown in 75 mL flask in Dulbecco's Minumal Essential Medium (DMEM) in 5% C0 ₂ at 37 °C. The cell culture medium was supplemented with 10% fetal bovine serum (FBS) and 1% the antibiotic primocin. The cell culture medium was replenished every two days and the cells were passaged once they reached 80% confluence. Primary mouse hepatocytes (PMH) were isolated and cultured as previously described.

[0094] For U87-Luc cell culture experiments. The cells were plated in an 8-chamber slide (Cultureslide, BD) were treated with 1-Α1-ΤΪ02 suspended in PBS over a range of 2 ng/mL to 2 mg/mL. A primary stock solution (6.3 mg 1-Al-Ti0 ₂ in 1 mL PBS) was prepared. The primary stock solution was further diluted to prepare secondary and tertiary stock solutions. The various amount of stock solutions were added to the eight-well glass slide plated with cells to give the aforementioned range of concentrations. The final volume for each well is 300 μί. After treatment, the treated cells and controls were incubated in the dark in 5% C0 ₂ at 37 °C for a period of 24, 48, and 72 h. The cells were imaged using the cooled IVIS® animal imaging system (Xenogen, Alameda, CA USA) linked to a PC running with Living Image™ software (Xenogen) along with IGOR (Wavemetrics, Seattle, WA, USA) under Microsoft® Windows® 2000. This system yields high signal-to-noise images of luciferase signals emerging from the cells. Before imaging, 0.5 mL of 150 mg/mL luciferin in normal saline was added to each well. An integration time of 1 min with binning of 5 min was used for luminescent image acquisition. The signal intensity was quantified as the flux of all detected photon counts within each well using the Livinglmage software package. All experiments were performed in triplicate.

[0095] For PMH cell culture experiments, the cells were plated in a 6-chamber slide (Cultureslide, BD). After three hours, media was exchanged (DMEM-F12) and the cells were treated with 1-Al-Ti0 ₂ suspended in PBS over a range of 0.3 ng/mL to 0.3 mg/mL. A primary stock solution (6.3 mg 1-Al-Ti0 ₂ in 1 mL PBS) was prepared. The primary stock solution was further diluted to prepare secondary and tertiary stock solutions. The various amount of stock solutions were added to the eight- well glass slide plated with cells to give the aforementioned range of concentrations. The final volume for each well is 2000 μΐ _^ . After 24 or 48 h of treatment, cells were double stained with Hoechst 33258 (8 mg/mL) and Sytox Green (1 mmol/L). Quantitation of total and necrotic cells (Sytox Green positive) was performed by counting cells in at least 5 different fields using ImageJ, as previously described. All experiments were done in triplicate.

[0096] In vitro confocal fluorescence microscopy. The U87-Luc cells were seeded at 20,000 cells per well on an 8-chamber slide (Cultureslide, BD) and allowed to grow overnight. Cells were washed with PBS and were incubated in serum free media mixed 1 : 1 with 1-Al-Ti0 ₂ for 24, 48, and 72 h at 37 °C over the concentration range similar to the U87-Luc cell viability assay (2 ng/mL to 2 mg/mL). Cells were then washed 3 ^X with PBS and stained with Hoechst 33258 and FM® 1-43FX stains. The cells were chilled on iced and then imaged without being fixed using a Zeiss LSM 710 inverted confocal microscope.

[0097] Electronic absorption spectra for 1, 1-Al, and 1-Ga was obtained in degassed toluene. Solutions reveal the signature Soret and Q-bands for these tetrapyrrolic macrocycles (Figure 1). The electronic absorption data for the chlorosulfonated corroles are also given in Table 3.

[0098] Table 3. Electronic spectroscopic data for chlorosulfonated corroles 1, 1-Al, and 1-Ga in toluene solution

"The measurements were performed in degassed toluene.

*The maximum absorption wavelengths are reported for both Soret (S) and

Q-bands (Q).

cThe relative emission quantum yields were determined using

tetraphenylporphyrin as a standard.

[0099] The electronic absorption spectra of the colloidal suspensions of 1-Ti0 ₂ , 1-Al- Ti0 ₂ , and 1-Ga-Ti0 ₂ nanoconjugates in PBS pH 7.4 reveal maximum absorptions centered around 425 and 600 nm for the Soret and Q-bands, respectively (Table 4).

[0100] Table 4. Electronic absorption, vibrational, and X-ray photoelectron spectroscopic data for corrole-Ti0 ₂ nanoconjugates 1-Ti0 ₂ , 1-Al-Ti0 ₂ , and 1-Ga-Ti0 ₂

[0101] These peak maxima are in agreement with the spectroscopic properties of the corresponding molecular corrole (Table 3). The Soret band splitting for 1-Ti0 ₂ is similar to the splitting observed for its amphiphilic molecular counterpart 2,17-bissulfonato-5,10,15- tris(pentafluorophenyl) corrole in an aqueous solution at physiologic pH, supporting the presence of the sulfonate linkage on the corrole anchored to Ti0 ₂ surfaces. The splitting pattern, however, was not observed for the metalloconjugates 1-Al-Ti0 ₂ and 1-Ga-Ti0 ₂ , owning to the presence of metal bound to deprotonated nitrogen atoms.

[0102] Characterization of the fine green powder of 1-Ti0 ₂ , 1-Al-Ti0 ₂ , and 1-Ga-Ti0 ₂ with FT-IR spectroscopy reveals vibrational absorption bands around 1180-1250 cm ¹ assigned to the symmetric stretching of S0 ₂ groups as well as those around 1400-1450 cm ¹ assigned to asymmetric stretching of S0 ₂ groups of covalent sulfonates. The presence of these vibrational signatures suggests that the corroles are covalently attached to the surface of Ti0 ₂ through a sulfonate linkage. The vibrational frequencies for these Ti0 ₂ -corrole nanoconjugates are listed in Table 2. X-ray photoelectron spectroscopy was performed to study the elemental presence of the surface of the nanoparticle conjugates (Table 4). High-resolution scans for the spectra of the conjugates revealed F(ls) binding energy peaks between 688 and 691 eV, suggesting the presence of corresponding pentafluorophenyl corroles attached to the Ti0 ₂ surface. See Figure 10.

[0103] Confocal fluorescence microscopy images of aggregates of the nanoconjugates 1-Ti0 ₂ , 1-Al-Ti0 ₂ , and 1-Ga-Ti0 ₂ in the solid state (Figure 1) were taken with the samples illuminated at λ _βχ = 405 nm and the λ _βΏ1 recorded from 508 to 722 nm. The images for 1-Al-Ti0 ₂ and 1-Ga-Ti0 ₂ (Figures le and lh) exhibit fluorescence areas on the nanoparticles compared to the relatively darker image for 1-Ti0 ₂ . The fluorescence signals observed with various intensities across the Ti0 ₂ samples for 1-Al-Ti0 ₂ and 1-Ga-Ti0 ₂ also suggest that the Ti0 ₂ surfaces are not evenly functionalized because of material aggregation. Selected fluorescence areas (white circles) on all three images, spectral profiles representing the nanoconjugates 1-Ti0 ₂ , 1-Al-Ti0 ₂ , and 1-Ga-Ti0 ₂ were obtained (Figures lc, If, and li). These spectral profiles and fluorescence signal intensities are in agreement with the fluorescence spectra (Figures la, Id, and lg) obtained from the molecular corroles 1, 1-Al, and 1-Ga.

[0104] The nanoconjugate 1-Al-Ti0 ₂ was chosen as a candidate for cellular uptake and cytotoxic effect studies. The TEM images of Ti0 ₂ (Figure 2) show the average particle size to be 29 nm, post-corrole functionalization, albeit, they appear to aggregate. Images were taken for both before and after surface functionalization as well as for both before and after H ₂ 0 ₂ -etching.

Absorption measurements of the particles embedded in a transparent polymer matrix, facilitated with the use of an integrating sphere, indicate nearly identical absorption features in the molecular and conjugated species. These experiments afforded an approximate loading of 1-Al on the surfaces of ca. 10-40 mg/g Ti0 ₂ . (Figure 2).

[0105] Treatment of the luciferase-transfected glioblastoma cell U87-Luc with a wide range of 1-Al-Ti0 ₂ concentrations (2 ng/mL to 2 mg/mL) reveals internalization of these nanocojugates over a period of 24, 48, and 72 h as shown by the confocal fluorescence microscopic (CFM) images (Figure 4).

[0106] The CFM images were taken after the cells were stained with the nuclear and cell membrane dyes, and washed with the media solution several times to remove the excess dyes and 1-Α1-ΤΪ02 nanoconjugates. The nucleus labeled with a Hoechst stain is seen in bluish purple (λεχ = 405 nm, λειη = 460 nm). The membrane seen in green is labeled with the dye FM® 1-43FX (λεχ = 488 nm, λειη = 580 nm). The nanoconjugate 1-Al-Ti0 ₂ is observed in red (λεχ = 405 nm, λειη = 634 nm).

[0107] The Z-stacked confocal fluorescence microscopic (CFM) images (Figure 5) of U87-Luc cells treated with similar concentrations (2 ng/mL to 2 mg/mL) of 1-Al-Ti0 ₂ for 48 and 72 h from three different perspectives are also shown (Figure 5). The Z-stacked CFM images of individual cells were taken at 0.5-μιη slice intervals from top to bottom.

[0108] The 1-Al-Ti0 ₂ nanoconstruct could also be internalized through endocytosis. Based on the confocal fluorescence images, the nanomaterials l-Al-modified Ti0 ₂ is suspended in the cytosol as opposed to the modified Ti0 ₂ labeled with alizarin red S, which showed perinuclear localization in HeLa cells. These findings suggest a distribution pattern of the Ti0 ₂ nanoconjugates within the cells similar to another study, in which 1-D Ti0 ₂ nanorods and nanoparticles labeled with fluorescein thiocyanate were internalized into HeLa cells after a given period of time. The internalization of 1-Al-Ti0 ₂ into glioblastoma cells can also be observed even at a very low concentration range (< /zg/mL).

[0109] Ti0 ₂ nanoparticles exhibit various degrees of cytotoxic activities upon photoactivation by UV-Vis light leading to formation of reactive oxygen species. To best study and understand the cytotoxic effect of the 1-Al-Ti0 ₂ conjugate that is not related to the photocatalytic property of Ti0 ₂ on cell death, the glioblastoma cell U87-Luc was treated in the absence of UV-Vis irradiation with the same range of 1-Al-Ti0 ₂ concentrations (2 ng/mL to 2 mg/mL) as in the cell internalization studies. The cells were incubated over a period of 24, 48, and 72 h prior to bio luminescence cell viability assays. Based on the bio luminescence signal of the firefly luciferin from living U87-Luc cells, which is related to the level of cellular ATP, the cytotoxic assay shows that the nanoconjugate 1-Al-Ti0 ₂ has essentially no cytotoxic effect on the glioblastoma cells after 24 h of treatment (Figure 6) and, therefore, could be considered biocompatible. On the other hand, the cytotoxic effect becomes more apparent as the cells were exposed to the corrole-Ti0 ₂ nanoparticles for extended periods of time at higher concentrations (> 200 /zg/mL). For example, only ca. 65% and ca. 30% of the bio luminescence signals from the live cells were observed after the 48-h and 72-h treatments at 2 mg/mL, respectively. This viability study of the U87-Luc cells treated with 1-Al-Ti0 ₂ is also consistent with a study performed on mouse fibroblast cells, using the MTT assay, showing that the cytotoxic effects of Ti0 ₂ at various concentrations (3 to 600 /zg/mL) were negligible after 24 h of treatment whereas the 48-h treatment of these cells with the nanoparticle showed decrease in cell viability at higher concentrations. Another study on the cytotoxicity effect of unmodified 1-D and 3-D Ti0 ₂ on HeLa cell also show that these nanoparticles were relatively nontoxic at concentrations up to 125 /zg/mL in the absence of light.

[0110] Additionally, to compare the cytotoxic effect of the nanoconjugate 1-Al-Ti0 ₂ on cancer and normal cells, mouse primary hepatocytes (MPH) were treated with 1-Al-Ti0 ₂ in various concentrations (0.3 ng/mL to 0.3 mg/mL) for 24 and 48 h (Figure 7). It was observed that 1-Al-Ti0 ₂ was also essentially nontoxic up to 3 μg/mL after both 24 and 48 h of treatment. Only at higher concentrations were the ratios of the live cells dropped below 80%. The MPH behave similarly after 24-h and 48-h treatments with various doses of 1-Al-Ti0 ₂ , suggesting that low 1- Al-Ti0 ₂ concentrations have minimal cytotoxic effects on the viability of these normal cells. While the trend at high concentrations were not observed for the glioblastoma U870-Luc cells treated with 1-Al-Ti0 ₂ , it is expected that normal cells, especially primary cells, are less tolerant towards exogenous non-native agents. Nonetheless, the intense fluorescence exhibited by 1-Al would allow for the use of the nanoconjugate 1-Al-Ti0 ₂ as an optical imaging agent observable by confocal fluorescence microscopy even at low concentrations (20-200 ng/mL) below the cytotoxic thresholds for both the cancer and normal cells observed in the studies.

4-(chlorosulfonyl)Benzoic Acid + Ti0 ₂ (Anatase)

[0111] Added Ti0 ₂ (0.1099 g) and 4-(chlorosulfonyl)Benzoic Acid (0.0171 g) to scintillation vial. Pumped into dry box. Added anhydrous Pyridine (3 mL) and heated to 120 °C for 1 hr, under an inert atmosphere. Allowed to cool to room temperature, then added 2 mL H ₂ 0, which then centrifuged down. Washed and centrifuged with acetone, acetone, water, and acetone. Pumped down on high vacuum line to afford product for Infrared Spectroscopy.

Biphenyl-4-sulfonyl Chloride + Ti0 ₂ (Anatase) [0112] Added Ti0 ₂ (0.1253 g) and Biphenyl-4-sulfonyl Chloride (0.0208 g) to scintillation vial. Pumped into dry box. Added anhydrous Pyridine (3 mL) and heated to 120 °C for 1 hr, under an inert atmosphere. Allowed to cool to room temperature, then added 2 mL H ₂ 0, which then centrifuged down. Washed and centrifuged with acetone, acetone, water, and acetone. Pumped down on high vacuum line to afford product for Infrared Spectroscopy.

4'-chlorobiphenyl-4-sulfonyl Chloride + Ti0 ₂ (Anatase)

[0113] Added Ti0 ₂ (0.1205 g) and 4'-chlorobiphenyl-4-sulfonyl Chloride (0.0210g) to scintillation vial. Pumped into dry box. Added anhydrous Pyridine (3 mL) and heated to 120 °C for 1 hr, under an inert atmosphere. Allowed to cool to room temperature, then added 2 mL H ₂ 0, which then centrifuged down. Washed and centrifuged with acetone, acetone, water, and acetone. Pumped down on high vacuum line to afford product for Infrared Spectroscopy.

Chlorsulfonyl Isocyante +Ti0 ₂ (Anatase)

[0114] Added Ti0 ₂ (0.1205 g) to scintillation vial. Pumped into dry box. Added 100 Chlorsulfonyl Isocyante. Added anhydrous Pyridine (3 mL) and heated to 120 °C for 1 hr, under an inert atmosphere. Allowed to cool to room temperature, then added 2 mL H ₂ 0, which then centrifuged down. Washed and centrifuged with acetone, acetone, water, and acetone. Pumped down on high vacuum line to afford product for Infrared Spectroscopy.

Chlorsulfonyl Isocyante +Ti0 ₂ (Anatase)

[0115] Added Ti0 ₂ (0.1269 g) to scintillation vial. Pumped into dry box. Added 400 Chlorsulfonyl Isocyante. Heated to 120 °C for 1 hr, under an inert atmosphere. Allowed to cool to room temperature, then added 4 mL H ₂ 0, which was then centrifuged down. Washed and centrifuged with acetone, acetone, water, and acetone. Pumped down on high vacuum line to afford product for Infrared Spectroscopy.

[0116] According to the methods described herein, other Cl-S0 ₂ -containing substrates can also be employed, such as, for example, 2-pentyl sulfonyl chloride, 3,3,3-trifluoropropane-l- sulfonyl chloride, methyl(chlorosulfonyl) acetate, and the like.