WO2009038659A2 - Organically modified silica nanoparticles with covalently incorporated photosensitizers for drug delivery in photodynamic therapy - Google Patents

Abstract

Nanoparticles containing covalently linked photosensitizer molecules that overcome the drawback of premature release and thus enhance the outcome of PDT. [Silica-based nanoparticles are provided containing at least one covalently linked photosensitizer. The photosensitizer is preferably a tetrapyrrole-based compounds related to porphyrins, chlorins, bacteriochlorins, benzochlorins, benzoporphyrin derivatives, pheophorbides including pyropheophorbides, and phthalocyanines, naphthanocyanines with and without fused ring systems and derivatives of all the above. The nanoparticle may also include covalently linked imaging agents, e.g. radionuclides, magnetic resonance (MR) and fluorescence imaging agents. The imaging agents and photosensitizers may be at a periphery (surface) of the nanoparticles to increase efficiency. Target-specific nanoparticles may be provided by incorporating biotargeting molecules such as specific antibodies at the surface that react with particular ligands to obtain target specificity. Diagnostic agents may be present in the antibody in addition to imaging agents and tumor specific photosensitizers.

Description

ORGANICALLY MODIFIED SILICA NANOPARTICLES WITH COVALENTLY INCORPORATED PHOTOSENSITIZERS FOR DRUG DELIVERY IN PHOTODYNAMIC THERAPY

BACKGROUND OF THE INVENTION

[0001] The present invention relates to the field of nanoparticle mediated drug delivery in photodynamic therapy. Photodynamic therapy (PDT), a light-activated treatment for cancer and other diseases, has emerged as one of the important areas in biophotonics research. PDT utilizes light-sensitive drugs or photosensitizers (PS), which are preferentially localized in malignant tissues upon systemic administration. The therapeutic effect is activated by the photoexcitation of the localized photosensitizers and the subsequent generation of cytotoxic species, such as singlet oxygen (1O2), free radicals or peroxides, which lead to selective and irreversible destruction of the diseased tissues without damaging adjacent healthy ones.

[0002] In spite of the advantages over current treatments including surgery, radiation therapy and chemotherapy, PDT still has problems to be resolved for a more general clinical acceptance. One of the major challenges in PDT is the preparation of stable pharmaceutical formulations of photosensitizers for systemic administration. Since most existing photosensitizers are poorly water soluble, they aggregate easily under physiological conditions and thus cannot be simply injected intravenously. Moreover, even with water- soluble photosensitizers, the accumulation selectivity for diseased tissues is not high enough for clinical use. [0003] Photodynamic therapy (PDT) is based on the concept that certain therapeutic molecules called photosensitizers (photosensitizer) can be preferentially localized in malignant tissues, and when these photosensitizers are activated with appropriate wavelength of light, they pass on their excess energy to surrounding molecular oxygen resulting in the generation of reactive oxygen species (ROS), such as free radicals and singlet oxygen (¹O₂), which are toxic to cells and tissues.

[0004] PDT is a non-invasive treatment and used for several types of cancers, and its advantage lies in the inherent dual selectivity. First, selectivity is achieved by a preferential localization of the photosensitizer in target tissue (e.g. cancer), and second, the photoirradiation and subsequent photodynamic action can be limited to a specific area. Since the photosensitizer is non-toxic without light exposure, only the irradiated areas will be affected, even if the photosensitizer does infiltrate normal tissues.

[0005] Although PDT is emerging as a choice of treatment for many cancer patients, because of the hydrophobic nature of the most of the photosensitizers, searches are still on for developing an ideal photosensitizer formulation that can be easily injectable in vivo. Numerous approaches have been proposed to achieve not only stable aqueous dispersion but also site-specific and time-controlled delivery of therapeutic agents, often using a biocompatible delivery vehicle. Colloidal carriers for photosensitizers, such as oil- dispersions, liposomes, low-density lipoproteins, polymeric micelles, and recently ceramic nanoparticles are examples of delivery shuttles for photosensitizer molecules some of which may offer benefits from rendering aqueous stability and appropriate size for passive targeting to tumor tissues by the "enhanced permeability and retention" (EPR) effect, offering a possibility of bioconjugation approaches to enhance bioavailability as well as tumor targeting and offering a possibility of actively targeting tumor tissues by appropriate surface functionalization.

[0006] In PDT the release of the photosensitizer drugs is not a prerequisite for their therapeutic action (unlike in conventional chemotherapy), and the premature release of the photosensitizer molecules from carrier vehicles while in systemic circulation results in reduced efficacy of treatment. [0007] Nanoparticles made of an organically modified silica complexed with polynucleotides have been described in co-pending United States priority application 11/195,066. That patent application does not, however, suggest anything concerning nanoparticles made of an organically modified silica complexed with a photodynamic agent.

BRIEF DESCRIPTION OF THE INVENTION

[0008] In accordance with the invention, nanoparticles containing covalently linked photosensitizer molecules are provided to overcome the drawback of their premature release and thus enhance the outcome of PDT.

[0009] In accordance with the invention, silica-based nanoparticles are provided containing at least one covalently linked photosensitizer. The photosensitizer is preferably a tetrapyrrole-based compounds related to porphyrins, chlorins, bacteriochlorins, benzochlorins, benzoporphyrin derivatives, pheophorbides including pyropheophorbides, and phthalocyanines, naphthanocyanines with and without fused ring systems and derivatives of all the above. [0010] The nanoparticle may also include covalently linked imaging agents, e.g. radionuclides, magnetic resonance (MR) and fluorescence imaging agents. The imaging agents and photosensitizers may be at a periphery (surface) of the nanoparticles to increase efficiency. [0011] Target-specific nanoparticles may be provided by incorporating biotargeting molecules such as specific antibodies at the surface that react with particular ligands to obtain target specificity. Diagnostic agents may be present in the antibody in addition to imaging agents and tumor specific photosensitizers as previously and subsequently discussed. [0012] In general, the nanoparticle of the invention has the structural formula:

where the ring represents a silicone polymer matrix, where R₄ is (Ri)_n-R₂ -(Rs)_n where Ri is a labeled photosensitizer (IP) or unlabeled photosensitizer (P), cyanine dye, SPECT imaging agent, PET imaging agent, MR imaging agent or fluorescent imaging agent at least partially available at a surface of the silicone polymer matrix; R₂ is -O-, -COO-, -NR₅ or -NH- connected to the silicone polymer matrix directly or through an intermediate group, n is 0 or 1; provided that at least one n is 1 and R3 is cyanine dye, SPECT, PET, MR or fluorescent imaging agent, linked targeting agent RGD, F3 peptide, carbohydrate or folic acid or labeled photosensitizer (IP) or unlabeled photosensitizer (P) embedded in the silicone polymer matrix, and R5 is lower alkyl of from 1 to 5 carbon atoms where a plurality of Ri groups, R₃ groups or mixtures thereof are photosensitizer and/or labeled photosensitizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure IA shows graphic results of spin-filtration of various formulations of

PS/nanoparticles. The graph shows the relative optical densities (read at 663 nm, the long- wavelength absorbance peak for IP) of the 'filtrate' and 'retentate' fractions, as well as the non-filtered 'original' samples, for cell lines NY-362 through NY-365. The non-silylated photosensitizer 3-iodobenzyl-pyro, or EP (compound I, Scheme 1), is used as the control, both dissolved in Tween-80 micelles as well as encaphotosensitizerulated in ORMOSIL nanoparticles. [0014] Figure IB shows a TEM image of NY-363. [0015] Figure 1C shows a scheme 1 for synthesis of the precursor 3-iodobenzylpyro- silane (EPS) shown as compound II. The non-silylated photosensitizer 3-iodobenzyl-pyro starting compound, or IP (compound I), is used as the control, both dissolved in Tween-80 micelles as well as encapsulated in ORMOSEL nanoparticles. [0016] Figure 2 shows emission of EP upon UV irradiation following TLC of EP- conjugated (lane 1) and encapsulated (lane 2) ORMOSlL nanoparticles. Lane 3 shows the same for EP/1 % Tween-80.

[0017] Figure 3A shows absorption spectra of the "micelle free" nanoparticle samples (retentate collected after spin-filtration and resuspended), as well as non-filtered micellar suspensions of EP/Tween-80 and NY-362 (100% of EPS, no VTES). Fluorescence was obtained on exciting the molecule at 514 nm.

[0018] Figure 3B shows fluorescence spectra of the "micelle free" nanoparticle samples (retentate collected after spin-filtration and resuspended), as well as non-filtered micellar suspensions of EP/Tween-80 and NY-362 (100% of EPS, no VTES). Fluorescence was obtained on exciting the molecule at 514 nm. [0019] Figure 4A shows results on singlet oxygen production by "micelle-free" suspensions of nanoparticles which were obtained by singlet oxygen phosphorescence spectroscopy, showing remarkable similarity to those obtained following the same trend: EP/Tween-80 micellar suspension demonstrated higher ¹O₂ generation than NY-363, NY-364, NY-365; whereas, intensitiy for NY-362 is lower. [0020] Figure 4B shows results on singlet oxygen production by "micelle-free" suspensions of nanoparticles, obtained using an ADPA (anthracenedipropionic acid) bleaching method. Non-spin-filtered micellar suspensions of EP/Tween-80 and NY-362 (100% of Iphotosensitizer, no VTES) were used as controls. Rose Bengal (RB) in methanol was used as a reference standard for the ¹O₂ phosphorescence measurements. This means that singlet oxygen generated within nanoparticles is mostly deactivated outside nanoparticles, causing bleaching of ADPA. Irradiation with 514 nm, applied laser power was 5 times higher for the bleaching experiment than for spectra acquisition. [0021] Figure 5 shows decays of the emission from Iphotosensitizer/VTES nanoparticle and IP Tween-80 suspensions at 1270 nm.

[0022] Figure 6 shows combined comparative photomicrographs Figure 6 of Colon-

26 cells treated overnight with NY-363 (A), NY-364 (B), NY-365 (C). Transmission (above) and fluorescence (below) channels are shown. Confocal pinhole and PMT gain remained same during imaging. [0023] Figure 7 shows a nonoparticle 10 having R4 groups where the ring represents a silicone polymer matrix. R₄ is (Rt)_n-R₂ -(Rs)_n . Ri is a labeled photosensitizer (IP) or unlabeled photosensitizer (P), cyanine dye, SPECT imaging agent, PET imaging agent, MR imaging agent or fluorescent imaging agent at least partially available at a surface of the silicone polymer matrix. R₂ is -O-, -COO-, -NR5 or -NH- connected to the silicone polymer matrix directly or through an intermediate group, n is 0 or 1; provided that at least one n is 1. R3 is cyanine dye, SPECT, PET, MR or fluorescent imaging agent, linked targeting agent RGD, F3 peptide, carbohydrate or folic acid or labeled photosensitizer (IP) or unlabeled photosensitizer (P) embedded in the silicone polymer matrix. R₅ is lower alkyl of from 1 to 5 carbon atoms where a plurality of Ri groups, R₂ groups or mixtures thereof are photosensitizer and/or labeled photosensitizer.

[0024] Figure 8A shows a structure where P is a photosensitizer, e.g. pophyrins, chlorins, bacteriochlorins, phthalocyanines with and without fused structures, R = OH, COOH, NH₂ groups, and/or R= imaging agent, e.g. PET, MR, or fluorescence imaging agents and/or R = targeting agent, e.g. RGD, F3 peptides, carbohydrates and folic acid. [0025] Figure 8B shows a structure where X = I¹²⁴-labeled photosensitizer (IP), cyanine dye, SPECT imaging agent, MR imaging agent and P = photosensitizer. [0026] Figure 8C shows a structure where X = I¹²⁴-labeled photosensitizer (IP), cyanine dye, SPECT imaging agent, MR imaging agent, R = OH, COOH, or NH₂ groups that may be linked by reaction with targetingents, e.g. RGD, F3 peptides, carbohydrates and folic acid. DETAILED DESCRIPTION OF THE INVENTION

[0027] In accordance with the invention, nanoparticles containing covalently linked photosensitizer molecules are provided to overcome the drawback of their premature release and thus enhance the outcome of PDT. [0028] As previously discussed, in accordance with the invention, silica-based nanoparticles are provided containing at least one covalently linked photosensitizer. The photosensitizer is preferably a tetrapyrrole-based compounds related to porphyrins, chlorins, bacteriochlorins, benzochlorins, benzoporphyrin derivatives, pheophorbides including pyropheophorbides. [0029] Specific ezamples of such photosensitizers may, for example be found in U.S.

Patent Nos. 4,649,151; 4,866,168; 4,932,934; 4,961,920; 5,002,962; 5,015,463; 5,028,621; 5,093,349; 5,145,863; 5,171,741; 5,173,504; 5,190,966; 5,198,460; 5,225,433; 5,257,970; 5,314,905; 5,459,159; 5,498,710; 5,591,847; 5,770,730; 5,952,366; 6,624,187 and 6,849,607, and phthalocyanines, naphthanocyanines with and without fused ring systems and derivatives of all the above.

[0030] The nanoparticle may also include covalently linked imaging agents, e.g. radionuclides, magnetic resonance (MR) and fluorescence imaging agents. The imaging agents and photosensitizers may be at a periphery (surface) of the nanoparticles to increase efficiency. [0031] Target-specific nanoparticles may be provided by incorporating biotargeting molecules such as specific antibodies at the surface that react with particular ligands to obtain target specificity. Diagnostic agents may be present in the antibody in addition to imaging agents and tumor specific photosensitizers as previously and subsequently discussed.

[0032] In general, the nanoparticle of the invention has the structural formula:

where the ring represents a silicone polymer matrix.

[0033] R₄ is (Ri)_n-R₂ -(Rs)_n where Ri is a labeled photosensitizer (IP) or unlabeled photosensitizer (P), cyanine dye, SPECT (single proton emission computed tomography) imaging agent, PET (positron emission tomography) imaging agent, MR imaging agent or fluorescent imaging agent at least partially available at a surface of the silicone polymer matrix.

[0034] At least one Ri or R₃ group may be a tetrapyrollic photosensitizer, e.g. porphyrins, chlorins, bacteriochlorins, benzochlorins, benzoporphyrins, pheophorbides including pyropheophorbides, and derivatives thereof. [0035] R₂ is -O-, -COO-, -NR5 or -NH- connected to the silicone polymer matrix directly or through an intermediate group. The intermediate group may, for example, be subtituted or unsubstituted alkylene or phenylene. The alkylene or phenylene may be substituted with at least one hydroxy, carboxy, amino, sulfo, alkylester, alkylether, heterocyclo, or halo group. [0036] n is 0 or 1 ; provided that at least one n is 1. [0037] R3 is cyanine dye, SPECT, PET, MR or fluorescent imaging agent, linked targeting agent RGD, F3 peptide, carbohydrate or folic acid or labeled photosensitizer (IP) or unlabeled photosensitizer (P) embedded in the silicone polymer matrix. RGD is a peptide that contains the Arg-Gly-Asp attachment site that recognizes v3 and v5 integrin receptors that play a role in angiogenesis, vascular intima thickening and proliferation of malignant tumors.

[0038] R₅ is lower alkyl of from 1 to 5 carbon atoms. A plurality of Ri groups, R3 groups or mixtures thereof are photosensitizer and/or labeled photosensitizer. [0039] The Ri or R₃ group may be phthalocyanine, naphthanocyanine and derivatives thereof and may also be a radionuclide or MR or fluorescencent imaging agent. A plurality of Ri groups are preferably photosensitizers located at peripheral positions on the nanoparticle and a plurality of R₃ groups are imaging agents located at peripheral positions on the nanoparticle. [0040] The nanoparticle are desirably provided with biotargeting molecules following suitable surface functionalization to obtain target-specific nanoparticles. Examples of such biotargeting molecules are antibodys and the suitable surface functionalization for the antibody is a ligand, e.g. RGD and F3 peptide.. [0041] The nanoparticle may further include at least one diagnostic agent. [0042] "Photosensitiers" (PS) as used herein means any material that can enter or attach to a cell or portion thereof and be activated by eletromagnetic radiation, usually light, to destroy the cell or significantly alter its activity.

[0043] "nanoparticles made of an organically modified silica" as used herein refers to nanoparticles made from silica that has been organically modified to self organize into polysicone nanoparticles upon precipitation from solution. Preferred organically modified silica nanoparticles are ORMOSIL nano particles usually made by inclusion of a vinyltriethoxysilane in a sufactant solution followed by precipitation with ammonia or other amine, e.g. 3 aminopropyltriethoxy silane. In the first case the nanoparticle has surface -OH groups and in the second case has surface amino groups. [0044] The silane (silicone) matrix is formed by self reaction of hydroxy silanes by dehydration to form a polymeric silicone matrix of silcon atoms interconnected by oxygen atoms. The starting silanes have the formula R₄Si where R is independently at each occurrence an alkyl, alkylene, hydroxy or alkoxy group, provided that at least two of said R groups are hydroxy groups. The other R groups are usually hydroxy, alkoxy or an alkyl group substituted with an alkoxy, carboxy, hydroxyl, amino or mercapto group. The silanes and R groups are selected such that they will form nanoparticles having a size of less than 200nm, preferably less than 100 nm and most preferably less than 50 nm. Particles of a size less than 20 nm are most desirable in most circumstances. The silanes are selected so that the nanoparticles will have hydroxyl, amino, mercapto and/or carboxy groups exposed at its surface.

[0045] The silane is desirably selected from the group consisting of vinyltrimethoxysilane, vinyltriethoxysilane, vinylytriacetosilane, γ- glycidoxypropyltrimethoxysilane, γ-methacryloxypropyltrimethoxysilane, γ- aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, γ- mercaptopropyltrimethoxysilane, γ-3,4-epoxycyclohexyltrimethoxysilane and phenyltrimethoxysilane.

[0046] The invention further includes a method for forming nanoparticles having covalently bonded photosensitizer. This accomplished by providing reactive intermediate structures on the the nanoparticle, either by providing them on the nanoparticle precursor or by adding them subsequent to nanoparticle formation. [0047] A specific method for forming such nanoparticles includes the steps of: a) forming a uniform medium comprising from about 70 to about 80 weight percent of a lower alcohol selected from isopropanol, n-butanol, isobutanol and n-pentanol, from about 20 to about 30 weight percent of DMSO, from about 2 to about 3 percent water and from about 0.05 to about 0.15 percent of sufficient surfactant to maintain a dispersion; b) uniformly incorporating one or more silanes, as above described wherein the amount of silane or mixtures of silanes is about the maximum permitted for stability; c) adding sufficient reactive basic compound to form nanoparticles having reactive hydroxyl, amino, mercapto and/or carboxy groups exposed at their surface; d) dialyzing the nanoparticles through a membrane having a pore size of from about 0.1 to about 0.3 μM; e) during step b) or prior to step d), reacting a photosensitizer with a reactive group on the surface of the nanoparticles.

[0048] The surfactant used in the method is usually a polyoxyethylene sorbitan monooleate or sodium dioctyl sulfosucinate and the silane usually includes: vinyltrimethoxysilane, vinyltriethoxysilane, vinylytriacetosilane, γ- glycidoxypropyltrimethoxysilane, γ-methacryloxypropyltrimethoxysilane, γ- aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, γ- mercaptopropyltrimethoxysilane, γ-3,4-epoxycyclohexyltrimethoxysilane and phenyltrimethoxysilane.

[0049] The silane is preferably vinyltriethoxysilane or phenyltrimethoxysilane and the basic compound is usually ammonia or 3-aminopropylethoxysilane. It should; however be understood that essentially any base may be used provided that it if it is a strong base, e.g. an alkali hydroxide, it is sufficiently diluted.

[0050] Preferred photosensitizers are preferentially absorbed or adsorbed by cells that require destruction or significant alteration, e.g. cells of hyperproliferative tissue such as tumor cells, hypervascularization such as found in macular degeneration and hyperepidermal debilitating skin diseases. Selectivity can be further enhanced by incorporating with nanoparticles in accordance with the present invention, targeting agents such as an monoclonal antibodies, integrin-antagonists or carbohydrates which have high affinity for target tissue (mainly cancer). [0051] Preferred photosensitizers are tetrapyrrole-based compounds related to porphyrins, chlorins, bacteriochlorins, benzochlorins, benzoporphyrin derivatives, pheophorbides including pyropheophorbides, and phthalocyanines and, naphthanocyanines with and without fused ring systems and derivatives of all the above. [0052] A desirable photosensitizer for many applications is a tumor avid tetrapyrollic photosensitizer, that may be complexed with an element X where X is a metal selected from the group consisting of Zn, In, Ga, Al, or Cu or a radioisotope labeled moiety wherein the radioisotope is selected from the group consisting of ¹¹C, ¹⁸F, ⁶⁴Cu, ¹²⁴I, ⁹⁹Tc, ¹¹¹In and GdIII that may be used in a method for diagnosing, imaging and/or treating hyperproliferative tissue such as tumors and other uncontrolled growth tissues such as found in macular degeneration.

[0053] More particularly, the photosensitizer may have the generic formula:

In the case of the presence of a tetrapyrollic photosensitizer, it usually has the structural formula:

and its complexes with X where:

R₁ is -CH=CH₂, -CH₂CH₃, -CHO, -COOH, or

where R₉ = -OR₁0 where Ri₀ is lower alkyl of 1 through 8 carbon atoms, -(CH₂-O)_nCH₃, -(CH₂)₂CO₂CH₃, -(CH₂)₂CONHphenyleneCH₂DTPA,

-CH₂CH₂CONH(CONHphenyleneCH₂DTPA)₂ , -CH₂R₁, or \ , or a

O=C

^R 1 i ^"N ~^R i 1

fluorescent dye moiety; R₂, R_2a, R_3, R₃₃, Ri, Rs, Rsa, R7, and R_7a are independently hydrogen, lower alkyl or substituted lower alkyl or two R₂, R_2a, R₃, R_3a, R5, Rsa, R7, and R_7a groups on adjacent carbon atoms may be taken together to form a covalent bond or two R₂, R_2a, R₃, R_3a, R₅, R_5a, R₇, and R_7a groups on the same carbon atom may form a double bond to a divalent pendant group; R₂ and R₃ may together form a 5 or 6 membered heterocyclic ring containing oxygen, nitrogen or sulfur; R₆ is -CH₂-_, -NRn- or a covalent bond; Rs is -(CH₂)₂CO₂CH₃, -(CH₂)₂CONHphenyleneCH₂DTPA,

-CH₂CH₂CONH(CONHphenyleneCH₂DTPA)₂, -CH₂Rn Or ₀_J, where

^R 1 i ^"N ^{' R} i 1

R_n is -CH₂CONH-RGD-Phe-Lys, -CH₂NHCO-RGD-PhC-LyS, a fluorescent dye moiety, or -CH₂CONHCH₂CH₂SO₂NHCH(CO₂)CH₂NHCOPhenylOCH₂CH₂NHcycloCNH(CH₂)₃N; and polynuclide complexes thereof; provided that the compound contains at least one integrin antagonist selected from the group consisting of -CH₂CONH-RGD-Phe-Lys, -CH₂NHCO- RGD-Phe-Lys and -CH₂CONHCH₂CH₂SO₂NHCH(CO₂)CH₂NHCOPhenylOCH₂CH₂NHcycloCNH(CH₂)₃N, where X is a metal selected from the group consisting of Zn, In, Ga, Al, or Cu or a radioisotope labeled moiety wherein the radioisotope is selected from the group consisting of ¹¹C, ¹⁸F, ⁶⁴Cu, ¹²⁴I, ⁹⁹Tc, ¹¹¹In and GdIII .

[0054] The complexes with X are readily made simply by heating the compound with a salt of X such as a chloride. The complex will form as a chelate of a -DTPA moiety, when present, or within the tetrapyrollic structure between the nitrogen atoms of the amine structure or both. Examples of such structures are:

M = 2H or

M = In, Cu, Ga (with or without radioactive isotope)

and

M = 2H or

M = In, Cu, Ga (with or without radioactive isotope) Where X=M

[0055] In accordance with the invention a method is provided for the synthesis of organically modified silica (ORMOSIL) nanoparticles with a covalently linked photosensitizer molecule. The nanoconjugated photosensitizer retained its spectral and therapeutic properties, was uptaken by tumor cells in culture and could elicit PDT effect upon photoirradiaion of the targeted cells.

[0056] In accordance with the invention, it has been possible to prepare a highly stable aqueous formulation of organically modified silica (ORMOSIL) nanoparticles encaphotosensitizerulating (photosensitizer encapsulating) the hydrophobic photosensitizer, where the encaphotosensitizerulated photosensitizer is able to generate singlet oxygen upon photoactivation owing to the free diffusion of molecular oxygen across the ORMOSIL matrix. However, because of mesoporosity of the ORMOSIL matrix, encaphotosensitizerulation of photosensitizer does not exclude the photosensitizer release during in vivo circulation. In accordance with the present invention, nanoparticles with covalently incorporated photosensitizer eliminate the possibility of premature release of the photosensitizer molecules while being in circulation and ensure maximum delivery of the photosensitizer to the targeted site. [0057] In particular, ORMOSIL nanoparticles, where the photosensitizer molecule is covalently incorporated within the ORMOSIL nanoparticle matrix, have been synthesized and characterized. This has been achieved by the synthesis of iodobenzyl-pyro-silane (Iphotosensitizer), a precursor for ORMOSIL with the linked photosensitizer iodobenzylpyropheophorbide (IP). Highly monodispersed aqueous dispersion of ORMOSIL nanoparticles (average diameter ~20 nm) were synthesized upon co-precipitation of Iphotosensitizer with the commonly used ORMOSIL precursor vinyltriethoxysilane (VTES). This synthesis is carried out in the non-polar core of Tween-80/water microemulsion media. In this microemulsion media, ORMOSIL nanoparticles can readily be synthesized with the combination of Iphotosensitizer and VTES. Photophysical study has demonstrated that the spectroscopic and functional (generation of cytotoxic singlet oxygen) properties of the photosensitizer are preserved in their 'nanoconjugated' state. These nanoparticles are found to be uptaken by tumor cells in culture, thus demonstrating the potential of these nanoparticles for PDT of cancer. [0058] Confocal bioimaging studies have revealed that the in vitro cellular uptake between these IP-conjugated nanoparticles is weaker when compared to that of the free IP (i.e. IP in Tween-80).This weak cellular uptake is also reflected in the in vitro PDT efficacy of the IP-conjugated nanoparticles, which is again found to be weaker than that of the free IP. This weak cellular uptake, however, can be substantially improved by modifying the surface of these nanoparticles. The surfaces of these nanoparticles may, however, be modified using bioconjugation approaches to improve their biocompatibility and biotargeting efficiency. The conjugated IP may also be modified with radiolabeled probes (e.g. 1-124) in an effort to combine the feasibiliy of positron-emission tomographic (PET) imaging along with PDT for these nanoparticles. [0059] In vitro experiments have revealed that these nanoparticles are avidly uptaken by tumor cells, demonstrating the potential for using them as drug-carriers in PDT. [0060] The materials used in the examples discussed herein are commercially available and found in many laboratories throughout the world and/or are readily prepared by those skilled in the art. ORMOSIL precursor vinyltriethoxysilane (VTES) was purchased from Sigma-Aldrich. Microfuge membrane-filters (NANOSEP IOOK OMEGA) are a product of Pall Corporation. N-Ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride, 4- dimethylamino pyridine and 4-(triethoxysilyl)-aniline were purchased from Aldrich and used without further purification. 9,10-Anthracenedipropionic acid, disodium salt (ADPA) was purchased from Invitrogen. Colon-26 cells were cultured according to manufacturer's instructions. Unless otherwise mentioned, all cell culture products were obtained from Invitrogen

[0061] Synthesis of Iodobenzyl-pyro-Silane (Iphotosensitizer): The synthesis of

Iphotosensitizer is shown in Figure 1C (Scheme 1). First, 3-iodobenzyl-pyro (Compound I, 50.0 mg, 0.065 mmol) was taken in a dry round bottom flask (RBF) (100 ml) and dissolved in dry dichloromethane (30 ml). To this, 4-(triethoxysilyl)-aniline (19.9 mg, 0.078 mmol), N- Ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (24.9 mg, 0.13 mmol) and 4- dimethylamino pyridine (15.8 mg, 0.13 mmol) were added and the resultant mixture was stirred for 14 hr at room temperature under N₂ atmosphere. Reaction mixture was then diluted with dichloromethane (100 ml) and washed with brine (50 ml). Then, the organic layer was separated, dried over sodium sulfate and concentrated. Finally, product (Iphotosensitizer, Compound II) was purified over silica gel plate using 2.5% methanol-dichloromethane as mobile phase. Yield: 35.0 mg (53.4%). NMR spectra were recorded on a Bruker DRX 400 MHz spectrometer at 303K in CDCl₃ solution and referenced to residual CHCU (7.26 ppm). ¹HNMR (400Mhz, CDCl₃): δ 9.80 (splitted singlet, IH, meso-H), 8.78 (splitted singlet, IH, meso-H), 8.55 (splitted singlet, IH, meso-H), 7.81 (d, IH, Ar-H, J = 17.6 Hz), 7.65 (t, IH, Ar-H, J = 8.0 Hz), 7.57 (m, IH, Ar-H), 7.50 (m, 2H, Ar-H), 7.34-7.29 (m, 3H, Ar-H), 7.08 (m, IH, Ar-H), 6.00 (m, IH, CH₃CHOAr), 5.34 (d, IH, 15'-CH₂, J = 19.6 Hz), 5.10 (d, IH, 15'-CH₂, J = 19.6 Hz), 4.72 (m, IH, H-17), 4.59 (m, 2H, OCH₂-Ar), 4.41 (m, IH, H-18), 3.83 (q, 6H, SiOCH₂-CH₃, J = 7.2 Hz), 3.55 (m, IH, 8-CH₂CH₃), 3.37 (m, IH, 8-CH₂CH₃), 3.34 (splitted singlet, 3H, ring-CH₃), 3.24 (splitted singlet, 3H, ring-CH₃), 2.81 (m, IH, 17²-CHA 2.72 (m, IH, 17^CH₂), 2.66 (splitted singlet, 3H, ring-CH₃), 2.36 (m, IH, 17'-CH₂), 2.24 (m, IH, 17'-CH₂), 2.21 (m, 3H, CH₃CH-OAr), 1.81 (d, 3H, 18-CH₃, J = 7.2 Hz), 1.53 (m, 3H, 8- CH₂CH₃), 1.20 (t, 9H, SiOCH₂-CH₃, J = 6.8 Hz), 0.45 (brs, IH, NH), -1.53 (brs, IH, NH). EMS: 1007 (M⁺+l).

[0062] Synthesis and characterization of the covalently linked iodobenzylether-pyro nanoparticles. In general, the nanoparticles were synthesized by the alkaline hydrolysis and polycondensation of the organo-trialkoxysilane precursors within the non-polar core of Tween-80/water microemulsion. Briefly, to 10 ml of 2% aqueous Tween-80 solution, 300 μL of co-surfactant 1-butanol was dissolved. To this solution, 40 μL of a solution (10 mM in DMSO) of Iphotosensitizer (Compound II, Scheme 1) was dissolved by simple magnetic stirring. Next, 0 or 40 or 80 or 160 μL of VTES was added dropwise and the resulting mixture was magnetically stirred for one hour. At this stage, 10DL of aqueous ammonia was added and the resulting solution was magnetically stirred overnight for the formation of the nanoparticles. In order to study the difference of conjugated photosensitizer in ORMOSIL, as opposed to encaphotosensitizerulated photosensitizer in ORMOSIL, we also synthesized photosensitizer encaphotosensitizerulated nanoparticles by an identical procedure as described above, except that 40 μL of 10 mM DMSO solution of IP (Compound I, Scheme 1) was used instead of Iphotosensitizer Next, the nanoparticles were dialyzed overnight against distilled water using a cellulose membrane of cut-off pore size of 12-14 kD for the removal of unreacted molecules. The dialysate containing the IP-conjugated ORMOSIL nanoparticles was sterile filtered (0.2 uM membrane) and was stored at 4⁰C for further use. Table 1 represents the amounts of the Iphotosensitizer and VTES used in the various formulations.

Table 1. Formulations of the Iphotosensitizer nanoparticles [0063] To separate the Tween-80 micelles (and any associated photosensitizer) from the nanoparticles, the dialyzed dispersions were filtered in a microfuge membrane-filter (NANOSEP IOOK OMEGA, Pall Corporation, USA) by centrifuging at 14,000 rpm for 30 minutes (spin-filtration). Tween-80 micelles and their associated photosensitizer molecules flowed through this membrane and are collected in the lower tube (fliltrate), while nanoparticles got embedded in the membrane and could be subsequently extracted by adding water and sonicating/vortexing briefly (retentate). The amount of photosensitizer associated with each fraction could be estimated by reading their optical density at 663 nm (the long wavelength absorption peak for IP/Iphotosensitizer). All subsequent studies with the nanoparticles were carried out with the micelle-free 'retentate' fraction, unless otherwise mentioned.

[0064] Thin-layer chromatography (TLC) was done on ANALTECH pre-coated silica gel GF PE sheets (Cat. 159017, layer thickness 0.25 mm). Preparative TLC plates used for the purification (ANALTECH pre-coated silica gel GF glass plate, Cat. 02013, layer thickness 1.0 mm). Dichloromethane was dried over P₂Os under N₂ atmosphere. [0065] Characterization of size, shape and functionality of the nanoparticles.

Transmission electron microscopy (TEM) was performed to determine the size and shape of the prepared nanoparticles, using a JEOL JEM-100cx microscope at an accelerating voltage of 80 kV. UV-visible absorption spectra were acquired using a Shimadzu UV-3600 spectrophotometer, in a quartz cuvette with 1 cm path length. Fluorescence spectra were recorded on a Fluorolog-3 spectrofluorometer (Jobin Yvon, Longjumeau, France). Generation of singlet oxygen (¹O₂) was detected by its phosphorescence emission peaked at 1270 nm.^8"10 A SPEX 270M Spectrometer (Jobin Yvon) equipped with a Hamamatsu ER-PMT was used for recording singlet oxygen phosphorescence. The sample solution in a quartz cuvette was placed directly in front of the entrance slit of the spectrometer and the emission signal was collected at 90-degrees relative to the exciting laser beam. An additional longpass filters (a 950LP filter and a 538AELP filter, both from Omega Optical) were used to attenuate the scattered light and fluorescence from the samples. ¹O₂ phosphorescence decays at 1270 nm were acquired using Infinium oscilloscope (Hewlett-Packard) coupled to the output of the PMT. A second harmonic (532 nm) from nanosecond pulsed Nd:YAG laser (Lotis TII, Belarus) operating at 20 Hz was used as the excitation source.

[0066] Chemical oxidation of ADPA in the nanoparticle water dispersions was used as an independent method to characterize singlet oxygen generation efficiency.⁸ In this case, a decrease in the absorbance of the ADPA added to nanoparticle water suspension was monitored as a function of time, following irradiation with 514 nm. [0067] In Vitro Studies with Tumor Cells: Nanoparticle Uptake and Imaging. For studying nanoparticles uptake and imaging, Colon-26 cells were used, maintained in RPMI- 1640 media with 10% fetal bovine serum (FBS) and appropriate antibiotic. The cells at a confluency of 70-75 % were treated overnight with the nanoparticles at a final photosensitizer concentration of 2 DM. Next day, the treated cells were washed thoroughly with PBS and then directly imaged using a confocal laser scanning microscope (MRC- 1024, Bio-Rad, Richmond, CA). A water immersion objective lens (Nikon, Fluor-60X, NA=LO) was used for cell imaging. A Ti:sapphire laser (Tsunami from Spectra-Physics) pumped by a diode- pumped solid state laser (Millenia, Spectra Physics) was used as a source of excitation. The Ti:sapphire output, tuned to 830 nm, was frequency doubled by second harmonic generation (SHG) in a β-barium borate (β-BBO) crystal to obtain the 415-nm light, and was coupled into a single mode fiber for delivery into the confocal scan head. A long-pass filter, 585 LP (585 nm), and an additional band pass filter with transmission at 680±15 nm (Chroma 680/30) were used as emission filters for fluorescence imaging. [0068] Figures IA and IB show the relative optical densities (read at 663 nm, the long-wavelength absorbance peak for IP) of the 'filtrate' and 'retentate' fractions, as well as the non-filtered 'original' samples, for NY-362 through NY-365. The non-silylated photosensitizer 3-iodobenzyl-pyro, or EP (compound I, Scheme 1), is used as the control, both dissolved in Tween-80 micelles as well as encaphotosensitizerulated in ORMOSIL nanoparticles.

[0069] From the Figure IA, it is evident that while NY-362 does not form any nanoparticles and most of the Iphotosensitizer is associated with the Tween-80 micelles which are collected in the filtrate, almost 80-90% of the Iphotosensitizer is associated with the nanoparticles in NY-363 through NY-365, which can be collected as the 'retentate'. Inability of pure Iphotosensitizer to form the nanoparticles is evidently associated with the steric hindrances due to bulky EP moieties, and nanoparticles are formed by combining both Iphotosensitizer and VTES precursors. A formation of the rigid, spherical and monodisperse nanoparticles with size about 20 nm for NY-363 is shown by TEM (Fig.l,B). It is worth noting that while TEM of NY-362 showed no formation of nanoparticles, thus confirming inability of Iphotosensitizer alone to form nanoparticles, NY-364 and NY-365 both formed the same-sized nanoparticles as NY-363 (data not shown), showing that the size of the nanoparticles is unaffected by the amount of the precursor used.

[0070] In order to confirm that EP is conjugated with the nanoparticles, and not merely physically associated, we have run both IP-conjugated and EP- encaphotosensitizerulated nanoparticles, as well as EP/micelles on silica-thin layer chromatography (Silica-TLC) plates in organic media ((Rf. ~0.5 in 10% Methanol/Dichloromethane). It can be seen that while the EP encaphotosensitizerulated in the nanoparticles (Lane 2) runs similar to EP in Tween-80 micelles (Lane 3) in the direction of the solvent front, the IP conjugated with the nanoparticles (NY-363) remains at the bottom, with the nanoparticles (Lane 1). This means that organic media is not able to wash away the IP molecules from the NY-363, indicating irreversible linkage. We obtained similar data for NY-364 and NY-365 (data not shown). In sharp contrast, encaphotosensitizerulated IP molecules can be easily extracted from the nanoparticles by organic solvents. [0071] Photophysical properties. Absorption and fluorescence of the IP covalently incorporated into nanoparticle matrix are similar to that of encaphotosensitizerulated in the Tween-80 micelles (Figures 3A and 3B). Whereas samples of NY-363, NY-364, NY-365 have almost identical absorption and fluorescence spectra, while NY-362 (non-spin-filtered) shows little decrease in the fluorescence intensity. This correlates with little broadening of long-wave absorption band and can originate from the interaction of the Iphotosensitizer chromohores in tween-80 micelles (self-aggregation).

[0072] Aggregation of the photosensitizer chromophores is usually manifested both in decrease of fluorescence intensity and singlet oxygen generation.¹¹' ¹² Figure 4A presents emission spectra of singlet oxygen sensitized by samples of suspensions with absorbance and fluorescence shown in Figure 2. To distinguish singlet oxygen phosphorescence, which is known to have extremely low yield in water,¹³ we have used methanol solution of Rose Bengal as a standard for obtaining singlet oxygen phosphorescence. As seen in Figure 4A, IP chromophores incorporated within nanoparticles are capable of generating singlet oxygen with yield comparable to that of ¹O₂ generated by Tween-80 micelles. Intensity of the singlet oxygen emission sensitized in all suspensions of nanoparticles / micelles correlates with fluorescence intensity (Figure 3B). Intensity of ¹O₂ emission as well as fluorescence intensity that was almost identical for NY-363, NY-364, NY-365. IP/Tween-80 micellar suspension shows slightly higher fluorescence and ¹O₂ emission intensities, whereas intensity for NY-362 (non-spin-filtered) is lower. [0073] Correlation of the fluorescence and ¹O₂ emission intensities confirms aggregation affecting singlet oxygen generation. It is worth noting that the amount of singlet oxygen generated by photosensitizer (EP) in surfactant (Tween-80) micelles does depend on the relative amount of surfactant, which protects hydrophobic photosensitizer molecules from aggregation. Absence of difference in absorption/fluorescence/singlet oxygen generation for the samples NY-363, NY-364, NY-365 shows that aggregation effect does not manifest itself in the micelle-free Iphotosensitizer/VTES nanoparticles and relative content of Iphotosensitizer within nanoparticles can be further increased to obtain higher ¹O₂ generation for more efficient PDT action. Singlet oxygen phosphorescence steady-state spectroscopy well characterizes singlet oxygen generation in a homogeneous medium. However, in a heterogeneous medium (i.e. water dispersion of a nanoparticles with embedded photosensitizer), question arises how observed ¹O₂ phosphorescence intensity will correlate with a phototoxic efficiency of the generated ¹O₂, because it could be, in principle, mostly deactivated within nanoparticles due to the limited lifetime of ¹O₂. [0074] Singlet oxygen mediated bleaching of the disodium salt of 9,10- anthracenedipropionic acid (ADPA) was used as an independent method for investigating the functional effect of the generated ¹O₂ outside nanoparticles. Here, we have added ADPA to the suspension of nanoparticles and recorded the time-dependent reduction of the ADPA absorption peak at 400 nm upon continuous irradiation with laser light (514 nm). The slope of the curves obtained is an indication of the functional efficiency of generated singlet oxygen (more the slope, more is the efficiency).¹⁴

[0075] As seen in Figure 4B, results on singlet oxygen production, which were obtained with method of ADPA bleaching, showed remarkable similarity to those obtained by singlet oxygen phosphorescence spectroscopy (Figure 4B), following the same trend: IP/Tween-80 micellar suspension demonstrated higher ¹O₂ generation then NY-363, NY-364, NY-365, whereas intensitiy for NY-362 is lower. This means that singlet oxygen generated within nanoparticles is mostly deactivated outside nanoparticles, causing bleaching of ADPA. In this case, lifetime of the singlet oxygen generated within nanoparticles should be determined by the water environment and be around 4μs.¹³ [0076] Figure 5 is a graph showing decays of emission at 1270 nm. Signal obtained for the suspension of neat ORMOSIL nanoparticles (100% of VTES) was used as Instrument Response Function (IRF). Rose Bengal (RB) in methanol was used as a reference standard producing singlet oxygen. Figure 5 shows decays of the emission from

Iphotosensitizer/VTES nanoparticle and IP Tween-80 suspensions at 1270 nm. Along with fast decay component coming from the scattered excitation light, one can see slow component with characteristic lifetime in microsecond range, which is definitely from singlet oxygen phosphorescence decay. Decays of ¹O₂ emission sensitized by

Iphotosensitizer/VTES are very close to that sensitized by IP/Tween-80 micellar suspension and have average lifetime (τ) in the range of 4.5-5 μs. In Figure 5, decay of ¹O₂ emisssion sensitized by RB in methanol is also shown, demonstrating monoexponential fitting with τ wlOμs, which is characteristic lifetime for ¹O₂ in methanol.¹³ Rise time of the ¹O₂ emission sensitized by the nanoparticle / micellar suspensions is noticeably higher then for RB molecular solution, including time of diffusion of the molecular oxygen to the incorporated photosensitizer chromophores. However, since decay time is close enough to that for ¹O₂ in water and action of the singlet oxygen was demonstrated in the ADPA bleaching experiment, we consider that EP chromophores within Iphotosensitizer/VTES nanoparticles retain their functionality as photosensitizer for PDT. [0077] Cellular uptake. Since nanoparticles are capable of singlet oxygen production, which is indispensable condition for successful application in PDT, we have tested cellular uptake of the nanoparticles in vitro. Figure 6 presents confocal images of Colon-26 cells treated overnight with nanoparticles of NY-363, NY-364, NY-365 formulation. As one can see, there is significant uptake of the nanoparticles for all formulations. Since imaging conditions were maintained same (in particular, confocal pinhole and aperture for fluorescence imaging), on can see that fluorescence intensity for NY-364 is higher than for NY-365 and is the highest for NY-363, correspondingly to the amount of the IP moieties (N) within nanoparticles (NNY363> NNY364 > NNY365 ). This is understandable, assuming that the average amount of the nanoparticles uptaken by cells is similar for all formulations. Figure 6 is a plurality of micrographs showing cellular uptake Colon-26 cells treated overnight with NY-363 (A), NY-364 (B), NY-365 (C). Transmission (above) and fluorescence (below) channels are shown. Confocal pinhole and PMT gain remained same during imaging. [0078] A structural representation of a nanoparticle in accordance with the invnetion is shown in Figure 7. As shown in Figure 7, a nonoparticle 10 is shown having linked Ri and R₂ groups where Ri = labeled photosensitizer (IP) or unlabeled photosensitizer (P), cyanine dye, SPECT, or PET, MR or fluorescent imaging agent and R₂ is -OH, -COOH, -NH, cyanine dye, SPECT, PET, MR or fluorescent imaging agent, linked targeting agent RGD, F3 peptide, carbohydrate or folic acid or labeled photosensitizer (EP) or unlabeled photosensitizer (P) where a plurality of R₂ groups, R₂ groups or mixtures thereof are photosensitizer and/or labeled photosensitizer where Rj is at least 80 percent embedded within the nanoparticle and R3 is at least 40 percent exposed at the surface of the nanoparticle.

[0079] As described above, synthesis of highly monodispersed aqueous dispersion of the ORMOSIL nanoparticles with covalently incorporated photosensitizer molecule is shown. Photophysical characterization has shown that the spectroscopic and functional (generation of cytotoxic singlet oxygen) properties of the photosensitizer moieties are preserved in their 'nanoconjugated' state. These nanoparticles are also avidly uptaken by tumor cells in culture, thus demonstrating the potential of these nanoparticles for PDT. In addition, it is also possible to chemically replace the iodine atom of the iodinated photosensitizer (EP) with a radiolabeled Iodine atom (e.g. 1-124, 125 etc), thus converting these nanoparticles as contrast agents for PET/SPECT imaging, while preserving their therapeutic functionality. [0080] Chemical modification of existing photosensitizers, to provide water solubility is described as is chemical conjugation of targeting group photosensitizer to existing photosensitizers, to increase the accumulation selectivity to diseased tissues.

[0081] Similarly, in accordance with the invention highly stable aqueous formulations of organically modified silica (ORMOSIL) nanoparticles encapsulating the hydrophobic photosensitizer HPPH [2-devinyl-2-(l-hexyloxyethyl)pyropheophorbide] have been made. This encaphotosensitizerulated photosensitizer is also able to generate singlet oxygen upon photoactivation owing to the free diffusion of molecular oxygen across the ORMOSIL matrix (Roy, I. et al., Ceramic-based nanoparticles entrapping water-insoluble photosensitizing anticancer drugs: a novel drug-carrier system for photodynamic therapy. J Am Chem Soc 2003, 125, (26), 7860-5). Again, because of mesoporosity of the ORMOSIL matrix, encaphotosensitizerulation of photosensitizer does not exclude the photosensitizer release, at least partially, during systemic circulation.

[0082] Again, in order to circumvent this problem, in accordance with the present invention, a modified formulation of the nanoparticles is provided with the photosensitizer molecule being covalently linked, instead of just being physically encaphotosensitizerulated. [0083] In accordance with the invention, nanoparticle conjugated (covalently linked) photosensitizers have been demonstrated having simple preparation that eliminate the possibility of premature release of the photosensitizer to unwanted sites in vivo. Additionally, the invention permits ease of active targeting by attaching targeting grouphotosensitizer on the particle surface [0084] As an additional option, the composite contains a covalently linked radioactive atoms for PET/SPECT imaging or magnetic resonance imaging contrast agents (i.e gadolinium).

Claims

What is claimed is:

1. A nano particle having the structural formula:

where the ring represents a silicone polymer matrix, where R₄ is (Ri)_n-R₂ -(Rs)_n where Ri is a labeled photosensitizer (IP) or unlabeled photosensitizer (P), cyanine dye, SPECT imaging agent, PET imaging agent, MR imaging agent or fluorescent imaging agent at least partially available at a surface of the silicone polymer matrix; R₂ is -O-, -COO-, -NR5 or -NH- connected to the silicone polymer matrix directly or through an intermediate group, n is 0 or 1; provided that at least one n is 1 and R₃ is cyanine dye, SPECT, PET, MR or fluorescent imaging agent, linked targeting agent RGD, F3 peptide, carbohydrate or folic acid or labeled photosensitizer (IP) or unlabeled photosensitizer (P) embedded in the silicone polymer matrix, and R5 is lower alkyl of from 1 to 5 carbon atoms where a plurality of Ri groups, R₃ groups or mixtures thereof are photosensitizer and/or labeled photosensitizer.

2. The nanoparticle of claim 1 where the intermediate group is subtituted or unsubstituted alkylene or phenylene.

3. The nanoparticle of claim 2 where the alkylene or phenylene is substituted with at least one hydroxy, carboxy, amino, sulfo, alkylester, alkylether, heterocyclo, or halo group.

4. The nanoparticle of claim 1 where at least one Ri or R₃ group is a tetrapyrollic photosensitizer selected from the group consisting of porphyrins, chlorins, bacteriochlorins, benzochlorins, benzoporphyrins, pheophorbides including pyropheophorbides, and derivatives thereof.

5. The nanoparticle of claim 1 where at least one Ri or R₃ group is selected from the group consisting of phthalocyanine, naphthanocyanine and derivatives thereof.

6. The nanoparticle of claim 1 where at least one of Ri and R₃ is a radionuclide MR or fluorescencent imaging agent.

7. A nanoparticle of claim 1 where a plurality of Ri groups are photosensitizers located at peripheral positions on the nanoparticle and a plurality of Ri groups are imaging agents located at peripheral positions on the nanoparticle.

8. A nanoparticle of claim 1 wherein it is provided with biotargeting molecules following suitable surface functionalization to obtain target-specific nanoparticles.

9. The nanoparticle of claim 8 wherein the biotargeting molecule is an antibody and the suitable surface functionalization is a ligand.

10. A nanoparticle of claim 1 further including at least one diagnostic agent.

11. A method for forming such nanoparticles includes the steps of : a) forming a uniform medium comprising from about 70 to about 80 weight percent of a lower alcohol selected from isopropanol, n-butanol, isobutanol and n-pentanol, from about 20 to about 30 weight percent of DMSO, from about 2 to about 3 percent water and from about 0.05 to about 0.15 percent of sufficient surfactant to maintain a dispersion; b) uniformly incorporating one or more silanes, as above described wherein the amount of silane or mixtures of silanes is about the maximum permitted for stability; c) adding sufficient reactive basic compound to form nanoparticles having reactive hydroxyl, amino, mercapto and/or carboxy groups exposed at their surface; d) dialyzing the nanoparticles through a membrane having a pore size of from about 0.1 to about 0.3 μM; e) during step b) or prior to step d), reacting a photosensitizer with a reactive group on the surface of the nanoparticles.

12. The method of claim 11 where the surfactant is a polyoxyethylene sorbitan monooleate or sodium dioctyl sulfosucinate.

13. The method of claim 11 where the silane is vinyltrimethoxysilane, vinyltriethoxysilane, vinylytriacetosilane, γ-glycidoxypropyltrimethoxysilane, γ- methacryloxypropyltrimethoxysilane, γ-aminopropyltrimethoxysilane, γ- aminopropyltriethoxysilane, γ-mercaptopropyltrimethoxy-silane, y-3,4- epoxycyclohexyltrimethoxysilane, phenyltrimethoxysilane or mixtures thereof.

14. The method of claim 1 where the silane is vinyltriethoxysilane or phenyltrimethoxysilane and the basic compound is ammonia or 3-aminopropylethoxysilane.

15. The method of claim 11 where the photosensitizer is a tetrapyrrole-based photosensitizing compound selected from the group consisting of porphyrins, chlorins, bacteriochlorins and derivatives thereof.

16. The method of claim 15 where the photosensitizer is selected from the group consisting of benzochlorins, benzoporphyrins, pheophorbides, pyropheophorbides, phthalocyanines, naphthanocyanines and derivative thereof.

17. The method of calim 11 where a targeting agent selected from the group consisting of monoclonal antibodies, integrin-antagonists and carbohydrates having high affinity for target tissue is reacted with at least one of the surface reactive groups.

18. A method for treating hyperproliferative tissue in an organism by infusing a compound according to claim 1 into the organism and exposing the hyperproliferative tissue to light at an activating wavelength of the photosensitizer.

19. A method for imaging hyperproliferative tissue in an organism by infusing a compound according to claim 1 having an incorporated imaging agent into the organism and imaging using the incorporated imaging agent.

20. The nanoparticle of claim 1 where the photosensitizer has the structure:

and its complexes with X where:

R, is -CH=CH₂, -CH₂CH₃, -CHO, -COOH, or

^H3^C\ /^R9

where R₉ = -ORi₀ where Rio is lower alkyl of 1 through 8 carbon atoms, -(CH₂-O)_nCH₃, -(CH₂)₂CO₂CH₃, -(CH₂)₂CONHphenyleneCH₂DTPA,

-CH₂CH₂CONH(CONHphenyleneCH₂DTPA)₂ , -CH₂Rn or

R

fluorescent dye moiety; R₂, R_2a, R₃, R_3a, R4, R5, Rsa, R7, and R_7a are independently hydrogen, lower alkyl or substituted lower alkyl or two R₂, R₂₃, R₃, R_3a, R5, Rs_a, R₇, and R₇₃ groups on adjacent carbon atoms may be taken together to form a covalent bond or two R₂, R_2a, R_3j R_3a, R₅, Rs_a, R₇, and R_7a groups on the same carbon atom may form a double bond to a divalent pendant group; R₂ and R₃ may together form a 5 or 6 membered heterocyclic ring containing oxygen, nitrogen or sulfur; R$ is -CH₂-, -NRn- or a covalent bond; Rg is -(CH₂)₂CO₂CH₃, -(CH₂)₂CONHphenyleneCH₂DTPA, -CH₂CH₂CONH(CONHphenyleneCH₂DTPA)₂, -CH₂Rn or _QJ_C where

^R 1 i ^"N^{" R} i 1

Rn is -CH₂CONH-RGD-Phe-Lys, -CH₂NHCO-RGD-PhC-LyS, a fluorescent dye moiety, or -CH₂CONHCH₂CH₂SO₂NHCH(CO₂)CH₂NHCOPhenylOCH₂CH₂NHcycloCNH(CH₂)₃N; and polynuclide complexes thereof; provided that the compound contains at least one integrin antagonist selected from the group consisting of -CH₂CONH-RGD-Phe-Lys, -CH₂NHCO-

RGD-Phe-Lys and

-CH₂CONHCH₂CH₂SO₂NHCH(CO₂)CH₂NHCOPhenylOCH₂CH₂NHcycloCNH(CH₂)₃N, where X is a metal selected from the group consisting of Zn, In, Ga, Al, or Cu or a radioisotope labeled moiety wherein the radioisotope is selected from the group consisting of ¹¹C, ¹⁸F, ⁶⁴Cu,

¹²⁴I₅ ⁹⁹Tc, ¹¹¹In and GdIII .

21. The nanoparticle of claim 1 where the photosensitizer is a tumor avid tetrapyrollic photosensitizer, a fluorescent dye, and an element X where X is a metal containing moiety selected from the group consisting of Zn, In, Ga, Al, or Cu or a radioisotope labeled moiety wherein the radioisotope is selected from the group consisting of ¹¹C, ¹⁸F, ⁶⁴Cu, ¹²⁴I, ⁹⁹Tc,

¹¹¹In and GdIII.