WO2009126832A2 - Methods of permeabilizing and adding agents to cells - Google Patents

Abstract

A method of using photosensitizers to permeabilizing cells, and translocating therapeutic and/or imaging agents into cells.

Description

METHODS OF PERMEABILIZING AND ADDING AGENTS TO CELLS CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to United States Provisional Application No. 61/043,725, filed April 9, 2008, entitled "Methods of Permeabilizing and Adding Agents to Cells", the contents of which is hereby incorporated by reference herein in its entirety. FIELD

[001] Method of using photosensitizers to permeabilizing cells, and translocating therapeutic and/or imaging agents into cells, are described.

BACKGROUND

[002] Methods of introducing compounds, such as diagnostic and therapeutic compounds, to cells and tissues are widely known. In some instances, cells can be permeabilized by well-characterized molecular biology methods. Many of these methods, however, are not amenable to a clinical setting in which cells are permeabilized in a controlled manner to maintain cell viability. For example, techniques suitable for transferring of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, and the calcium phosphate precipitation method cannot be adapted to in vivo methods.

[003] Moreover, these methods generally result in a low efficiency of cell uptake into a cell. For example, electroporation (passing cells between electrodes accelerated to very high voltage) typically has approximately 10% efficiency for introducing compounds to a cell in vitro and is clearly not safe in vivo, providing limited usefulness.

[004] Controlling transport of therapeutic compounds through biological barriers provides one basis for selectively delivering the therapeutic and/or imaging agent to the intended target. Strategies for selective transport include use of a targeting component that directs the therapeutic/imaging agent to a specific cell surface molecule, which is then internalized via regulated cellular transport mechanisms. One method includes use of antibodies selective for a unique cell surface antigen or use of ligands selective for a receptor expressed on the surface of the targeted cell. For example, if a cell population expresses a unique cell surface marker, an antibody can be raised against the unique marker and the therapeutic agent linked to the antibody. Upon administration of the antibody-drug complex, the binding of the antibody to the cell surface marker could result in the delivery of relatively high concentration of the drug to the cell. U.S. Patent No. 5,527,527 describes use of antibodies against the transferrin receptor while Pardridge, W.M. et al., Pharm. Res. 12: 807- 816 (1995) describes use of human insulin receptors for this purpose. Inclusion of antibodies into drug delivery vehicles, such as liposomes, also allows targeting of the drug delivery vehicle to specific cellular targets (see, e.g., U.S. Patent No. 5,858,382). The same concept applies to use of ligands and homing peptides that bind to cell surface receptors (see, e.g., U.S. Patent No. 5,442,043; 4902,505; 4,801 ,575; and 6,576,239). For example, the botulinum neurotoxin heavy chain can target to cholinergic motor neurons and may be used to deliver compounds to these cells (U.S. Patent No. 6,670,322). Even in these cases, the therapeutic/imaging agent remains attached to the membrane and the cell machinery for recycling or turning over the membrane keeps the therapeutic/imaging agent bound to a membrane trafficking mechanism and not directly soluble in the cell cytoplasm. This often causes low efficiency of agent uptake by the cell.

[005] Selective targeting approaches, however, require restricted presence of the cell surface marker on the cells being targeted for therapy. General expression of the cell surface antigen or receptor on non-targeted cells makes such targeted delivery less desirable while absence of specific markers on the cell surface severely limit this delivery strategy to only certain types of conditions or diseases.

[006] Another strategy to enhance selectivity of a therapeutic agent is the use of an inactive compound, for example a prodrug, which is converted to the active form by chemical modification. In this approach, endogenous enzymes are exploited to convert the prodrug to the active compound. Endogenous enzyme systems useful in the prodrug strategy include oxidoreductases (e.g., aldehyde oxidase, amino acid oxidase, cytochrome P450 reductase, DT-diaphorase) transferases (e.g., thymidylate synthase, thymidine phosphorylase, glutathione S-transferase), hydrolases (e.g., carboxylesterase, alkaline phosphatase, 0- glucuronidase), and lyases. Selectivity is obtained if expression of the endogenous enzyme is restricted to the tissues or cells being targeted for therapy. Variations of this approach include the delivery of non-endogenous enzymes to the target cell via an antibody ("ADEPT" or antibody-dependent enzyme prodrug therapy; U.S. Pat. No. 4,975,278) or introducing the gene encoding the non-endogenous enzyme into the targeted cells (e.g., gene dependent enzyme-prodrug therapy; see, e.g., Melton, R. G. and Sherwood, R. E., J Natl Cancer Inst. 88(3-4): 153-65. (1996)). Depending on the cells or tissues being targeted, examples of non- endogenous enzymes used for prodrug activation include nitroreductase cytochrome P450, purine-nucleoside phosphorylase, thymidine kinase, alkaline phosphatase, D-glucuronidase, carboxypeptidase, and cytosine deaminase.

[007] The advantage of using non-endogenous enzymes is that conversion of the prodrug does not occur except in those cells targeted by the antibody-enzyme complex or in cells modified by introduction of the enzyme-encoding gene. The use of catalytic antibodies as a non-endogenous enzyme has extended this approach for unique prodrug substrates (see, e.g., U.S. Patent No. 6,702,705). These strategies are effective if the prodrug or activated compound is itself capable of entering the targeted cell. Lack of permeability of the compounds can limit the use of these techniques.

[008] Mechanical means of permeabilizing cells include, osmotic shock, mechanical shearing, ultrasound to rupture membranes usually results in low success rates and high rates of cell death, often greater than 50% cell loss. Done in vitro this procedure offers lower risk to the subject but the entire procedure of removing stem cells and manipulating them before returning them to the subject is a high-risk procedure.

[009] Methods of photodynamic therapy have been developed to destroy cells. A photosensitizer that has entered a cell or tissue can be excited by light of a specific wavelength of radiation. Energy transfer between the photosensitizer and a proximal oxygen molecule allows the photosensitizer to relax to its ground state, and create an excited singlet state oxygen molecule that reacts rapidly with nearby molecules to cause cell death through apoptosis or necrosis. Such therapy is used to treat cancer and other diseases. However, photosensitizers are often lipid-soluble, and do not enter the cell selectively. Moreover, these methods are designed to destroy cells by activation of the photosensitizer. For example, when hematoporphyrin (C₃₄H₃₈O₆N) is used in anti-tumor therapy, the hematoporphyrin readily diffuses through cell membranes of all cells in a specific location. Only one of four hematoporphyrin derivatives (HPD) is photodynamic. Light incident on the hemaptoporphorin induces cell death of and healthy cells. Doctors rely on increased metabolic activity of cancer cells to enhance accumulation of HPD to provide greater lethality to cancer cells than to normal cells when illumination provides phototoxicity to HPD.

[0010] Methods of selectively introducing photosensitizers to cells and methods of selectively introducing diagnostic or therapeutic agents to cells by permeabilizing are need. Methods of introducing photosensitizers to cells, while maintaining cell viability, are also needed.

[0011] Against this backdrop, the disclosure has been developed.

SUMMARY

[0012] In one aspect, a method of translocating a photosensitizer into a cell is provided. A photosensitizer is provided proximal to the cell. Radiation at an absorption wavelength of the photosensitizer is applied to the cell. The photosensitizer translocates across the cell membrane into the cell to form a photosensitizer-containing cell. The method according to claim 1 , wherein the photosensitizer is Lucifer yellow CH.

[0013] In another aspect, methods of translocating a therapeutic and/or imaging agent into a cell is provided. The photosensitizer is translocated into the cell to form a photosensitizer- containing cell. A therapeutic and/or imaging agent is provided proximal to the photosensitizer-containing cell. Radiation is applied at an absorption wavelength of the photosensitizer to rupture the cell membrane from the inside and allow the therapeutic and/or imaging agent to translocate directly into the cell cytoplasm.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 depicts confluent cells that covered the entire area of the culture dish.

[0015] Figure 1B depicts Lucifer yellow-containing cells after activation with light radiation.

[0016] Figure 1C depicts the red-dye labeled albumin containing cells after activation of Lucifer yellow-containing cells.

[0017] Figure 2 depicts and electron micrograph showing the photosensitizer Lucifer yellow incorporated into the cell.

[0018] Figures 3A-3C depict the viability of photosensitizer-containing cells.

[0019] Figure 4 depicts a magnetic field measurement showing translocation of phenyl- tertiary butyl nitrone (PBM) in the presence of oxygen, and no translocation in the absence of oxygen in Lucifer yellow-containing cells.

DETAILED DESCRIPTION

[0020] In one aspect, the present disclosure provides methods for the controlled delivery of photosensitizers and/or therapeutic/imaging agents into target cells. The compositions herein exploit the ability of classes of photosensitizers that permeabilize cells to macromolecules while maintaining cell viability. Light activation of photosensitizers proximal to a cell results in the translocation of the photosensitizer into the cell. Subsequently, light activation of photosensitizers in the cell results in translocation of therapeutic and/or imaging agents such as small organic molecules, proteins, or nucleic acids into the cell. By light activating photosensitizer-containing cells, delivery of a therapeutic and/or imaging agentis generally confined to target cells.

[0021] In various aspects, the disclosed methods provide direct entry of agents into the cytoplasm without using toxic or low-yield permeabilization methods such as electroporation. The methods do not require use of targeting components such as cell targeting sequences or a viral vector to obtain entry to the cytoplasm.

Photosensitizers

[0022] As referred to herein, a "photosensitizer" is a chemical compound that absorbs light radiation and transfers its energy to other molecules. In various embodiments, photosensitizers maintain cell viability when the photosensitizer in the cell is activated. By "activate" or "activation" is meant applying light radiation to a photosensitizer at an absorbing wavelength of the photosensitizer. The absorption wavelength can be any wavelength of the photosensitizer within the absorption spectrum. In various embodiments, the photosensitizer can be the free base of the compound, or a salt form. The absorption wavelength can be in any radiation spectrum, including the visible spectrum, ultraviolet spectrum, or infrared spectrum.

[0023] In various embodiments, the photosensitizers described herein are not membrane permeable in the absence of light activation. The photosensitizers generally have a relatively hydrophilic solubility relative to cell membranes, and are not soluble in the lipid portion of a cell membrane in the absence of a stimulus such as light radiation. The cell membrane can thus be controlled by light activation, without unintended translocation across the cell membrane.

[0024] A non-limiting list of exemplary photosensitizers that can be used as described herein is provided in Table 1.

Table 1

[0025] In various embodiments, many photosensitizers can transfer energy to dissolved oxygen and generate a singlet oxygen excited state. Without wishing to be limited to a particular theory or mechanism of action, in various embodiments, photosensitizers produce oxide radicals (superoxide, hydroxide radicals, singlet oxygen etc.). Oxide radicals are highly reactive species that chemically oxidize surrounding molecules. Without wishing to be limited to a particular theory or mechanism, when the photosensitizer is proximal to a cell membrane (whether inside or outside the cell), light activation damages the structure forming holes in the cell membrane. The pores are large enough to allow of proteins larger than albumen, including enzymes, signaling molecules and nucleic acids across the membrane and directly into the cell cytoplasm without passing through a membrane bound organelle recycling mechanism. In certain embodiments, the pores are large enough to allow translocation of photosensitizers or agents through the pore by diffusion. In various aspects, the hole in the membrane persists for sufficient time to allow compounds to enter the cytoplasm of the cell, before reannealing and without resulting in cell death.

[0026] In certain embodiments, the photosensitizer is stable under physiological conditions for extended periods of time. For example, certain photosensitizers have a permanent negative charge at physiological pH. As a non-limiting example, Lucifer yellow has a permanent negative charge from pH 0-10 inside cells in vitro. Lucifer yellow as a photosensitizer can thus stay in the cytoplasm for a period of many days without apparent loss to the surround and without toxicity to the cell.

[0027] In various embodiments, photosensitizers can be linked covalently to a targeting agent. The photosensitizer can be lined to the targeting agent covalently, and can be accomplished by methods known to those of skill in the art. The linkage can be direct, or indirect, such as via a linker.

[0028] Targeting agents can include any number of compounds known in the art. In certain situations, the targeting agent specifically binds to a particular biological target. Nonlimiting examples of biological targets include tumor cells, bacteria, viruses, cell surface proteins, cell surface receptors, cell surface polysaccharides, extracellular matrix proteins, intracellular proteins and intracellular nucleic acids.

[0029] The targeting agents described herein are not limited to any particular targeting agent, and a variety of targeting agents can be used. The targeting agents can be, for example, various specific ligands, such as antibodies, monoclonal antibodies and their fragments, folate, mannose, galactose and other mono-, di-, and oligosaccharides, and RGD peptide. Examples of such targeting agents include, but are not limited to, nucleic acids

(e.g., RNA and DNA), polypeptides (e.g., receptor ligands, signal peptides, avidin, Protein A, and antigen binding proteins), polysaccharides, biotin, hydrophobic groups, hydrophilic groups, drugs, and any organic molecules that bind to receptors. In some instances, a photoactivator described herein can be linked to one, two, or more of a variety of targeting agents. For example, when two or more targeting agents are used, the targeting agents can be similar or dissimilar. Utilization of more than one targeting agent in a particular nanoparticle can allow the targeting of multiple biological targets or can increase the affinity for a particular target.

[0030] In some instances, the targeting agents are antigen binding proteins or antibodies or binding portions thereof. Antibodies can be generated to allow for the specific targeting of antigens or immunogens (e.g., tumor, tissue, or pathogen specific antigens) on various biological targets (e.g., pathogens, tumor cells, normal tissue). Such antibodies include, but are not limited to, polyclonal antibodies; monoclonal antibodies or antigen binding fragments thereof; modified antibodies such as chimeric antibodies, reshaped antibodies, humanized antibodies, or fragments thereof (e.g., Fv, Fab¹, Fab, F(ab')2); or biosynthetic antibodies, e.g., single chain antibodies, single domain antibodies (DAB), Fvs, or single chain Fvs (scFv).

[0031] Methods of making and using polyclonal and monoclonal antibodies are well known in the art, e.g., in Harlow et ah, Using Antibodies: A Laboratory Manual: Portable Protocol I. Cold Spring Harbor Laboratory (December 1 , 1998). Methods for making modified antibodies and antibody fragments (e.g., chimeric antibodies, reshaped antibodies, humanized antibodies, or fragments thereof, e.g., Fab¹, Fab, F(ab')2 fragments); or biosynthetic antibodies (e.g., single chain antibodies, single domain antibodies (DABs), Fv, single chain Fv (scFv), and the like), are known in the art and can be found, e.g. , in Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives, Springer Verlag (December 15, 2000; 1st edition). In some instances, the antibodies recognize tumor specific epitopes (e.g., TAG-72 (Kjeldsen et al, Cancer Res., 48:2214-2220 (1988); U.S. 5,892,020; 5,892,019; and 5,512,443); human carcinoma antigen (U.S. 5,693,763; 5,545,530; and 5,808,005); TPI and TP3 antigens from osteocarcinoma cells (U.S. 5,855,866); Thomsen-Friedenreich (TF) antigen from adenocarcinoma cells (U.S. 5,110,911); "KC -4 antigen" from human prostrate adenocarcinoma (U.S. 4,708,930 and 4,743,543); a human colorectal cancer antigen (U.S. 4,921 ,789); CA125 antigen from cystadenocarcinoma (U.S. 4,921 ,790); DF3 antigen from human breast carcinoma (U.S. 4,963,484 and 5,053,489); a human breast tumor antigen (U.S. 4,939,240); p97 antigen of human melanoma (U.S. 4,918,164); carcinoma or orosomucoid-related antigen (CORA)

(U.S. 4,914,021 ); a human pulmonary carcinoma antigen that reacts with human squamous cell lung carcinoma but not with human small cell lung carcinoma (U.S. 4,892,935); T and Tn haptens in glycoproteins of human breast carcinoma (Springer et ah, Carbohydr. Res., 178:271- 292 (1988)), MSA breast carcinoma glycoprotein (Tjandra et al, Br. J. Surg., 75:811-817 (1988)); MFGM breast carcinoma antigen (Ishida et al, Tumor Biol, 10: 12-22 (1989)); DU-PAN-2 pancreatic carcinoma antigen (Lan et al, Cancer Res., 45:305-310 (1985)); CA125 ovarian carcinoma antigen (Hanisch et ah, Carbohydr. Res., 178:29-47 (1988)); and YH206 lung carcinoma antigen (Hinoda et al, Cancer J., 42:653-658 (1988)). For example, to target breast cancer cells, the nanoparticles can be modified with folic acid, EGF, FGF, and antibodies (or antibody fragments) to the tumor-associated antigens MUC 1 , cMet receptor and CD56 (NCAM).

[0032] Other antibodies that can be used recognize specific pathogens (e.g., Legionella peomophilia, Mycobacterium tuberculosis, Clostridium tetani, Hemophilus influenzae, Neisseria gonorrhoeae, Treponema pallidum, Bacillus anthracis, Vibrio cholerae, Borrelia burgdorferi, Comebacterium diphtheria, Staphylococcus aureus, human papilloma virus, human immunodeficiency virus, rubella virus, and polio virus).

[0033] In some instances, the targeting agents include a signal peptide. These peptides can be chemically synthesized or cloned, expressed and purified using known techniques. Signal peptides can be used to target the nanoparticles described herein to a discreet region within a cell. In some situations, specific amino acid sequences are responsible for targeting the nanoparticles into cellular organelles and compartments. For example, the signal peptides can direct a nanoparticle described herein into mitochondria. In other examples, a nuclear localization signal is used.

[0034] In other instances, the targeting agent is a nucleic acid (e.g., RNA or DNA). In some examples, the nucleic acid targeting agents are designed to hybridize by base pairing to a particular nucleic acid (e.g., chromosomal DNA, mRNA, or ribosomal RNA). In other situations, the nucleic acids bind a ligand or biological target. For example, the nucleic acid can bind reverse transcriptase, Rev or Tat proteins of HIV (Tuerk et al, Gene, 137(l):33-9 (1993)); human nerve growth factor (Binkley et al, Nuc. Acids Res., 23(16):3198-205 (1995)); or vascular endothelial growth factor (Jellinek et al, Biochem., 83(34): 10450-6 (1994)). Nucleic acids that bind ligands can be identified by known methods, such as the SELEX procedure (see, e.g., U.S. 5,475,096; 5,270,163; and 5,475,096; and WO 97/38134; WO 98/33941 ; and WO 99/07724). The targeting agents can also be aptamers that bind to particular sequences.

[0035] The targeting agents can recognize a variety of epitopes on biological targets (e.g., pathogens, tumor cells, or normal cells). For example, in some instances, the targeting agent can be sialic acid to target HIV (Wies et al , Nature, 333:426 (1988)), influenza (White et al, Cell, 56:725 (1989)), Chlamydia (Infect. Immunol, 57:2378 (1989)), Neisseria meningitidis, Streptococcus suis, Salmonella, mumps, newcastle, reovirus, Sendai virus, and myxovirus; and 9-OAC sialic acid to target coronavirus, encephalomyelitis virus, and rotavirus; non-sialic acid glycoproteins to target cytomegalovirus (Virology, 176:337 (1990)) and measles virus (Virology, 172:386 (1989)); CD4 (Khatzman et al, Nature, 312:763 (1985)), vasoactive intestinal peptide (Sacerdote et al, J. of Neuroscience Research, 18: 102 (1987)), and peptide T (Ruff et al, FEBS Letters, 211 : 17 (1987)) to target HIV; epidermal growth factor to target vaccinia (Epstein et al , Nature, 318: 663 (1985)); acetylcholine receptor to target rabies (Lentz et al, Science 215: 182 (1982)); Cd3 complement receptor to target Epstein- Barr virus (Carel et al, J. Biol. Chem., 265: 12293 (1990)); .beta, -adrenergic receptor to target reovirus (Co et al, Proc. Natl. Acad. ScL USA, 82: 1494 (1985)); ICAM-I (Marlin et al, Nature, 344:70 (1990)), N-CAM, and myelin-associated glycoprotein MAb (Shephey et al, Proc. Natl. Acad. ScL USA, 85:7743 (1988)) to target rhinovirus; polio virus receptor to target polio virus (Mendelsohn et al, Cell, 56:855 (1989)); fibroblast growth factor receptor to target herpes virus (Kaner et al, Science, 248: 1410 (1990)); oligomannose to target Escherichia coli; and ganglioside GMI to target Neisseria meningitides.

[0036] In other embodiments, the targeting agent may be an aptamer.

Importing Photosensitizers to Cells

[0037] Importing a photosensitizer into a target cell or tissue can be a two step process. First, the photosensitizer is administered to a target cell or tissue. Second, light radiation is applied to the cell to allow translocation the photosensitizer across the target cell membrane. In various embodiments, excess photosensitizer can be removed from the surrounding target cell (e.g., removed from the organ or subject), and eliminated from the animal while the photosensitizer stays inside target cells for later manipulation.

[0038] "Target" cells include any cell being targeted for delivery of a photosensitizer, or delivery of a therapeutic and/or imaging agent. Such cells include, among others, hyperproliferative cells that are de-differentiated, immortalized, neoplastic, malignant, metastatic or transformed. Examples include, but are not limited to cancer cells such as sarcoma cells, leukemia cells, carcinoma cells, basal cells, or adenocarcinoma cells.

Specified cancer cells include, but are not limited to, breast cancer cells, lung cancer cells, brain cancer cells, hepatoma cells, liver cancer cells, pancreatic carcinoma cells, oesophageal carcinoma cells, bladder cancer cells, gastrointestinal cancer cells, ovarian cancer cells, skin cancer cells, prostate cancer cells, and gastric cancer cells. Likewise, "target" tissues include any cell being targeted for delivery of a photosensitizer or delivery of a therapeutic and/or imaging agent.

[0039] The photosensitizer may be administered in a convenient manner such as by systemic injection (subcutaneous, intravenous, etc.), tissue or cell specific injection, oral administration, parenterally, intraperitoneally, inhalation, transdermal application, or rectal administration. In various embodiments, the photosensitizer can be injected directly to a specific cell, cells or tissue. For example, methods of administering compounds such as cells or viral vectors directly to tumors have been accomplished in other contexts. For example, cells have been administered to a tumor site by injection Rodriguez-Madoz et al., Molecular Therapy (2005) 12, 153-163, incorporated by reference herein in its entirety.

[0040] In various embodiments, the photosensitizer can be provided to contact a desired tissue (e.g., a tumor tissue), or proximal to the location of a desired cell or tissue. By "proximal to" is meant within an effective distance of the cell or tissue, such that the photosensitizer will reach the cell or tissue directly and can translocate across the cell membrane with the application of light radiation. For example, a photosensitizer that generates singlet oxygen when photo-excited in a water solution will have limited effect on distant cells because singlet oxygen will be quenched by the water solvent withing a matter of microseconds, allowing the energetic oxygen species to diffuse only a matter of a few microns from the site where it was generated.

[0041] In certain embodiments, the photosensitizers can be administered to a subject in a biologically compatible form suitable for pharmaceutical administration in vivo. The term "subject" includes living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Administration of a therapeutic and/or imaging agent as described herein can be in any pharmacological form, optionally in combination with an additional cell therapeutic agent, or a pharmaceutically acceptable carrier.

[0042] Administering the photosensitizer can be accomplished in a specific dosage and/or for periods of time necessary to achieve import into the cell. As used herein, "dosage" and "dose" are used interchangeably. A dosage of a photosensitizer may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the photosensitizer to be administered to the cytoplasm of a cell, or cells in a tissue. A dosage regimen may be adjusted to provide a specific amount of photosensitizer to a specific cell.

[0043] In one embodiment, a photosensitizer dose is administered at a high concentration, but confined to the immediate vicinity of the target cells. Activation induces cell membrane rupture, and the photosensitizer enters a cell at a high concentration. Excess photosensitizer not inside the cell can be removed, for example by rinsing, flushing, or allowing the subject to carry a restricted volume of photosensizer away and out of the subject. At a later stage, a desired agent may be imported into the target cell. [0044] The concentration of photosensitizer may be selected to translocate into target cells at a greater rate.

[0045] The dose of photosensitizer can be calibrated to the amount of photosensitizer desired for diffusion into the cell. For example, standard curves can be used to determine the intensity and amount of activation time to attain cell permeabilization.

[0046] The dosage unit form may be dependent on the characterisitics of a specific photosensitizer. The specific dose can be readily calculated by one of ordinary skill in the art, e.g., according to the approximate body weight or body surface area of the subject or the volume of body space occupied, size of a tumor or proximity to the point of introduction and delivery. The dose may be calculated dependent on the particular route of administration selected.

[0047] The photosensitizer may also be administered in a pharmaceutically acceptable formulation. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils, ionic liquids, or nonpolar solvents that extend oxide radical lifetimes such as bromonaphthalene, propionic acid, diphenyl sulfide, ionic liquids, etc.

Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0048] In the case of photosensitizers that may be toxic to cells or tissues, the photosensitizer dosage can be selected to minimize toxic effects while maintaining translocation into the membrane. The toxicity of the photosensitizer can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxicity and cell uptake (administration and cell uptake) is the dosage index and can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While photosensitizers that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of desired cells and/or tissues in order to minimize potential damage to other cells or tissues and, thereby, reduce side effects.

[0049] In other embodiments, photosensitizers may be administered to a specific cell, cells or group of tissues. For example, the photosensitizer can be administered by injection or microinjection to a cell or group of cells. The photosensitizer can migrate between cells across gap junctions. [0050] In certain embodiments, a plurality of photosensitizers may be added to the cell. In certain variations, different photosensitizers may have different properties, such as absorption wavelength or efficiency of active oxide generation.

Translocating a photosensitizer across a target cell membrane

[0051] After the photosensitizer is administered to the cell, light radiation is applied to photosensitizer to translocated across the cell membrane. Light radiation can be applied in any method known in the art. In various embodiments, the light radiation can be directed to specific tissues or cells in a localized manner to facilitate translocation for specific cells without affecting adjoining cells.

[0052] Light radiation may be applied by any method known in the art. In various embodiments, light radiation may be applied to the photosensitizer-containing cell or tissue by an endoscopes or fiber optic catheter. Any method of applying radiation known in the art may be provided.

Radiation may be applied to internal organs, as well as larger tumor tissues or metastatic tumor tissue. A specific wavelength of radiation may be provided to specific cell or group of cells.

[0053] Photosensitizer-containing cells can maintain viability. By "viability" is meant the cells are in a living state. In various aspects, cell viability may be maintained for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 21 days, or 30 days.

Administering a therapeutic and/or imaging agent to a photosensitizer-containing cell or tissue

[0054] A therapeutic and/or imaging agent is then administered to a photosensitizer- containing cell or tissue. Agents can include any compound known in the art. In particular embodiments, the therapeutic/imaging agent can be a diagnostic or therapeutic agent. In various embodiments, the therapeutic/imaging agent can be a small organic molecules, macrocylic compounds, nucleotides, nucleic acids, peptides, proteins, and carbohydrates, as described herein below.

[0055] Agents may be translocated into the cell by applying light radiation to the photosensitizer-containing cell. The light radiation may be applied by any method known in the art, as described above for translocating photosensitizers into the cell. In various embodiments, light radiation may be applied to the photosensitizer-containing cell or tissue by an endoscope or fiber optic catheter. Radiation may be applied to cells in internal organs, as well as larger tumor tissues or metastatic tumor tissue. Without being limited to a specific theory or mechanism of action, activation of the photosensitizer in the cell creates pores in the cell membrane from inside the cell. The therapeutic/imaging agent can then enter the cell.

[0056] The photosensitizer-containing cell can be used to translocate a therapeutic and/or imaging agent into a target cell or target tissue. Agents include, among others, small organic molecules, macrocylic compounds, nucleotides, nucleic acids, peptides, proteins, and carbohydrates.

[0057] Translocation of the therapeutic/imaging agent into the cell can be accomplished with higher efficiency than other methods of cell import. In various embodiments, the therapeutic/imaging agent can be translocated into the cell with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% translocation. The viability of the cell may be maintained as described herein.

Types of Agents

[0058] In various aspects, the methods described herein may include a number of different types of agents.

Small Organic Molecules

[0059] In one aspect, the therapeutic/imaging agent comprises a small organic molecule. As used herein, small organic molecules refers to molecules of about 200 to about 2500 daltons, although it may be larger depending on the compound. The organic compounds typically comprise functional groups, for interacting covalently or non-covalently with biological molecules. Functional groups include, for example, amines, carbonyl, hydroxyl, or carboxyl groups. The organic compounds often comprise cyclical carbon or heterocyclic structures, and/aromatic or polyaromatic structure substituted with one or more functional groups. Such compounds may be antibiotics, small organic molecule drugs, nucleotides, amino acids, saccharides, fatty acids, steroids, dye molecules (see, e.g., Conn's Biological Stains, 10^th Ed. (Horobin, R.W.and Kiernan, J.A.), BIOS Scientific Publishers, Oxford, UK (2002), incorporated herein by reference), and derivatives thereof. Small organic molecules also encompass haptens recognized by antibodies or other proteins, and include, by way of example and not limitation, digoxigenin, dinitrophenol, biotin, oestradiol, fluorescein isothiocyanate (FITC), 3-nitro-4-hydroxy-5-iodophenylacetic acid (NIP), and the like.

[0060] A wide variety of agents, including bioactive compounds, flurochromes, dyes, metals and metal chelates may be delivered into the cell by use of the compositions described herein. Nucleic acids may also be delivered into the nucleus. "Bioactive" refers to a compound having a physiological effect on the cell as compared to a cell not exposed to the compound. A physiological effect is a change in a biological process, including, by way of example and not limitation, DNA replication and repair, recombination, transcription, translation, secretion, membrane turnover, cell adhesion, signal transduction, and the like. A bioactive compound includes pharmaceutical compounds.

[0061] Bioactive compounds suitable for delivery by the compositions herein, include, among others, chemotherapeutic compounds, including by way of example and not limitation, vinblastine, bleomycin, taxol, cis-platin, adriamycin, and mitomycin. Exemplary chemotherapeutic agents suitable for the present purposes are compounds acting on DNA synthesis and stability. For example, antineoplastic agents of the anthracyclin class of compounds act by causing strand breaks in the DNA and are used as standard therapy against cancer. Exemplary anti-neoplastic agents of this class are daunorubicin and doxorubicin. Providing antineoplastic agents proximal to the cell, the therapeutic/imaging agents may be translocated into the cell upon activation of the photosensitizer.

[0062] Other classes of antitumor agents are the enediyne family of antibiotics, representative members of which include calicheamicins, neocarzinostatin, esperamincins, dynemicins, kedarcidin, and maduropeptin (see, e.g., Smith, A.L. and Nicolaou, K.C., J. Med. Chem. 39:2103-2117 (1996)). Similar to doxorubicin and daunorubicin, the antitumor activity of these agents resides in their ability to create strand breaks in the cellular DNA.

[0063] In a further embodiment, the compounds are small molecule modulators of telomerase activity. These include, by way of example and not limitation, alterperynol, a fungal metabolite capable of inhibiting telomerase activity (Togashi, K. et al., Oncol. Res. 10:449-453 ((1998)); isothiazolone derivatives (Hayakawa, N. et al., Biochemistry 38:11501-11507 (1999)); rhodacyanine derivatives (Naasani, I. et al. , Cancer Res.

59:4004^011 (1999)); rubromycin (Ueno, T. et al., Biochemistry 39:5995-6002 (2000)); diazaphilonic acid (Tabata, Y. et al., J. Antibiot. 52:412-414 (1999)); 9-Hydroxyellipticine (Sato, N. et al., FEBS Lett. 441 :318-321 (1998)); and others known in the art.

[0064] In another embodiment, the small molecules comprise reporter compounds, particularly fluorescent, phosphorescent, radioactive labels, and detectable ligands. Useful fluorescent compounds include, by way of example and not limitation, fluorescein, rhodamine, TRITC, Coumadin, Cy5, ethidium bromide, DAPI, and the like. Suitable fluorescent compounds are described in Haughland, R.P., Handbook of Fluorescent Probes and Research Chemicals Eugene, 9^th Ed., Molecular Probes, OR (2003); incorporated herein by reference in its entirety). [0065] Agents can be modified to carry the radioactive molecule. Radioactive compounds are useful as signals (e.g., tracers) or used to provide a therapeutic effect by specific delivery to a cell targeted (e.g., in the form of radiopharmaceutical preparations). Radioactive nuclides include, by way of example and not limitation, ³H, ¹⁴C, ³²P, ³⁵S, ⁵¹Cr, ⁵⁷Co ⁵⁹Fe, ⁶⁷Ga, ⁸²Rb, ⁸⁹Sr, ⁹⁹Tc, ¹¹¹In, ¹²³1, ¹²⁵1, ¹²⁹1, ¹³¹I₁ and ¹⁸⁶Re.

[0066] In yet a further embodiment, the small organic molecules are chelating ligands, or macrocyclic organic chelating molecules, particularly metal chelating compounds used to image intracellular ion concentrations or used as contrast agents for medical imaging purposes. Chelating ligands are ligands that can bind with more than one donor atom to the same central metal ion. Chelators or their complexes have found applications as MRI contrast agents, radiopharmaceutical applications, and luminescent probes. Chelating compounds useful for assessing intracellular ion concentrations may be voltage sensitive dyes and non-voltage sensitive dyes. Exemplary dye molecules for measuring intracellular ion levels include, by way of example and not limitation, Quin-2; Fluo-3; Fura-Red; Calcium Green; Calcium Orange 550 580; Calcium Crimson; Rhod-2 550 575; SPQ; SPA; MQAE; Fura-2; Mag-Fura-2; Mag-Fura-5; Di-4-ANEPPS; Di-8-ANEPPS; BCECF; SNAFL-1 ; SBFI; and SBFI.

[0067] In another embodiment, the ligands are chelating ligands that bind paramagnetic, superparamagnetic or ferromagnetic metals. These are useful as contrast agents for medical imaging and for delivery of radioactive metals to selected cells. Metal chelating ligands, include, by way of example and not limitation, diethylenetriaminepenta acetic acid (DTPA); diethylenetriaminepenta acetic acid bis(methylamide); macrocyclic tetraamine 1 ,4,7,10-tetraazacyclododecane-N,N¹,N",N"'-tetraacetic acid (DOTA); and porphyrins (see, e.g., The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging, Merbach A.E. and Toth E. ,Ed., Wiley lnterscience (2001)). Paramagnetic metal ions, which are detectable in their chelated form by magnetic resonance imaging, include, for example, iron(lll), gadolinium(lll), manganese (Il and III), chromium(lll), copper(ll), dysprosium(lll), terbium(lll), holmium (III), erbium (III), and europium (III). Paramagnetic metal ions particularly useful as magnetic resonance imaging contrast agents comprise iron(lll) and gadolinium(lll) metal complexes. Other paramagnetic, superparamagnetic or ferromagnetic are well known to those skilled in the art.

[0068] In another embodiment, the metal-chelate comprises a radioactive metal. Radioactive metals may be used for diagnosis or therapy based on delivery of small doses of radiation to a specific site in the body. Targeted metalloradiopharmaceuticals are constructed by attaching the radioactive metal ion to a metal chelating ligand, such as those used for magnetic imaging, and targeted delivery of the chelate complex to cells. An exemplary radioactive metal chelate complex is DTPA (see, e.g., U.S. Patent No. 6,010,679, incorporated herein by reference in its entirety).

Nucleic Acids

[0069] In various embodiments, the therapeutic/imaging agent can be a nucleic acid or nucleic acid containing compound. The nucleic acid can have an associated nuclear localization signal to deliver agents into the target cell nucleus.

[0070] In one aspect, the therapeutic/imaging agents comprise nucleic acids, including oligonucleotides and polynucleotides. By "nucleic acid" or "oligonucleotide" or "polynucleotide refers to at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds. However, in some cases, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage, S.L. et al., Tetrahedron 49:1925-63 (1993); Letsinger, R.L. et al., J. Org. Chem. 35: 3800-03 (1970); Sprinzl, M. et al., Eur. J. Biochem. 81 :579-89 (1977); Letsinger, R.L. et al., Nucleic Acids Res. 14:3487-99 (1986); Sawai et al., Chem. Lett. 805 (1984); Letsinger, R.L. et al., J. Am. Chem. Soc. 110: 4470 (1988)), phosphorothioate (Mag, M. et al., Nucleic Acids Res. 19: 1437-41 (1991); and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111 : 2321 (1989)), O- methylphophoroamidite linkages (see, e.g., Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press (1991)), and peptide nucleic acid backbones and linkages (Egholm, M., Am. Chem. Soc. 114: 1895-97 (1992); Meier et al., Chem. Int. Ed. Engl. 31 :1008 (1992); Egholm, M., Nature 365: 566-68 (1993); Carlsson, C. et al., Nature 380: 207 (1996)). Other analog nucleic acids include those with positive backbones (Dempcy, R.O. et al., Proc. Natl. Acad. Sci. USA 92: 6097-101 (1995)); non-ionic backbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30: 423 (1991); Letsinger, R.L. et al., J. Am. Chem. Soc. 110: 4470 (1988); and Letsinger, R.L. et al., Nucleoside & Nucleotide 13: 1597 (1994)). All publications are hereby expressly incorporated by reference.

[0071] The nucleic acids may be single stranded or double stranded, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or hybrid, where the nucleic acid contains any combination of deoxyribo- and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, and any of known base analogs, including, but not limited to, 4- acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2- thiouracil, 5 carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracils,5-methoxyaminomethyl-2- thiouracil, beta-D-maninosylqueosine, δ'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2- methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5-methyl uracil, N-uracil-5-oxyacetic acid methylester, uracil-5- oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0072] In one aspect the nucleic acids comprise functional nucleic acids. By "functional nucleic acid" refers to any nucleic acid that is bioactive. A functional nucleic acid may have enzymatic function, regulate transcription of nucleic acids, regulate the translation of an mRNA so as to interfere with the expression encoded protein, or affect other physiological processes in the cell. Functional nucleic acids include, by way of example and not limitation, ribozymes, antisense nucleic acids, decoy oligonucleotide nucleic acids, and interfering RNAs (RNAi). These also include short interfering RNA (siRNA).

[0073] In one embodiment, the nucleic acids comprise anti-sense nucleic acids. As used herein, "anti-sense nucleic acids" comprise nucleic acids, particularly in the form of oligonucleotides, characterized as hybridizing to the corresponding complementary or substantially complementary nucleic acid strand to inhibit expression of the gene encoded by the complementary strand.

[0074] Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule. Alternatively, the antisense molecule is a synthetic oligonucleotide. Antisense oligonucleotides will generally be at least about 7, usually at least about 12, more usually at least about 20 nucleotides in length, and not more than about 500, usually not more than about 50, more usually not more than about 35 nucleotides in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. Generally, short oligonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see, e.g., Wagner et al., Nature Biotechnol. 14:840-844 (1996)).

[0075] Selection of a specific sequence for the oligonucleotide may use an empirical method, where several candidate sequences are assayed for inhibition of expression of the target gene in an in vitro or animal model. A combination of sequences may also be used, where several regions of the mRNA sequence are selected for antisense complementation. The antisense nucleic acids may be directed to any expressed protein, including, by way of example and not limitation, to transcription factors, kinases, phosphorylases, telomerases, receptors, etc.

[0076] In one embodiment, the antisense nucleic acids are directed against telomerase (Norton, J. C. et al., Nat. Biotechonol. 14 , 615-619 (1969); Pitts, A.E. and Corey, D.R., Proc. Natl. Acad. ScL U.S.A. 95, 11549-11554 (1998); Elayadi, A.N. et al., Nucleic Acids Res. 29, 1683-1689 (2001 ); Tao, M. et al., FEBS Lett. 454, 312-316 ((1999)). In another embodiment, the antisense oligonucleotides are directed against receptors and components of cell signaling pathways. Various antisense oligonucleotides have been developed against cell signaling components. Exemplary antisense nucleic acids, include, by way of example and not limitation, the antisense nucleic acid directed against RaM (Mullen, P. et al., CHn Cancer Res. 10(6):2100-2108 (2004), vascular endothelial zinc finger 1 (Vezfi ), a zinc finger transcription factor expressed in endothelial cells (ECs) during vascular development (Miyashita, H. et al., Arterioscler Thromb Vase Biol, epublication, Mar 18 (2004)); phosphorothioate antisense oligonucleotides to beta-catenin (Veeramachaneni, N. K. et al., J Thorac Cardiovasc Surg. 127(1):92-8 (2004)); and antisense oligonucleotides to Statδ transcription factors (Xi, S. et al., Cancer Res. 63(20):6763-71 (2003)). It is to be understood that other antisense nucleic acids may be delivered into cells by the compositions described herein.

[0077] In another aspect, the nucleic acids are decoy oligonucleotides (ODN). The basis of the ODN decoy approach involves introducing into the cell a competing synthetic, transcription factor-specific consensus sequences or sequences that interact with other nucleic acid binding proteins. These synthetic decoys "compete" for binding of the protein (e.g., transcription factor) with consensus sequences in target genes. If delivered into the cell in sufficient concentrations these "decoys" have the potential to attenuate the binding of the nucleic acid binding protein, for example binding of transcription factors to promoter regions of target genes and thus attenuate the function of the protein to regulate the expression of its target gene(s). Generally, the decoy nucleic acids will comprises a minimal sequence bound by the nucleic acid binding protein. Transfected at high concentrations these decoys are shown to block activities of the nucleic acid binding proteins (see, e.g., Mann, MJ. and Dzau, V.J., J Clin Invest. 106(9): 1071 -5 (2000)).

[0078] Thus in one embodiment, the sequences of the decoy nucleic acids are the sequences bound by a transcription factor. Exemplary ODN nucleic acid sequences have been described for transcription factors, including, by way of example and not limitation, NF- kB (nuclear factor-kappaB) (Sharrna, H.W. et al., Anticancer Res. 16(1):61-9 (1996)); transcription factor E2F (Morishita, R. et al., Proc Natl Acad Sci USA 92(13):5855-9 (1995)); negative regulatory element (NRE) for the renin gene; angiotensinogen gene-activating element (AGE) for the angiotensinogen gene; and for the TERT Site C repressor protein, which inhibits expression of telomerase (U.S. Patent No. 6,686,159; Morishita et al., Circ. Res. 82 (10): 1023-8 (1998)).

[0079] In another embodiment, the decoy nucleic acid comprises a sequence bound by a viral protein involved in viral gene expression and replication. Exemplary nucleic acids for modulating viral activity included HIV TAR sequence, which regulates tat; HIV RRE sequence, which regulates rev to inhibit replication of the HIV virus (Sullenger, B.A. et al., Ce// 63(3):601-8 (1990); Lee, S.W. et al., J Virol. 68 (12): 8254-8264 (1994)); and ICP4 of herpes simplex virus type 1 required for viral replication (Clusel, C. et al., Gene Expr. 4(6):301-9 (1995)).

[0080] In another aspect, the nucleic acids for delivery into a target cell using the compositions of the present invention are interfering RNAs. RNAi, interfering RNA, or dsRNA mediated interference refers to double stranded RNAs capable of inducing RNA interference or RNA silencing (Bosher, J. M. et al., Nat. Cell Biol. 2: E31-36 (2000)). Introducing double stranded RNA can trigger specific degradation of homologous RNA sequences, generally within the region of identity of the dsRNA (Zamore, P. D. et. al., Ce// 101 : 25-33 (1997)). This provides a basis for silencing expression of genes, thus permitting a method for altering the phenotype of cells. The dsRNA may comprise synthetic RNA made by known chemical synthetic methods or by in vitro transcription of nucleic acid templates carrying promoters (e.g., T7 or SP6 promoters). The double stranded regions of the RNAi molecule are generally about 10 -500 base pairs or more, preferably 15 -200 base pairs, and most preferably 20-100 base pairs (see, e.g., Elbashir, S. M. et al., Genes Dev. 15(2): 188-200 (2001)).

[0081] RNAi sequences have been described for silencing gene expression in numerous organisms from plants, nematodes, trypanosomes, insects, and mammals. Exemplary RNAi sequences are described for cell surface receptor proteins integrins D3 and D1 (Billy, E. et al., Proc Natl Acad Sci USA 98(25): 14428-33 (2001 )); lamin B1 , lamin B2, NUP153, GAS41 , ARC21 , cytoplasmic dynein, the protein kinase cdk1 and D- and D-actin (Harborth, J. et al., J Cell Sci. 114:4557-65 (2001 )); DNMT-1 , which plays an role in CpG methylation and control of gene expression (Sui, G. et al., Proc Natl Acad Sci USA 99(8):5515-20 (2002)); D- arrestin (Sun, Y. et al., J. Biol. Chem. 277(51 ):49212-9 (2002); checkpoint kinase Chk-1 involved in regulating cell cycle progression in response to double-strand DNA breaks (Zhao, H. et al., Proc Natl Acad Sci USA 99(23): 14795-800 (2002); hepatitis C virus replication using HCV specific RNAi sequences (Kapadia, S.B. et al., Proc Natl Acad Sci USA 100(4):2014-8 (2003)); homeobox transcription factor Rax involved in rat retina development (Matsuda, T. and Cepko, C.L., Proc Natl Acad Sci USA 101(1): 16-22 (2004)); and ubiquitin conjugating enzyme (Habelhah, H. et al., EMBO J 23(2):322-32 (2004)).

[0082] In yet a further embodiment, the compositions are used to deliver ribozymes or DNAzymes. Ribozymes and DNAzymes are nucleic acids capable of catalyzing cleavage of target nucleic acids in a sequence specific manner. Ribozymes include, among others, hammerhead ribozymes, hairpin ribozymes, and hepatitis delta virus ribozymes (Tuschl, T., Curr. Opin. Struct. Biol. 5:296-302 (1995)); Usman N., Curr. Opin. Struct. Biol. 6: 527-33 (1996)); Chowrira B.M. et al., Biochemistry 30: 8518-22 (1991)); Perrotta AT. et al., Biochemistry 3:16-21 (1992)). As with antisense nucleic acids, nucleic acids catalyzing cleavage of target nucleic acids may be directed to a variety of expressed nucleic acids, including those of pathogenic organisms or cellular genes (see, e.g., Jackson, W. H. et al., Biochem. Biophys. Res. Commun. 245:81-84 (1998)). Catalytic DNA, or DNAzymes are RNA-cleaving DNA, which may offer a higher catalytic efficiency, specificity, and inherently greater stability than a typical ribozyme (Sun, L.Q. et al., Pharmacol. Rev. 52, 325-347 (2000)).

[0083] It is to be understood that the person of ordinary skill in the art with the guidance provided herein can deliver nucleic acids other than those described above. For example, the nucleic acids may be candidate nucleic acids for use in screens for bioactive nucleic acid sequences.

Proteins

[0084] In another embodiment, the therapeutic/imaging agents comprise proteins. As used herein, a protein includes oligopeptides, peptides, and polypeptides. By "protein" herein is meant at least two covalently attached amino acids, which may be naturally occurring amino acids or synthetic peptidomimetic structures. The protein may be composed of naturally occurring and synthetic amino acids, including amino acids of (R) or (S) stereo configuration. Proteins including non-naturally occurring amino acids may be synthesized or in some cases, made by recombinant techniques (van Hest, J. C. et al., FEBS Lett. 428: 68-70 (1998); and Tang et al., Abstr. Pap. Am. Chem. S218: U138-U138 Part 2 (1999)), both of which are expressly incorporated by reference herein).

[0085] In one aspect, the therapeutic/imaging agents are peptide tags. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 (Field et al., MoI. Cell. Biol. 8:2159-2165 (1988)); the c- myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., MoI. Cell. Biol. 5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering 3:547-553 (1990)). Other tag polypeptides include the Flag-peptide (Hopp et al., BioTechnology 6:1204-1210 (1988)); the KT3 epitope peptide (Martin et al., Science 255:192-194 (1992)); tubulin epitope peptide (Skinner et al., J. Biol. Chem. 266:15163-15166 (1991)); and the T7 gene 10 protein peptide tag (Lutz- Freyermuth et al., Proc. Natl. Acad. ScL USA 87:6393-6397 (1990)).

[0086] In another aspect, the proteins comprise detectable enzymes or other reporter proteins. Enzymes and reporter proteins include, by way of example and not limitation, green fluorescent protein (Chalfie, M. et al., Science 263: 802-05 (1994)); Enhanced GFP (Clontech; Genbank Accession Number U55762 ); blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; Stauber, R. H., Biotechniques 24: 462-71 (1998); Heim, R. et al., Curr. Biol. 6: 178-82 (1996)), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303), Anemonia majano fluorescent protein (amFP486, Matz, M.V., Nat. Biotech. 17: 969-73 ((1999)), Zoanthus fluorescent proteins (zFP506 and zFP538; Matz, supra), Discosoma fluorescent protein (dsFP483, drFP583; Matz, supra), Clavularia fluorescent protein (cFP484; Matz, supra); luciferase (for example, firefly luciferase, Kennedy, H.J. et al., J. Biol. Chem. 274: 13281-91 (1999); Renilla reniformis luciferase (Lorenz, W.W., J Biolumin. Chemilumin. 11 : 31-37 (1996)); Renilla muelleri luciferase (U.S. Patent No. 6,232,107); _/ff-galactosidase (Nolan, G. et al., Proc. Natl. Acad. ScL USA 85: 2603-07 (1988)); D-glucouronidase (Jefferson, R.A. et al. EMBO J. 6: 3901-07 (1987); Gallager, S., GUS Protocols: Using the GUS Gene as a reporter of gene expression, Academic Press, Inc. (1992)); and alkaline phosphatase (Cullen, B.R. et al., Methods Enzymol. 216: 362-68 (1992)).

[0087] In another embodiment, the proteins comprise toxins that cause cell death, or impair cell survival when introduced into a cell. A suitable toxin is Campylobacter toxin CDT (Lara- Tejero, M., Science 290:354-57 (2000)). Expression of the CdtB subunit, which has homology to nucleases, causes cell cycle arrest and ultimately cell death. Another exemplary toxin is diptheria toxin (and similar Pseudomonas exotoxin), which functions by ADP ribosylating ef-2 (elongation factor 2) molecule in the cell and preventing translation. Expression of the diptheria toxin A subunit induces cell death in cells expressing the toxin fragment. Other useful toxins include cholera toxin and pertussis toxin (catalytic subunit-A ADP ribosylates the G protein regulating adenylate cyclase), pierisin from cabbage butterflys, inducers of apoptosis in mammalian cells (Watanabe, M., Proc. Natl. Acad. ScL USA 96:10608-13 (1999)), phospholipase snake venom toxins (Diaz, C. et al., Arch. Biochem. Biophys. 391 :56-64 (2001)), ribosome inactivating toxins (e.g., ricin A chain, Gluck, A. et al., J. MoI. Biol. 226:411-24 (1992)); and nigrin (Munoz, R. et al., Cancer Lett. 167: 163-69 ((2001)).

[0088] In yet a further embodiment, the protein is a protein domain, or peptide mimic thereof, that interacts with other biological molecules. A protein-interaction domain refers to a protein region or sequence that interacts with other biomolecules, including other proteins, nucleic acids, lipids, etc. These protein domains frequently act to provide regions that induce formation of specific multiprotein complexes for recruiting and confining proteins to appropriate cellular locations or affect specificity of interaction with target ligands. Protein- interaction domains comprise modules or micro-domains ranging about 20-150 amino acids that can be expressed in isolation and bind to their physiological partners. Many different interaction domains are known, most of which fall into classes related by sequence or ligand binding properties. Accordingly, the interaction domains may comprise proteins that are members of these classes of protein domains and their relevant binding partners. These include, among others, SH2 domains (src homology domain 2), SH3 domain (src homology domain 3), PTB domain (phosphotyrosine binding domain), FHA domain (forkedhead associated domain), WW domain, 14-3-3 domain, pleckstrin homology domain, C1 domain, C2 domain, FYVE domain (Fab-1 , YGL023, Vps27, and EEA1 ), death domain, death effector domain, caspase recruitment domain, Bcl-2 homology domain, bromo domain, chromatin organization modifier domain, F box domain, hect domain, ring domain (Zn+2 finger binding domain), PDZ domain (PSD-95, discs large, and zona occludens domain), sterile a motif domain, ankyrin domain, arm domain (armadillo repeat motif), WD 40 domain and EF-hand (calretinin), PUB domain (Suzuki T. et al., Biochem. Biophys. Res. Commun. 287: 1083-87 (2001 )), nucleotide binding domain, Y Box binding domain, H. G. domain, all of which are well known in the art. Since protein interaction domains are pervasive in cellular regulation, such as signal transduction cascades and transcription factors, introduction of protein interaction domains acting in a specific regulatory pathway may provide a basis for inactivating or activating such pathways in normal and diseased cells.

[0089] It is to be understood that other protein compounds may be translocated into the cell.

Administration and Dose

[0090] The therapeutic/imaging agent can be administered to a subject in a biologically compatible form suitable for pharmaceutical administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the therapeutic/imaging agent to be administered in which any toxic effects are outweighed by the desired diagnostic or therapeutic effect of the therapeutic/imaging agent. [0091] Administration of a therapeutically effective amount of a therapeutic agent is meant an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically active amount of a therapeutic and/or imaging agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic/imaging agent to elicit a desired response in the individual. A dosage regime may be adjusted to provide an optimum therapeutic or diagnostic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of a therapeutic or diagnostic situation.

[0092] Like the photosensitizer, the therapeutic/imaging agent may be administered in a convenient manner such as by systemic injection (subcutaneous, intravenous, etc.), tissue specific oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the therapeutic/imaging agent may be coated in a material to protect the therapeutic/imaging agent from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic/imaging agent.

[0093] The adjuvant may be administered as a composition with other compounds to limit inactivation of the therapeutic/imaging agent.

[0094] In some embodiments, it may be necessary to coat agents such as proteins and nucleic acids with, or co-administer the therapeutic/imaging agent with, a material to prevent inactivation of the therapeutic/imaging agent. A therapeutic and/or imaging agent may be administered in a composition in an appropriate carrier, diluent or adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Exemplary adjuvants include alum, resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol 7:27).

[0095] Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0096] In some embodiments, a pharmaceutical composition suitable for injectable use includes sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition will preferably be sterile and fluid to the extent that easy syringability exists. The composition may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a therapeutic and/or imaging agent which delays absorption, for example, aluminum monostearate and gelatin.

[0097] Sterile injectable solutions can be prepared by incorporating one or more agents, together or separately with additional immune response stimulating agents or immunosupressants, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the therapeutic/imaging agent into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0098] When a therapeutic and/or imaging agent comprising a protein is suitably protected, as described above, the protein may be orally administered, for example, with an inert diluent or an assimilable edible carrier. As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active agent, use thereof in the therapeutic compositions is contemplated. Supplementary compounds can also be incorporated into the compositions.

[0099] Compositions of agents may be prepared in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms may depend on (a) the unique characteristics of the therapeutic/imaging agent(s) and the particular therapeutic effect to be achieved, and/or (b) the limitations inherent in the art of compounding such a therapeutic and/or imaging agent for the treatment of sensitivity in individuals.

[00100] The specific dose can be readily calculated by one of ordinary skill in the art, e.g., according to the approximate body weight or body surface area of the subject or the volume of body space to be occupied. The dose may also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in assay preparations of target cells. Exact dosages are determined in conjunction with standard dose-response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the chosen route of administration.

[00101] The toxicity and therapeutic efficacy of the therapeutic/imaging agents described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Agents exhibiting large therapeutic indices are preferred. While agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00102] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test agent which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00103] The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an antibody can include a single treatment or, preferably, can include a series of treatments.

[00104] Where appropriate, gene delivery agents such as, e.g. integration sequences can also be employed. Numerous integration sequences are known in the art (see for example Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol., 165:341- 357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. MoI. Biol., 150:467- 486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), FIp (Broach, et al, Cell, 29:227-234, 1982) R (Matsuzaki, et al, J. Bacteriology, 172:610-618, 1990), φC31 (see for example Groth et al., J. MoI. Biol. 335:667-678, 2004), sleeping beauty, transposases of the mariner family (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, and lentiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003).

[00105] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. Kits for practice of the instant invention are also provided. For example, such a kit comprises a human cell, viral vector or composition comprising a cell and/or viral vector, together with a means for administering the cell, viral vector or composition, e.g., one or more syringes. The kit can come packaged with instructions for use.

Examples

[00106] The following non-limiting examples illustrate aspects of the disclosure, and are not intended to be limiting thereto.

Example 1 [00107] Figure 1 A shows confluent cells that cover the entire area of the culture dish.

[00108] Culture media was rinsed from the cells and replaced by saline solution. The photosensitizer Lucifer yellow in saline was added to the cell surface and a diaphragm was imaged onto the cells. Lucifer yellow was activated with light radiation in the absorption wavelength. The Lucifer yellow entered the cells and filled them with a bright fluorescent glow.

[00109] All cells in which radiation was not applied had darker nuclei. Only the outline of the cells can be seen but the cells that were photoactivated while the diaphragm was closed have been filled with photsensitizer. If extracellular photosensitizer is washed out by rinsing with clear saline, only the stained cells were visible. Photosensitizer remained trapped in the target cells is depicted in Figure 1 B.

[00110] Figure 1 C depicts the addition of a red-dye labeled protein (rhodamine labeled albumen). In the absence of photoactivation, the protein will not enter the cells. Activation with light radiation in the absorption wavelength of Lucifer yellow allows for uptake of the dye-labeled protein. Without being limited to a specific theory or mechanism, activation with light radiation in the absorption wavelength of Lucifer yellow in those same cells generates holes in the membranes large enough to allow albumen to enter and remain trapped in the cells without migrating to any other cells in the same field.

[00111] Figure 1C shows that the red-dye labeled albumin containing cells appears larger than the field of Lucifer yellow-labeled cells, but the expanded field is no larger than the diffusion distance for singlet oxygen with a lifetime of 5 μs (microseconds) before being quenched by water. Therefore, the number of cells stained red involved only those cells marked with the photosensitizer plus the diffusion distance of less than 3 microns from those cells.

Example 2

[00112] Figure 2 depicts an electron micrograph showing the results of another experiment in which the photosensitizer was incorporated into the cell.

[00113] The photosensitizer Lucifer yellow was translocated into the cell. At a later time, a cell containing the photosensitizer Lucifer yellow was activated in the presence of the enzyme Horseradish Peroxidase. The cells were fixed with glutaraldehyde so all diffusion and cell reaction or transfer was stopped. [00114] Only the cell containing the photosensitizer took up the enzyme which was later developed in the presence of diamino-benzidine (DAB). DAB monomer polymerizes when in the presence of reactive oxygen. Only the polymer and not the monomer is osmiophyllic and stains black when treated with osmium tetroxide. Osmium is appears black in the electron beam.

[00115] The electron micrograph of Figure 2 depicts three cells, one colored black and the other two lacking any black color inside. An exploded view of the area where the cells meet and contact one another is shown to the right. The arrows point to small vesicles on the cell surfaces where enzyme is being engulfed by the membrane as the cell would take up any protein from the outside.

[00116] By contrast, the stained cell shows that in addition to the vesicular uptake, the entire cytoplasm is black indicating that the enzyme went directly into the cytoplasm of the cell. The amount of enzyme incorporated is increased significantly as compared to the two cells that were outside the activation field (i.e. activation light did not reach the cells) when the photosensitizer was present.

[00117] These two examples show first that a large protein albumen (molecular weight >140kDa) enters the cells on command. Uptake was limited to those cells with photosensitizer present. Morover, the therapeutic/imaging agent (enzyme HRP in Figure 2) entered directly into the cytoplasm of the target cell.

Example 3

[00118] Adult human stem cells were grown to confluence. The photosensitizer Lucifer yellow was applied to the cells and a diaphragm was imaged onto the cells. Photoactivation by applying light radiation continued for 3 minutes (one minute was enough to see nuclei begin to stain). The second patch of cells was activated by light radiation a second time. The cells were then returned to culture media and the incubator.

[00119] Figure 3 is a low magnification image of the same cells taken 17 days after the experiment, shwoing that there is a slow cytoplasmic exchange of photosensitizers with cells that were not initially stained, showing that there was a slow cytoplasmic exchange of photosensitizer with neighboring cells that were not initially stained, forming a subtle greening halo around the target cells and further indicating the cells were alive. This exchange did not appreciably dilute the photosensitizer in the target cells.

[00120] Cells were monitored and photographed over subsequent weeks. Figure 3B shows the same cells 33 days after the experiment. There was no evident loss of photosensitizer from any of the cells and there was no loss of cell numbers or cell position since the cells were already confluent at the time of the experiment. The experiment shows that 100% of target cells were stained with the photosensitizer and that 33 days later no stained cells had been lost. Other experiments with the standard cell viability test showed these cells continued to exclude ethidium bromide, indicating that the cells were still alive.

[00121] Similar experiments were conducted on cultured cells with the exception that the saline used to rinse the cells of growth media had been equilibrated with nitrogen gas bubbled through the solution in order to drive all the oxygen out (data not shown). The cells were exposed to a nitrogen atmosphere. The nitrogen equilibrated saline was applied and a glass coverslip was applied to the cells to keep oxygen out. Activation in the presence of the photosensitizer did not permeabilize any cells. However, restoring oxygen to the saline and repeating the experiment with the same cells resulted in complete permeabilization as the previous experiments showed.

Example 4

[00122] An aqueous solution of the photosensitizer Lucifer yellow and a radical trap, phenyl- tertiary butyl nitrone (PBN), were combined. PBN is paramagnetic when a radical is trapped by the compound. When the solution was prepared in a dry box with no oxygen present, activation of the photosensitizer did not generate any reactive oxygen species shown by the green line. However, introduction of oxygen followed by activation by light radiation generated a paramagnetic signal shown by the spectral line in Figure 4 at the characteristic magnetic field for PBN (field in Gauss-x axis). The amplitude was graphed against time of activation and showed a proportional increase in signal amplitude.

[00123] This experiments is performed on living animals. Activation of the photosensitizer caused the cells to take up the dye. Nonactivated cells did not take up photosensitizer. Photoreceptor cells did not take up the photosensitizer. Photoreceptor cells in the retina takes up the photosensitizer, then use the intracellular process of pigment granule migration to test cell viability which remained intact. Excess photosensitizer is removed from the animal's body by the kidney. Animals live their normal lifetime without apparent detriment.

Claims

We claim:

1. A method of translocating a photosensitizer into a cell comprising: a) providing a photosensitizer proximal to the cell; and b) applying radiation at the absorption wavelength of the photosensitizer, wherein said translocates the membrane of said cell to form a photosensitizer-containing cell.

2. The method according to claim 1 , wherein the photosensitizer is Lucifer yellow.

3. The method according to one of claims 1 or 2, wherein the cell remains viable for at least 30 days.

4. A method according to one of claims 1-3, wherein applying radiation to said photosensitizer generates one or more oxide radicals.

5. The method according to one of claims 1-4, wherein the photosensitizer is linked to a targeting agent.

6. A method according to one of claims 1-4, wherein more than 90% of said photosensitizer-containing cells maintain viability.

7. The method according to one of claim 1-6, comprising providing an therapeutic and/or imaging agent to said cell, wherein the therapeutic and/or imaging agent translocates the membrane of said cell.

8. A method of translocating a therapeutic and/or imaging agent into a cell comprising: a) translocating a photosensitizer into a cell according to claim 1 ; b) providing a therapeutic and/or imaging agent proximal to the photosensitizer- containing cell; and c) applying radiation at an absorption wavelength of the photosensitizer to permeabilize translocate said therapeutic and/or imaging agent into the cell.

9. A method of claim one of claims 7 or 8, wherein the therapeutic/imaging agent is a nucleic acid or protein.

10. A composition comprising: a photosensitizer, and a therapeutic and/or imaging agent.

11. The composition according to claim 10, wherein the photosensitizer is Lucifer yellow.

12. The method according to one of claims 10 or 11 , wherein the cell remains viable for at least 30 days.

14. The method according to one of claims 10-12, wherein the photosensitizer is linked to a targeting agent.