WO2008088917A2

WO2008088917A2 - Methods of using pyridinium and thiazolium compounds as reagents, diagnostic compounds and therapeutic agents

Info

Publication number: WO2008088917A2
Application number: PCT/US2008/000794
Authority: WO
Inventors: Richard B. Klein; Jeffrey L. Selph; John J. Partridge
Original assignee: Mycosol Inc
Current assignee: Mycosol Inc
Priority date: 2007-01-19
Filing date: 2008-01-22
Publication date: 2008-07-24
Anticipated expiration: 2009-07-19
Also published as: WO2008088917A3

Abstract

Methods of using pyridinium and thiazolium compounds, homologs, analogs and derivatives thereof as reagents, diagnostic tools and therapeutic agents are provided.

Description

METHODS OF USING PYRIDINIUM AND THIAZOLIUM COMPOUNDS AS REAGENTS, DIAGNOSTIC COMPOUNDS AND THERAPEUTIC AGENTS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States Provisional Application Serial No. 60/881,348, filed on January 19, 2007, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of using to pyridinium and thiazolium compounds, homologs, analogs and derivatives thereof as reagents, diagnostic tools and therapeutic agents. More particularly, the present invention relates to the use of these compounds to target biological components and to function as dyes, stains and/or fluorescent stains.

BACKGROUND OF THE INVENTION

The need to identify, mark, isolate, modify or otherwise determine analytes, cells, microorganisms, amino acids or nucleic acid sequences (for example multiple pathogens or multiple genes or multiple genetic variants) alone and in blood or in other biological fluids or tissues, and study, diagnose and/or treat various diseases and disorders has become increasingly apparent in many branches of medicine. Many multi-analyte assays, such as assays that detect multiple nucleic acid sequences, involve multiple steps, have poor sensitivity, a limited dynamic range (typically on the order of 2 to 100-fold differences) and/or often utilize sophisticated instrumentation. There is also a continuing need in the assay art for labels with the following features:

(i) high fluorescent intensity (for detection in small quantities), (ii) adequate separation between the absorption and emission frequencies, (iii) desirable solubility, (iv) ability to be readily linked to other molecules, (v) stability towards harsh conditions and high temperatures, (vi) a symmetric, nearly Gaussian emission lineshape for deconvolution of multiple colors, and/or (vii) compatibility with automated analysis. SUMMARY OF THE INVENTION

Embodiments of the present invention provide novel uses of pyridinium compounds, such as stilbazium compounds, homologs, analogs and derivatives thereof and/or thiazolium compounds, homologs, analogs and derivatives thereof. These compounds can be used in cytological, histochemical, immunohistochemical, immunocytochemical and chemical applications such as cell staining, cell tracking, cell selection, cell imaging, used in laser applications, and in particular, used in microscopic imaging, cell tracking experiments and flow cytometry analysis. Accordingly, these compounds can be used as reagents, diagnostic tools or therapeutic agents. The compounds can also be formulated in liposomes, pegylated, encapsulated and/or microencapsulated for use in such applications.

Embodiments of the present invention include methods of identifying, differentiating, diagnosing, evaluating and/or studying a disease or disorder, or abnormality, or lack thereof, including contacting a cell with a compound having the following structure:

or a solvate thereof, wherein X^' is an anionic salt; Ri₁ R₂, R₃, or R₄ are the same or different and independently selected from the group consisting of methyl, ethyl, Ci_-I0 alkyl (linear or branched) and alkenes (linear or branched), or wherein when Ri and R₂ or when R₃ and R₄ are taken together with the nitrogen atom to which they are attached, they form substituted or unsubstituted pyrrolidino or piperidino rings; R₅ is an organometallic compound; R₅ may be (CH₂)_n-MR₆, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₆ is a selected from the group consisting of propyl, butyl, or any alkyl compound; R₅ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C2-₅ alkyne substituted polyethylene glycol, or

or a solvate thereof wherein X^* is an anionic salt; R₁ and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, Ci_-10 alkyl (linear or branched) and alkenes (linear or branched), or wherein R₁ and R₂ may be taken together with the nitrogen atom to which they are attached form substituted or unsubstituted pyrrolidino or piperidino rings; R₃ is selected from the group consisting of methyl, ethyl, Q- io alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n- butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₃ is an organometallic compound; R₃ is (CH₂)_n-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound; or R₃ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol, wherein contact with the cell allows (a) detection of a biomarker, or (b) viewing of abnormal cellular structure, wherein detection of a biomarker and/or viewing of abnormal cellular structure are associated with characteristics of the disease or disorder. In some embodiments of the present invention, contact with a cell allows (a) detection and/or identification of a target cell, (b) detection and/or identification of target organelles within that cell, (c) detection of a biomarker, or (d) viewing of normal or abnormal cellular structure, wherein such detection and/or identification are associated with characteristics of a healthy cell or of a disease or disorder.

Embodiments of the present invention further provide methods of evaluating physiological processes and/or functions including administering compounds described herein. Embodiments of the present invention also provide methods of facilitating delivery of an agent such as an antibody or therapeutic moiety to a target of interest to a subject in an amount effective to monitor and/or treat a disease or disorder afflicting the subject.

Embodiments of the present invention provide methods of using the compounds described herein in laser applications and methods of identifying, studying, evaluating and/or treating diseases and disorders using lasers. In some embodiments, the administration of the compound provides identification, alteration, or destruction of the target cells or tissue by application of laser or ultrasonic energy or other outside stimulus to the compound.

Embodiments of the present invention further provide assays for determining the presence, absence or health of a cell, analyte, nucleic acid or microorganism in a sample and probes including a ligand or antibody and a pyridinium or thiazolium compound, analog, homolog or derivative thereof.

Embodiments of the present invention provide methods of selecting an analyte that binds to a compound described herein. Embodiments of the present invention provide methods of determining whether a nucleic acid of interest interacts with a protein of interest in a cell or an in vitro sample.

Embodiments of the present invention further provide methods for determining the presence or absence or health of one or more target compounds in a sample.

Embodiments of the present invention further include kits for staining and/or modifying cells, analytes, nucleic acids and/or microorganisms, biological tissue and/or fluids and environmental liquids and identifying, diagnosing, studying, evaluating and/or treating diseases and disorders described herein or the absence thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Figure 1 presents chemical structures of exemplary compounds tested.

Figure 2. Figure 2 presents epifluorescence micrograph of 4Tl cells stained with Hoechst and compound B excited with a single wavelength.

Figure 3. Figure 3 presents the effect of compounds described herein on cell viability of 4Tl cells as compared to a leading cell stain, MitoTracker Red (MTR). Figure 4. Figure 9 presents the effect of compounds described herein on cell viability of 4Tl cells after long term exposure.

Figure 5. Figure 5 presents flow cytometry data from cell tracking experiment with compound B. Each row depicts cell-associated fluorescence as a function of time post staining for each concentration tested (last panel in each row shows data from preceding panels superimposed with untreated cells represented by the black curve). Each column depicts cell-associated fluorescence as a function of concentration tested for each time post staining (last panel in each column shows data from preceding panels superimposed with untreated cells represented by the black curve). Figure 6. Figure 6 presents 2-photon fluorescence micrographs collected with indicated excitation wavelengths in ran.

Figure 7. Figure 7 presents high-resolution 2-photon fluorescence micrographs collected with compound B at 910 nm excitation and indicated magnification. Figure 8. Figure 8 presents representative 2-photon fluorescence micrographs collected with additional compounds at 2OX magnification (NOTE: Raw images are shown without pseudo-coloring).

Figure 9. Figure 9 presents confocal fluorescence micrographs of 4Tl cells co- stained with compounds described herein and Mitofluor green (MFG). MitoTracker red (MTR) co-staining with MFG is shown for reference.

Figure 10. Figure 10 presents fluorescence micrographs of 4Tl cells stained with compounds described herein in the presence and absence of CCCP.

Figure 11. Figure 11 presents cell-associated fluorescence in 4Tl cells treated with compounds described herein versus MitoTracker Red (MTR).

DETAILED DESCRIPTION

The foregoing and other aspects of the present invention will now be described in more detail with respect to other embodiments described herein. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the embodiments of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Also, as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items. As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y." As used herein, phrases such as "from about X to Y" mean "from about X to about Y." Unless otherwise defined, all terms, including technical and scientific terms used in the description, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

"Alkyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.

"Loweralkyl" as used herein, is a subset of alkyl, and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso- butyl, tert-butyl, and the like. Alkyl and loweralkyl groups may be unsubstituted or substituted one or more times with halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, alkyl-S(O)_m, haloalkyl-S(O)_m, alkenyl-S(O)_m, alkynyl- S(O)_n,, cycloalkyl-S(O)_m, cycloalkylalkyl-S(O)_m, aryl-S(O)_m, arylalkyl-S(O)_m, heterocyclo- S(O)_n,, heterocycloalkyl-S(O)_m, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m=0,l or 2. "Alkoxy," as used herein alone or as part of another group, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.

"Acyl" or "Alkanoyl" as used herein alone or as part of another group, refers to a - C(O)R radical, where R is any suitable substituent such as alkyl, alkenyl, alkynyl, aryl, alkylaryl, etc.

"Cell" as used herein refers to a basic component of a living or fixed organism and includes organelles. Thus, detecting the presence of a cell, assaying a cell, staining a cell, etc. can refer to a whole cell, a portion of a cell such as the cytoplasm, or at least one organelle of the cell. According to embodiments of the present invention, cells may be plant or animal cells. As recognized by one skilled in the art, "organelles" as used herein refer to cellular components or structures suspended in the cytoplasm including those providing a boundary therefor and having specialized functions. Organelles include, but are not limited to, the nucleus, smooth and/or rough endoplasmic reticulum, centrosome, cytoskeleton, cell wall, cell membrane, flagella, cilia, chloroplast, mitochondria, golgi apparatus, ribosome, lysosome, centriole, acrosome, glyoxysome, secretory vesicle, peroxisome, vacuole, melanosome, myofibril and parenthesome.

"Dye" or "stain" as used herein refers to a colored substance that has an affinity to the substrate to which it is being applied. The color can be temporary, semi-permanent or permanent.

"Fluorescent stain" or "fluorescent dye" as used herein refers to coloration that requires exposure to excitation light of a particular wavelength resulting in the emission of light. "Analyte" as used herein refers to the substance or chemical constituent that undergoes analysis. For example, an analyte can be a molecule, protein, chemical substance, etc. that can be detected as a result of biological, chemical or clinical testing to evaluate the same. Analytes can include, but are not limited to, ions; metabolites such as glucose and urea; trace metabolites such as hormones, drugs, steroid hormones; gases such as respiratory gases, anesthetic gases, toxic gases and flammable gases; toxic vapors; proteins and nucleic acids; antigens and antibodies and microorganisms.

"Nucleic acid" as used herein refers to an oligonucleotide, nucleotide, or polynucleotide, and to DNA or RNA or chimeras thereof, single stranded or double-stranded, and can be fully or partially synthetic or naturally occurring. Nucleic acids can include modified nucleotides or nucleotide analogs. Further, the nucleic acids can be from any species of origin, including plant species or mammalian species such as human, non-human primate, mouse, rat, rabbit, cattle, goat, sheep, horse, pig, dog, cat, etc. In some embodiments, the nucleic acid is an isolated nucleic acid. As used herein, an "isolated" nucleic acid means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism, for example, the cell structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid.

"Lipid vesicle" as used herein refers to structures including amphiphiles, for example, surfactants or phospholipids, characterized by the presence of an internal void. The internal void can be filled with any appropriate material such as a liquid, aqueous solution, gas, gel, solid material or mixture thereof. Lipid vesicles include, but are not limited to, liposomes, helices, discs, tubes, tori, hexagonal, phase structures, micelles, gel phases, reverse micelles, bicelles, microemulsions, emulsions and combinations thereof. "Microorganism" as used herein refers to microscopic organisms that can exist as a single cell or cell clusters.

"Laser" (Light Amplification by Stimulated Emission of Radiation) as used herein refers to a device that controls the manner that energized atoms release photons, i.e., light energy for purposes which range from pure visualization, to excitation/emission, to alteration or destruction of a target. Some examples of lasers include chemical lasers and dye lasers. As used herein, a "chemical laser" is a laser that obtains energy from a chemical reaction. In a chemical laser, a suitable chemical reaction produces a stream of gas rich with excited atoms or molecules. Another gas can then be injected to this stream, and can react with those particles, producing an excited molecule, or takes energy from the excited particle. A powerful beam of coherent laser radiation can be produced. As used herein, a "dye laser" is a laser that employs a dye as a lasing medium, and can be in a liquid solution. The dye can be used in a varied range of wavelengths. The dye lasers can be tunable lasers (allowing wavelength or frequency changes to be controlled by the operator) and pulsed lasers, which impart the ability to generate extremely short pulses of light. "Reagent" as used herein refers to a substance used in a chemical, biochemical or physiological reaction or process. In some embodiments of the present invention, "reagent" refers a to chemical substance of desired purity for use in chemical or biochemical analysis, chemical or biochemical reactions or physical testing. In general, purity standards for reagents are set by organizations such as ASTM International. Reagent further refers to research tools used in areas including, but not limited to, drug discovery and research, medical research, basic science research, applied science research and veterinary applications.

"Diagnostic" as used herein refers to identifying a state, condition, disease or marker therefor by performing a procedure that provides the opportuity to visualize and/or note signs or characteristics of the particular state, condition, diease or marker therefor. "Treat" as used herein refers to an action resulting in a reduction in the severity of the subject's condition or at least the condition is partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the condition and/or prevention or delay of the onset of the condition. Thus, the term "treat" can also refer to prophylaxis. Further, "treat" and "therapeutic" are used interchangeably herein.

As used herein, the term "effective amount" refers to an amount of a compound or composition that is sufficient to produce the desired effect, which can be a diagnostic, or therapeutic effect. The effective amount will vary with the application for which the compound or composition is being employed, the microorganism and/or the age and physical condition of the target or subject, the severity of the condition, the duration of the research, experiment, or treatment, the nature of any concurrent application or treatment, the delivery system or pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. An appropriate "effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. (See, for example for pharmaceutical applications, Remington, The Science And Practice of Pharmacy (9th Ed. 1995).

1. Compounds Compounds used in the present invention include pyridinium compounds such as stilbazium compounds, homologs, analogs and derivatives thereof as well as thiazolium compounds, homologs, analogs and derivatives thereof.

Stilbazium, homologs, analogs and derivatives thereof

Stilbazium iodide is a known anthelmintic, which is reported to be effective against roundworms, threadworms, and whipworms. U.S. Patent Nos. 3,075,975 and 3,085,935 recite methods of eradicating infestations of parasitic nematodes inhabiting the intestinal tract. Stilbazium chloride as well as other stilbazium salts, analogs and homologs thereof may exist as bright red to dark red or other colored compounds that may possess UV-visible chromophores and may further exhibit characteristic strong fluorescence. Embodiments of the present invention include the use of a compound having the following structure:

or a solvate thereof, wherein X^" is an anionic salt, wherein Ri, R₂, R₃, or R₄ are the same or different and independently selected from the group consisting of methyl, ethyl, Ci_-10 alkyl (linear or branched) and alkenes (linear or branched), or wherein when Ri and R₂ or when R₃ and R₄ are taken together with the nitrogen atom to which they are attached, they form substituted or unsubstituted pyrrolidino or piperidino rings. X^" can be selected from the group including fluoride, chloride, bromide, iodide halide, methanesulfonate (mesylate), p- toluenesulfonate (tosylate), napthylate, m-nitrobenzenesulfonate (nosylate), para- aminobenzoate, benzenesulfonate (besylate), lauryl sulfate, 2,4-dihydroxy benzophenone, or 2-(2-hydroxy-5'-methylphenyl) benzotriazole, R₅ is selected from the group consisting of methyl, ethyl, Ci_-I0 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties. R₅ may also be an organometallic compound such as organotin, organosilicon, or organogermanium. Additionally, R₅ may be (CH₂)_H-MR₆, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₆ is a selected from the group consisting of propyl, butyl, or any alkyl compound. R₅ may also be a polyalkylene glycol moiety comprising a C]_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol. The end-terminals of these polyethylene glycol moieties can be hydroxy, methoxy, ethoxy and acetyloxy. The amino moieties on the aromatic rings can be in either the ortho, meta or para position. The formulation can further include a solvent such as water, ethanol, isopropyl alcohol, propylene glycol (a diol), benzyl alcohol, glycerin, methanol, ethylene glycol and polyethylene glycols.

Embodiments of the present invention include use of a compound having the following structure:

or a solvate thereof, wherein X^" is an anionic salt, wherein Rj₁ R₂, R₃, or R₄ are the same or different and are independently selected from the group consisting of methyl, ethyl, C M_O alkyl (linear or branched) and alkenes (linear or branched), or wherein when Ri and R₂ or when R₃ and R₄ are taken together with the nitrogen atom to which they are attached, they form substituted or unsubstituted pyrrolidino or piperidino rings. R₅ is selected from the group consisting of methyl, ethyl, Cj.io alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties. X^" can be selected from the group including fluoride, chloride, bromide, iodide, halide, methanesulfonate (mesylate), p- toluenesulfonate (tosylate), napthylate, m-nitrobenzenesulfonate (nosylate), para- aminobenzoate, lauryl sulfate, 2,4-dihydroxy benzophenone, 2-(2-hydroxy-5'-methylphenyl) benzotriazole, or benzenesulfonate (besylate). R₅ may also be an organometallic compound such as organotin, organosilicon, or organogermanium. Additionally, R₅ may be (CH₂)_n- MR_ό, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₆ is a selected from the group consisting of propyl, butyl, or any alkyl compound. R₅ may also be a polyalkylene glycol moiety including a C_1-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol. The end- terminals of these polyethylene glycol moieties can be hydroxy, methoxy, ethoxy and acetyloxy. The present pyridinium compound is more commonly known as stilbazium. In a particular embodiment, the compound is 2,6,-bis (p-pyrrolidinostyryl) pyridine methiodide. The formulation can further include a solvent such as water, ethanol, isopropyl alcohol, propylene glycol (a diol), benzyl alcohol, glycerin, methanol, ethylene glycol and polyethylene glycols. Alternatively, the NR₁R₂ and NR₃R₄ moieties may be in various positions as evidenced in the compounds below. For example, in one embodiment, the NRiR₂ moiety is in one meta position:

In another embodiment, the NR₁R₂ and NR₃R₄ moieties are present in both meta positions:

wherein X^" may be an anionic salt, R)₁ R₂, R₃, or R₄ are the same or different and are independently selected from the group consisting of methyl, ethyl, C_MO alkyl (linear or branched), and alkenes (linear or branched), or wherein when Ri and R₂ or when R₃ and R₄ are taken together with the nitrogen atom to which they are attached, they form substituted or unsubstituted pyrrolidino or piperidino rings. R₅ is selected from the group consisting of methyl, ethyl, Ci-I₀ alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties. Additionally, R₅ can be (CH₂)_n-MR₆, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₆ is a selected from the group consisting of propyl, butyl, or any alkyl compound. R₅ may also be a polyalkylene glycol moiety including a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol. The end-terminals of these polyethylene glycol moieties can be hydroxy, methoxy, ethoxy and acetyloxy.

The compounds described herein are capable of existing as geometric isomers. AU such isomers, individually and as mixtures, are included within the scope of the present invention for their industrial uses. The E₁E isomer is one configuration of the invention, and both the cisoid and transoid 2,6-conformations of the E,E-configuration are possible. Additionally, the ortho, ortho conformation of the structure can be formed in addition to the para and meta structures illustrated above. The ortho conformation structure can include the same salts and moieties as disclosed above and throughout the application.

Some of the compounds employed in the present invention include l-ethyl-(E,-E)-2,6- bis[2-[4-(pyrrolidinyl)phenyl]ethenyl]pyridinium chloride, l-ethyl-(E,-E)-2,6-bis[p-(l- pyrrolidinostyryljpyridinium chloride, l-methyl-(E,-E)-2,6-bis[2-[4-

(pyrrolidinyl)phenyl]ethenyl]pyridinium chloride and l-methyl-(E,-E)-2,6-bis[p-(l- pyrrolidinostyryljpyridinium chloride in a suitable formulation as described herein.

Additionally, the present invention includes use of compounds having the following structure:

or a solvate thereof, wherein n is a number from 1 to 5, wherein Z can be present at multiple positions on the phenyl ring and is selected from the group consisting of C, N, O, S and halogen, wherein X^" is an anionic salt, wherein R_1, R₂, R₃, or R₄ are independently not present or are the same or different and selected from the group consisting of hydrogen, methyl, ethyl, Ci_-IO alkyl (linear or branched), alkenes (linear or branched), nitriles, benzenes, pyridines, benzothiophenes, trifluoroalkyls, difluoroalkyls, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties, or wherein when Ri and R₂ or when R₃ and R₄ are taken together with the nitrogen atom to which they are attached, they form substituted or unsubstituted pyrrolidino or piperidino rings. X^" can be selected from the group including fluoride, chloride, bromide, iodide, halide, methanesulfonate (mesylate), p- toluenesulfonate (tosylate), napthylate, m-nitrobenzenesulfonate (nosylate), para- aminobenzoate, benzenesulfonate (besylate), lauryl sulfate, 2,4-dihydroxy benzophenone, or 2-(2-hydroxy-5'-methylphenyl) benzotriazole. R₅ is selected from the group consisting of methyl, ethyl, Ci_-I0 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties. R₅ may also be an organometallic compound such as organotin, organosilicon, or organogermanium. Additionally, R₅ may be (CH₂)_n-MR₆, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₆ is a selected from the group consisting of propyl, butyl, or any alkyl compound, with the proviso that said compound is not 1 -ethyl-(Z,Z), (Z,E) or (E,Z) - 2,6-bis[2-[4-(pyrrolidinyl)phenyl]ethenyl]pyridinium chloride. R₅ may also be a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol. The end-terminals of these polyethylene glycol moieties can be hydroxy, methoxy, ethoxy and acetyloxy.

The present invention further includes the use of compounds described in U.S. Patent No. 7,220,761 to Klein et al. Generally, compounds according to the invention can be made according to any suitable method of organic chemistry. As a non-limiting example for guidance, see U.S. Patent No. 3,085,935.

Thiazolium, homologs, analogs and derivatives thereof

Compounds including a thiazole moiety such as thiazolium, thiazolium salts, analogs and homologs thereof may exist as pale yellow or yellow or other colored compounds that may possess UV-visible chromophores and may further exhibit characteristic strong fluorescence.

or a solvate thereof wherein the compound is substantially in the E, E configuration, or the compound can also be in the E,Z or Z,Z configuration. The amino moieties can be in either the ortho, meta or para positions. The anion X^" can be an anionic salt. The anion X^" can be fluoride, chloride, bromide, iodide, halide, methanesulfonate (mesylate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), napthylate, m- nitrobenzenesulfonate (nosylate), para-aminobenzoate, lauryl sulfate, 2,4-dihydroxy benzophenone, or 2-(2-hydroxy-5'-methylphenyl) benzotriazole. Rj and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, Ci_-10 alkyl (linear or branched) and alkenes (linear or branched), or wherein Ri and R₂ may be taken together with the nitrogen atom to which they are attached form substituted or unsubstituted pyrrolidino or piperidino rings. R₃ can be selected from the group consisting of methyl, ethyl, C_MO alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties. R₃ may also be an organometallic compound such as organotin, organosilicon, or organogermanium. Additionally, R₃ may be (CH₂)_n-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound. In some embodiments, R₃ is selected from the group consisting of methyl, ethyl, Ci-io alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i- propyl, n-butyl, i-butyl, an organometallic compound, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties. R₃ can also be a polyalkylene glycol moiety including a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol. The end-terminals of these polyethylene glycol moieties can be hydroxy, methoxy, ethoxy and acetyloxy. R₄ through R₈ are the same or different and may be selected from the group consisting of hydrogen, Ci_-10 alkyl (linear or branched), representative examples of alkyl including, but not limited to, n-propyl, i-propyl, n-butyl, i- butyl, alkenes (linear or branched), alkynes, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties, hydroxy, alkoxy, SCH₃, (Ci-C₃) alkylthio, SH, (Ci-C₃) haloalkoxy, (Ci-C₃) perhaloalkoxy, NH₂, NH(lower alkyl), N(lower alkyl)₂, halogen, (C₁-C₃) haloalkyl, (C₁-C₃) perhaloalkyl, -CF₃, -CH₂CF₃, -CF₂CF₃, -CN, -NC, -OCN, -SCN, -NO, -NO₂, -N₃, -S(O) (lower alkyl), -S(O) (aryl), -S(O)₂ (lower alkyl), -S(O)₂ (aryl), S(O)₂ (alkoxyl) , -S(O)₂(aryloxy), -S(O)NH₂; -S(O)₂NH-lower alkyl, -S(O)₂NH-aryl, -S(O)₂N- (lower alkyl)₂, -S(O)₂N-(aryl)₂, -C(O)R₃, -C(O)OR₃, -C(0)NR_aR_b, -C(NH)NR_aR_b, -OC(O)R₃, -SC(O)R₃, -OC(O)OR₃, -SC(O)OR₃, -0C(0)NR_aR_b, -SC(O)NR_aR_b, -0C(NH)NR_aR_b, -SC(NH)NR₃R_b, -[NHC(O)J_nR₃, -[NHC(O)J_nOR₃, -[NHC(O)J_nNR₃R₁, and -[NHC(NH)J_nNR₃R_b, wherein n is an integer from 1 to 5, and wherein R₃ and R_b can be the same or different and are independently selected from the group consisting of hydrogen, halogen, trifluoromethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamide, and any of R₄ through Rg together can form a fused ring. In some embodiments, the compound is thiazolium or a salt thereof.

According to some embodiments of the present invention, a suitable thiazolium compound used according to the present invention can be pegylated at at least four sites and/or can be PEGylated in many differing PEG lengths and molecular weights. In some embodiments, the PEG moiety is PEG₂oo through PEG₅₀Oo-

In some embodiments, the compounds have the following structure:

R is H, CH₃, C₂H₅ and CH₃CO IX.

In further embodiments, the compounds have the following structure:

O,S- ^■(CH₂)₁₀(CH₂)CH₃ X. As shown below in the Scheme 1, typically, commercially available substituted or unsubstituted phenyl methyl ketones (1) are reacted with bromine in a non-polar solvent to produce the corresponding phenacyl bromides (2). Reaction of the reactive alpha-bromo ketones (2) with commercially available thioacetamide (3) in an protic solvent such as methanol with heat afforded the 2-methyl-4-phenylthiazoles (4). N-Alkylation of thiazoles (4) with PEGylated alkyl halides such as MeO-PEGlOOO-Cl (5, Biolink Life Sciences, Inc., Cary, NC BLS- 106- 1000) in aprotic solvents such as dimethylformamide and heat readily formed the corresponding PEGylated products (6). Reaction of these thiazolium halides (6) with (N,N'-disubstituted)amino benzaldehydes (7) in a protic solvent such as methanol with a basic catalyst such as piperidine and heat then produces the desired 2-(N,N'- dialkylaminostyryl)-3-(PEGylated)alkyl-4-phenylthiazolium halides (8). This synthetic scheme can be carried out in accordance with procedures and modifications known to those skilled in the art such as those described in U.S. Patent Nos. 3,641,012; 3,851,060 and 3,883,658. Scheme 1

i phenyl methyl ketones 2 phenacyl bromides 3 thioacetamide

6

8 2-(dialkylaminostyryl)-3-PEGylated alkyl- 4-phenylthiazolium halides

Both pyridinium and thiazolium compounds, homologs, analogs and derivatives thereof are capable of existing as geometric isomers. All such isomers, individually and as mixtures, are included within the scope of the present invention for their industrial uses. The E, E isomer is one configuration of the invention, and both the cisoid and transoid 2,6- conformations of the E,E-configuration are possible. Additionally, the ortho conformation of the structure can be formed in addition to the para and meta structures illustrated above. The ortho conformation structure can include the same salts and moieties as disclosed above and throughout the application.

Additionally, the pyridinium and thiazolium compounds, homologs, analogs and derivatives thereof can be encapsulated, formulated in lipid vesicles such as liposomes, micelles and bicelles and/or pegylated (PEG). As used herein, "encapsulated" refers to a formulation of a compound according to the present invention confined by a material or matter. The material or matter can be synthetic or of natural origin. Accordingly, a lipid vesicle provides an exemplary mechanism for encapsulation. Moreover, as understood by one skilled in the art, compounds of the present invention can further be encapsulated by application of a coating surrounding the compound. Such coatings can include biomaterials and further include materials discussed below in reference to microcapsules.

The amount of the compound encapsulated or formulated in lipid vesicles and/or pegylated can be determined by one skilled in the art based upon the method for which the compound is employed. In general, the compound can be present in an amount in a range from about 1 weight percent to about 50 weight percent or more of the formulation. Such encapsulated, lipid vesicle formulations and pegylated formulations can be water soluble. Microencapsulation

According to some embodiments, compounds used in the present invention can be encapsulated in microcapsules. As used herein, the term "microcapsules" is intended to contemplate single molecules, encapsulated discrete particulate, multiparticulate, liquid multicore and homogeneously dissolved active components. The encapsulation method may provide either a water soluble or oil soluble active component encapsulated in a shell matrix of either a water or oil soluble material. The microencapsulated active component may be protected from oxidation (e.g., UV) and hydration, and may be released by melting, rupturing, biodegrading, or dissolving the surrounded shell matrix or by slow diffusion of the active component through the matrix. Microcapsules usually fall in the size range of between about 1 and 2000 microns, although smaller and larger sizes are known in the art.

Compounds used in the present invention may be placed in a microcapsule for distribution. They may also be dispersed in a polymeric material or held as a liquid. No particular limitation is imposed on the shape for holding the active ingredient. In other words, there are various forms for holding the active ingredient by a holding mixture. Specific examples include microcapsules in which the surface of the active ingredient has been covered with the holding mixture; and products processed into a desired shape, each being obtained by kneading, mixing or blending the active ingredient in the holding mixture or forming a uniform solution of the holding mixture and the active ingredient, dispersing the active ingredient in the holding mixture by the removal of the solvent or the like and then processing the dispersion into a desired shape such as single molecule, molecular chain, liquid, sheet, film, tape or sphere. In addition, these processed products having a surface covered with a barrier layer for controlling the release of the active ingredient and those coated with a binder or binding substance for improving applicability can be given as examples.

The container formed of a holding mixture which container has an active ingredient enclosed therein as a liquid phase to secure uniform release ability based on a time-dependent criteria, a degradation criteria, exposure to a specific external stimulus or target trigger criteria.

The compound remains inside the microcapsules while the composition is packaged and in storage, i.e., in a closed container due to the partial pressure of the pyridinium compound salt surrounding the microcapsules. The compound is chemically stable during storage and after application until released from or permeating the capsule walls.

Suitable fill stabilizers absorb ultraviolet radiation in the range of about 270-350 nanometers and convert it to a harmless form. They have a high absorption coefficient in the near ultraviolet portion of the spectrum (e.g. a log molar extinction coefficient of from about 2 to 5) but only minimal absorption in the visible portion of the spectrum. They do not exhibit any substantial chemical reaction with the isocyanate groups and primary amine groups of the shell forming compounds during the microencapsulation process. Among the compounds which can be used as fill stabilizers are substituted benzophenones such as 2,4-dihydroxy benzophenone, 2-hydroxy-4-methoxy benzophenone, 2-hydroxy-4-octyloxy benzophenone, etc.; the benzotriazoles such as 2-(2-hydroxy-5'-methylphenyl) benzotriazole, 2-(3',5'-diallyl- 2'-hydroxylphenyl)benzotriazole, etc.; substituted acrylates such as ethyl 2-cyano-3,3- diphenyl acrylate, 2-ethylhexyl-2-cyano-3,3-diphenyl acetate, etc.; salicylates such as phenyl salicylates, 5-butyl phenyl salicylate, etc.; and nickel organic compounds such as nickel bis (octylphenol) sulfide, etc. Additional examples of each of these classes of fill stabilizers may be found in Kirk-Othmer, Encyclopedia of Chemical Technology. The fill stabilizers may comprise up to 5 percent, and are generally from about 0.01 to 2 percent, by weight of the microcapsule composition.

Another embodiment of the present invention may include heat sensitive materials that can facilitate preservation stability, particularly in resistance to light, and/or release the compound when exposed to a specific temperature. Microcapsules having an ultraviolet absorber enclosed therein as discussed in greater detail below, which are applicable to various fields. Desirable constituents that may be present in a base material include materials that can absorb heat and protect an underlying material from overheating. Thermal energy is absorbed by the phase change of such materials without causing an increase in the temperature of these materials. Suitable phase change materials include paraffinic hydrocarbons, that is, straight chain hydrocarbons represented by the formula C_nH_n+2, where n can range from 13 to 28. Other compounds that are suitable for phase change materials are a PABA salt, 2,2-dimethyl-l,3-propane diol (DMP), 2-hydroxymethyl-2-methyl-l,3-propane diol (HMP) and similar compounds. Also useful are the fatty esters such as methyl palmitate. Phase change materials that can be used include paraffinic hydrocarbons.

The microcapsules can retain an ultraviolet absorber and/or microcapsule formulations can include suitable ultraviolet ray absorbing efficiency, exhibit resistance to being ruptured at a usual pressure or rupture upon exposure to a target stimulus.

Further, the present invention provides the use of microcapsules having an ultraviolet absorber and as required an organic solvent enclosed therein, which have capsule wall film of synthetic resin and mean particle size of about 0.1 to 3μm.

The following are examples of ultraviolet absorbers that may be used in the present invention. Phenyl salicylate, p-tert-butylphenyl salicylate, p-octylphenyl salicylate and like salicylic acid type ultraviolet absorbers; 2,4-dihydroxybenzophenone, 2-hydroxy-4- methoxybenzophenone, 2-hydroxy-4-octyloxybenzophenone, 2-hydroxy-4- dodecyloxybenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone, 2,2,'-dihydroxy-4,4'- dimethoxybenzophenone, 2-hydroxy-4-methoxy-5-sulfobenzophenone and like benzophenone type ultraviolet absorbers; 2-ethylhexyl 2-cyano-3,3-diphenyt-acrylate, ethyl 2-cyano-3,3-diphenylacrylate and like cyanoacrylate type ultraviolet absorbers; bis(2,2,6,6- tetramethyl-4-piperidyl) sebacate, bis(2,2,6,6-tetramethyl-4-piperidyl) succinate, bis( 1 ,2,2,6,6-pentamethyl-4-piperidyl) 2-(3 ,5-di-tert-butyl-4-hydroxybenzyl)-2-n-butyl malonate and like hindered amine type ultraviolet absorbers; 2-(2'- hydroxyphenyl)benzotriazole, 2-(2'-hydroxy-5'-methylphenyl)benzotriazole, 2- (2'-hydroxy-5 -tert-butylphenyl)benzotriazole, 2- (2'-hydroxy-3',5'-di-tert-butylphenyl)benzotriazole, 2- (2'- hydroxy-3'-tert-butyl-5'-methylphenyl)-5-chlorobenzotriazole, 2-(2'-hydroxy-3',5'-di-tert- butylphenyl)-5 -chlorobenzotriazole, 2-(2'-hydroxy-3 ',5 '-di-tert-butylphenyl)-5 -tert- butylbenzotriazole, 2-(2^l-hydroxy-3',5'-di-tert-amylphenyl)benzotriazole, 2-(2'-hydroxy-3',5'- di-tert-amylphenyl)-5-tert-amylbenzotriazole, 2-(2^l-hydroxy-3',5^l-di-tert-amylphenyl)-5- methoxybenzotriazole, 2-[2^l-hydroxy-3^I-(3",4",5",6"-tetrahydrophthalimido-methyl)-5^l- methylpheny l]benzotriazole, 2-(2'-hydroxy-5'-tert-octylphenyl)benzotriazole, 2-(2'-hydroxy- 3'-sec-butyl-5'-tert-butylphenyl)benzotriazole, 2-(2'-hydroxy-3'-tert-amyl-5'-phenoxyphenyl)- 5-methylbenzotriazole, 2-(2'-hydroxy-5'-n-dodecylphenyl)benzotriazole, 2-(2'-hydroxy-5'- sec-octyloxyphenyl)-5-phenylbenzotriazole, 2-(2'-hydroxy-3'-tert-amyl-5'-phenylphenyl)-5- methoxybenzotriazole, 2-[2'-hydroxy-3',5'-bis(α,α-dimethylbenzyl)phenyl]benzotriazole and like benzotriazole type ultraviolet absorbers which are solid at ordinary temperature; 2-(2'- Hydroxy-3'-dodecyl-5'-methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-undecyl-5'- methylphenyl)-benzotriazole, 2-(2'-hydroxy-3 '-tridecyl-5 '-methylphenyl)-benzotriazole, 2-(2'- hydroxy-3'-tetradecyl-5'-methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-pentadecyl-5'- methylphenyl)-benzotriazole, 2-(2'-hydroxy-3'-hexadecyl-5^l-methylphenyl)-benzotriazole, 2- [2'-hydroxy-4^l-(2"-ethylhexyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(2"- ethylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(2"-ethyloctyl)oxyphenyl]- benzotriazole, 2-[2'-hydroxy-4'-(2"-propyloctyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'- (2"-propylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(2"-propylhexyl)oxyphenyl]- benzotriazole, 2-[2'-hydroxy-4'-(l"-ethylhexyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'- (1 "-ethylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy-4'-(l "-ethyloctyl)oxyphenyl]- benzotriazole, 2-[2'-hydroxy-4'-(l"-propyloctyl)oxyphenyl] -benzotriazole, 2-[2'-hydroxy-4'- (1 "-propylheptyl)oxyphenyl]-benzotriazole, 2-[2'-hydroxy4'-(l "-propylhexyl)oxyphenyl]- benzotriazole, 2-(2'-hydroxy-3'-sec-butyl-5'-tert-butylphenyl-5-n-butylbenzotriazole, 2-(2'- hydroxy-3'-sec-butyl-5'-tert-butylphenyl) -5-tert-pentyl-benzotriazole, 2-(2'-hydroxy-3'-sec- butyl-5'-tert-butylphenyl)-5-n-pentyl-benzotriazole, 2-(2'-hydroxy-3'-sec-butyl-5'-tert- pentylphenyl)-5-tert-butylbenzotriazole , 2-(2'-hydroxy-3'-sec-butyl-5'-tert-pentylphenyl)-5- n-butylbenzotriazole, 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)-5-sec-butylbenzotriazole, 2-(2'- hydroxy-3',5'-di-tert-pentylphenyl)-5-sec-butylbenzotriazole, 2-(2'-hydroxy-3'-tert-butyl-5'- tert-pentylphenyl)-5-sec-butylbenzotriazole, 2-(2'-hydroxy-3',5'-di-sec-butylphenyl)-5- chlorobenzotriazole, 2-(2'-hydroxy-3',5'-di-sec-butylphenyl)-5-methoxybenzotriazole, 2-(2'- hydroxy-3',5'-di-sec-butylphenyl)-5-tert-butylbenzotriazole, 2-(2'-hydroxy-3',5'-di-sec- butylphenyl)-5-n-butylbenzotriazole, octyl 5-tert-butyl-3-(5-chloro-2H-benzotriazole-2-yl)-4- hydroxybenzene-propionate, condensate of methyl 3-[3-tert-butyl-5-(2H-benzotriazole-2-yl)- 4-hydroxyphenyl]propionate and polyethylene glycol (molecular weight: about 300) and like benzotriazole type ultraviolet absorbers which are liquid at ordinary temperature. Of course, the ultraviolet absorber is not limited to thereabove and can be used as required in a mixture of at least two of them.

The microcapsules for use in the present invention can be prepared by various known methods. They are prepared generally by emulsifying and dispersing the core material (oily liquid) comprising an ultraviolet absorber and, if necessary, an organic solvent in an aqueous medium, and forming a wall film of high-molecular-weight substance around the resulting oily droplets. The present invention may also include the use of an organic solvent together with an ultraviolet absorber in some embodiments of the present invention. The organic solvent is not particularly limited and various hydrophobic solvents can be used which are used in a field of pressure sensitive manifold papers. Examples of organic solvents are tricresyl phosphate, octyldiphenyl phosphate and like phosphates, dibutyl phthalate, dioctyl phthalate and like phthalates, butyl oleate and like carboxylates, various fatty acid amides, diethylene glycol dibenzoate, monoisopropylnaphthalene, diisopropylnaphthalene and like alkylated naphthalenes, 1 -methyl- 1 -phenyl- 1 -tolylmethane, 1 -methyl- 1 -phenyl- 1 -xylylmethane, 1- phenyl-1-tolylmethane and like alkylated benzenes, isopropylbiphenyl and like alkylated biphenyls, trimethylolpropane triacrylate and like acrylates, ester of polyols and unsaturated carboxylic acids, chlorinated paraffin and kerosene. These solvents can be used individually or in a mixture of at least two of them. Among these hydrophobic media having a high boiling point, tricresyl phosphate and 1 -phenyl- 1 -tolylmethane are desirable since they exhibit high solubility in connection with the ultraviolet absorber to be used in the present invention. Generally, the lower the viscosity of the core material, the smaller is the particle size resulting from emulsification and the narrower is the particle size distribution, so that a solvent having a low boiling point is conjointly usable to lower the viscosity of the core material. Examples of such solvents having a low boiling point are ethyl acetate, butyl acetate, methylene chloride, etc. Additionally, an absorber may be utilized. An absorber should be selected which reduces the sensitivity of the microcapsule in those portions of its spectral sensitivity range which interfere with the exposure of microcapsules at other wavelengths (its inactive range) without overly reducing the sensitivity of the microcapsule in those portions of the spectral sensitivity range in which the microcapsule is intended to be exposed (its active range). In some cases it may be necessary to balance the absorption characteristics of the absorber in the active range and the inactive range to achieve optimum exposure characteristics. Generally, absorbers having an extinction coefficient greater than about 100/M cm in the inactive range and less than about 100,000/M cm in the active range of the microcapsule are used. When the absorber is directly incorporated into the photosensitive composition, ideally, it should not inhibit free radical polymerization, and it should not generate free radicals upon exposure.

Ultraviolet absorbers that may be desirable include those selected from hydroxybenzophenones, hydroxyphenylbenzo-triazoles and formamidines. The absorbers may be used alone or in combination to achieve the spectral sensitivity characteristics that are desired. Representative examples of useful hydroxybenzophenones are 2-hydroxy-4-n- octoxybenzophenone (UV-CHEK AM-300 from Ferro Chemical Division, Mark 1413 from Argus Chemical Division, Witco Chem. Corp., and Cyasorb UV-531 Light Absorber from American Cyanamid), 4-dodecyl-2-hydroxybenzophenone (Eastman Inhibitor DOBP from Eastman Kodak), 2-hydroxy-4-methoxybenzophenone (Cyasorb UV-9 Light Absorber from American Cyanamid), and 2,2'-dihydroxy-4-methoxybenzophenone (Cyasorb UV-24 Light Absorber from American Cyanamid). Representative examples of useful hydroxybenzophenyl benzotriazoles are 2-(2'-hydroxy-5'-methylphenyl)benzotriazole (Tinuvin P from Ciba-Geigy Additives Dept.), 2-(3',5'-ditert-butyl-2'hydroxyphenyl)-5- chlorobenzotriazole (Tinuvin 327 from Ciba-Geigy), and 2-(2-hydroxy-5-t- octylphenyl)benzotriazole (Cyasorb UV-541 1 Light Absorber from American Cyanamid). Representative examples of useful formamidines are described in U.S. Patent No. 4,021,471 and include N-(p-ethoxy-carbonylphenyl)-N'-ethyl-N'-phenylformamidine (Givsorb UV-2 from Givaudan Corp.). The optimum absorber and concentration of absorber for a particular application depends on both the absorption maximum and extinction coefficient of the absorber candidates and the spectral sensitivity characteristics of the associated photoinitiators.

Liposomal Formulations

As used herein, the term "liposome" refers to a structure including a lipid bilayer enclosing at least one aqueous compartment. The walls are prepared from lipid molecules, which have the tendency both to form bilayers and to minimize their surface area. The lipid molecules that comprise the liposome have hydrophilic and lipophilic portions. Upon exposure to water, the lipid molecules form a bilayer membrane wherein the lipid ends of the molecules in each layer are directed to the center of the membrane, and the opposing polar ends form the respective inner and outer surfaces of the bilayer membrane. Thus, each side of the membrane presents a hydrophilic surface while the interior of the membrane comprises a lipophilic medium.

Liposomes can be classified into several categories based on their overall size and the nature of the lamellar structure. The classifications include small unilamellar vesicles (SUV), multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and oligolamellar vesicles. SUVs range in diameter from approximately about 20 to 50 nanometers and can include a single lipid bilayer surrounding an aqueous compartment. A characteristic of SUVs is that a large amount of the total lipid, about 70%, is located in the outer layer of the bilayer. Where SUVs are single compartment vesicles of a fairly uniform size, MLVs vary greatly in diameter up to about 30,000 nanometers and are multicompartmental in their structure wherein the liposome bilayers can be typically organized as closed concentric lamellae with an aqueous layer separating each lamella from the next. Large unilamellar vesicles are so named because of their large diameter, which ranges from about 600 nanometers to 30 microns. Oligolamellar vesicles are intermediate liposomes having a larger aqueous space than MLVs and a smaller aqueous space than LUVs. Oligolamellar vesicles have more than one internal compartment and possibly several concentric lamellae, but they generally have fewer lamellae than MLVs.

A variety of methods for preparing liposomes are known in the art, several of which are described in Liposome Technology (Gregoriadis, G., editor, three volumes, CRC Press, Boca Raton 1984) or have been described by Lichtenberg and Barenholz in Methods of Biochemical Analysis, Volume 33, 337-462 (1988). Further methods of preparing liposomal formulations can be found in U.S. Patent Nos. 7,022,336; 6,989,153; 6,726,924; 6,355,267; 6,110,491; 6,007,838; 5,094,785 and 4,515,736. Liposomes are also well recognized as useful for encapsulating biologically active materials. Preparation methods particularly involving the encapsulation of DNA by liposomes, and methods that have a direct application to liposome-mediated transfection, have been described by Hug and Sleight in Biochimica and Biophysica Acta, 1097, 1-17 (1991).

The liposomes used in the present invention can be prepared from phospholipids, but other molecules of similar molecular shape and dimensions having both a hydrophobic and a hydrophilic moiety can be used. For the purposes of the present invention, all such suitable liposome-forming molecules will be referred to herein as lipids. One or more naturally occurring and/or synthetic lipid compounds may be used in the preparation of the liposomes.

Representative suitable phospholipids or lipid compounds for forming initial liposomes useful in the present invention include, but are not limited to, phospholipid-related materials such as phosphatidylcholine (lecithin), lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, phosphatidylcholine, and dipalmitoyl-phosphatidylglycerol. Additional nonphosphorous- containing lipids include, but are not limited to, stearylamine, dodecylamine, hexadecyl- amine, acetyl palmitate, glycerol ricinoleate, hexadecyl sterate, isopropyl myristate, amphoteric acrylic polymers, fatty acid, fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, diacylglycerolsuccinate, and the like. As understood by one skilled in the art, different lipids can be used with different properties, cationic, anionic or neutral, but the preparation method can remain the same regardless of which lipid combination is used. More specifically, once lipids have been selected for use in the liposome, they can be dissolved in an organic solvent to ensure complete mixing. The organic solvent can be removed by evaporation followed by drying and a lipid film remains of the homogenous lipid mixture. The lipid mixture can be frozen in cakes and dried. The lipid cakes can be stored frozen until hydration.

The addition of an aqueous medium and agitation of the container hydrate the lipid cake. The resulting product is a large, multilamellar vesicle. This structure can include concentric rings of lipid bilayers separated by water. The large, multilamellar vesicles can be downsized by the application of energy, either in the form of mechanical energy in the process of extrusion or by sonic energy in sonication. The hydrated lipid can be forced though a polycarbonate filter with progressively smaller pores to produce particles with a diameter of similar size to the pore. Before the final pore size is used, the lipid suspension may be subjected to several freeze-thaw cycles to ensure the final particles are homogenous in size. Final particle size is partly dependent on the lipid combination used. The mean particle size is reproducible from batch to batch. This process can produce large, unilamellar vesicles that can be reduced to small, unilamellar vesicles by the application of sonic energy from a sonicator. The particles in the test tube being sonicated can be removed by centrifugation. Mean size of the resulting vesicles can be influenced by composition, concentration, volume and temperature of the lipid mixture and duration, power, and tuning of the sonicator.

Specific liposome preparation methods include, but are not limited to, the hand- shaken method, sonication method, reverse-phase evaporation method, freeze-dried rehydration method, and the detergent depletion method. According to the hand-shaken method, in order to produce liposomes, lipid molecules are introduced into an aqueous environment. When dry lipid film is hydrated the lamellae swell and grow into myelin figures. Mechanical agitation, for example, vortexing, shaking, swirling or pipetting, causes myelin figures (thin lipid tubules) to break and reseal the exposed hydrophobic edges resulting in the formation of liposomes.

The sonication method can be used to prepare small unilamellar vesicles. Two exemplary sonication techniques include probe sonication and bath sonication. During probe sonication, the tip of a sonicator is directly immersed into the liposome dispersion. The dissipation of energy at the tip can result in local overheating, and therefore, the vessel may be immersed into an ice/water bath. During bath sonication, the liposome dispersion in a tube is placed into a bath sonicator. Material being sonicated can be kept in a sterile container, unlike the probe units, or under an inert atmosphere.

The reverse-phase evaporation method is based on the formation of inverted micelles. More specifically, inverted micelles are formed upon sonication of a mixture of a buffered aqueous phase, which includes contains the water-soluble molecules to be encapsulated into the liposomes and an organic phase in which the amphiphilic molecules are solubilized. The slow removal of the organic solvent leads to transformation of these inverted micelles into a gel-like and viscous state. During some point in this procedure, the gel state collapses and some of the inverted micelles disintegrate. The excess of phospholipids in the environment contributes to the formation of a complete bilayer around the remaining micelles, which results in formation of liposomes. Liposomes made by reverse phase evaporation method can be made from various lipid formulations and have a tendency to possess aqueous volume-to- lipid ratios that are four times higher than multilamellar liposomes or hand-shaken liposomes. During the freeze-dried rehydration method, freeze-dried liposomes are formed from preformed liposomes. During dehydration, the lipid bilayers and the materials to be encapsulated into the liposomes are brought into close contact. Upon reswelling, the chances for encapsulation of the adhered molecules increases. The aqueous phase is generally added in small portions to the dried materials. As a general rule, the total volume used for rehydration is less than the starting volume of the liposome dispersion.

The detergent depletion method can be used for preparation of a variety of liposomes and proteoliposome formulations. Detergents can be depleted from a mixed detergent-lipid micelles by various techniques that lead to the formation of homogeneous liposomes. In practice, lipids below their phase transition temperature can be used with this preparation method. However, not all detergents are suited for this method. Exemplary detergents include, but are not limited to, sodium cholate, alkyl(thio)glucoside, and alkyloxypolyethylenes. Mixed micelles are prepared by adding the concentrated detergent solution to multilamellar liposomes (the final concentration of the detergent should be well above the critical micelle concentration (CMC) of the detergent). The use of different detergents can result in different size distributions of the vesicles formed. Faster depletion rates can produce smaller size liposomes. The use of different detergents may also result in different ratios of large unilamellar vesicles/ oligolamellar vesicles/multilamellar vesicles. Micelles and Bicelles

As used herein, "micelle" refers to an aggregate of surfactant molecules dispersed in a liquid colloid. Micelles can be globular in shape, but may exist in other shapes including, but not limited to, ellipsoids, cylinders, bilayers, and vesicles. The shape of a micelle can be controlled largely by the molecular geometry of its surfactant molecules; however, shape also depends on the conditions (such as temperature or pH, and the type and concentration of any added salt). In a micelle, the hydrophobic tails of several surfactant molecules assemble into an oil-like core that has less contact with water. In contrast, surfactant monomers are surrounded by water molecules that create a "cage" of molecules connected by hydrogen bonds. In a nonpolar solvent, the hydrophilic groups form the core of the micelle, and the hydrophobic groups remain on the surface of the micelle (so-called reverse micelle).

Micelles may form when the concentration of surfactant is greater than the critical micelle concentration (CMC), and the temperature of the system is greater than the critical micelle temperature, or Krafft temperature. In water, the hydrophobic effect is the driving force for micelle formation, despite the fact that assembling surfactant molecules together reduces their entropy. Generally, above the CMC, the entropic penalty of assembling the surfactant molecules is less than the entropic penalty of the caging water molecules.

As used herein, the term "bicelle" refers to a bilayered mixed micelle. Bicelles can be characterized as a mixture of long-chain bilayer forming phospholipids and short-chain micelle forming lipids of detergents.

The preparation of micelles and bicelles are well known in the art. Further details regarding preparation of these structures can be found in U.S. Patent Nos. 6,897,297; 6,696,081 ; 6,586,559; 6,444,793;and 5,534,259.

PEGylation In general, attachment of polyalkylene moieties as described herein can be employed to reduce immunogenicity and/or extend the half-life of the native compounds discussed herein. Any conventional PEGylation method can be employed, provided that the PEGylated agent retains pharmaceutical activity. See also Schacht, E.H. et al. Poly(ethylene glycol) Chemistry and Biological Applications, American Chemical Society, San Francisco, CA 297- 315 (1997).

Polyalkylene glycol is a biocompatible polymer where, as used herein, polyalkylene glycol refers to straight or branched polyalkylene glycol polymers such as polyethylene glycol, polypropylene glycol, and polybutylene glycol, and further includes the monoalkylether of the polyalkylene glycol. In some embodiments of the present invention, the polyalkylene glycol polymer is a lower alkyl polyalkylene glycol moiety such as a polyethylene glycol moiety (PEG), a polypropylene glycol moiety, or a polybutylene glycol moiety. PEG has the formula HO(CH2CH2O)_nH, where n can range from about 1 to about 4000 or more. In some embodiments, n is 1 to 100, and in other embodiments, n is 5 to 30. PEG can range from average molecular weight of about 90 to about 180,000 or more. For example, an average molecular weight of about 300 can correspond to n is 5-6, an average molecular weight of about 2,300 can correspond to n is 50, an average molecular weight of about 13,300 can correspond to n is 300 and an average molecular weight of about 22,000 can correspond to n is 500. In some embodiments, the PEG moiety can be linear or branched. In further embodiments, PEG can be attached to groups such as hydroxyl, alkyl, aryl, acyl or ester. In some embodiments, PEG can be an alkoxy PEG, such as methoxy-PEG (or mPEG), where one terminus is a relatively inert alkoxy group, while the other terminus is a hydroxyl group.

PEG moieties are well known in the art and can be synthesized or are commercially available products that can be readily obtained. See, for example, http://www.biolinkonline.com/MPEG%20CATALOG.pdf.

According to some embodiments of the present invention, the pegylated compounds used in the present invention can be water soluble, soluble in isopropyl alcohol (IPA), ethanol (EtOH), dimethyl sulfoxide (DMSO) and methanol (MeOH), less sensitive to UV light than a non-pegylated counterpart and/or economical to synthesize.

Pyridinium compounds suitable for PEGylation include, but are not limited to those described herein. Moreover, suitable pyridinium compounds include those described in U.S. Patent Application Serial No.10/792,339, filed March 3, 2004, U.S. Application Serial No. 10/792,495, filed March 3, 2004, and U.S. Application Serial No. 10/792,496, filed March 3, 2004. A suitable pyridinium compound of the present invention can be pegylated at at least four sites (the 1-, 3-, 4- and 5- positions on the central pyridinium core) and/or can be PEGylated in many differing PEG lengths and molecular weights. In some embodiments, the PEG moiety is PEG₂₀₀ through PEG₅₀₀₀.

Thiazolium compounds suitable for PEGylation include, but are not limited to, those described herein. Moreover, suitable thiazolium compounds include those described in published PCT application WO 2006/065942. A suitable thiazolium compound used according to the present invention can be pegylated at at least four sites and/or can be PEGylated in many differing PEG lengths and molecular weights. In some embodiments, the PEG moiety is PEG₂₀₀ through PEG_5O00. Pegylated compounds used in the present invention can further exhibit improved solubility, enhanced bioavailability, improved stability, lower toxicity, decreased degradation and chemical sensitivities and/or increased conjugation potential to like molecules and other drug molecules. 2. Methods of Using Pyridinium and Thiazolium Compounds, Homologs, Analogs and Derivatives Thereof

Embodiments of the present invention provide methods of using pyridinium (for example, stilbazium) and/or thiazolium compounds, homologs, analogs and derivatives thereof as reagents, diagnostic tools or therapeutic agents. Reagents

According to embodiments of the present invention, these compounds can serve as targeting moieties, stains and/or dyes, fluorescent stains and/or fluorescent dyes. More specifically, pyridinium compounds such as stilbazium chloride as well as other stilbazium salts, analogs and homologs can exhibit classic staining properties and can bond to cloth, paper, wood, plastic, glass, metal and other substrates, as well as skin and related living or dead tissues, while retaining its red or pink or other coloration. Thiazolium compounds, salts thereof, analogs and homologs can function in a similar manner.

One mechanism of action may include a strong covalent bond being formed between the nucleophile (Nu:) and one of the stilbazium chloride side chains. The other side chain retains the chromophore and fluorophore as shown in the scheme below.

Scheme 2.

Stilbazium

Chlonde

The nucleophile may be a nucleic acid, a microorganism, an amino acid, a cell, or an analyte. Additionally, in particular embodiments, the nucleic acid, amino acid, cell, or analyte may be bound to a nucleophile such as OH, 0^", NH₂, and the like before the nucleophile is covalently bonded to stilbazium chloride, salts, analogs and homologs thereof. Thiazolium compounds, salts thereof, analogs and homologs may act in a similar manner.

The pyridinium and/or thiazolium compounds, homologs, analogs and derivatives thereof may be fast acting, staining the object of interest in less than 10 minutes, and in some embodiments, seconds. These compounds may be visible in bright field and/or under fluorescence. Moreover, these compounds may provide a non-toxic staining option for viable cells and can provide repeatable staining of living cells. Additionally, these compounds may be used in multiple assays of the same cultures. The pyridinium and/or thiazolium compounds, homologs, analogs and derivatives thereof may further provide detection of live cell mitotic division. These compounds are believed to be safe to users and user-friendly.

Additionally, these compounds may exhibit stability at room temperature for a period of time sufficient to allow appropriate assays to be performed.

Methods of screening cells treated with dyes and fluorescent reagents are well known in the art. There is a considerable body of literature related to genetic engineering of cells to produce fluorescent proteins, such as modified green fluorescent protein (GFP), as a reporter molecule. Some properties of wild-type GFP are disclosed by Morise et al. (Biochemistry 13 (1974), p. 2656-2662), and Ward et al. (Photochem. Photobiol. 31 (1980), p. 61 1-615).

The types of biochemical and molecular information now accessible through fluorescence-based reagents applied to cells include ion concentrations, membrane potential, specific translocations, enzyme activities, gene expression, as well as the presence, amounts and patterns of metabolites, proteins, lipids, carbohydrates, and nucleic acid sequences (DeBiasio et al., (1996) MoI. Biol. Cell. 7:1259; Giuliano et al., (1995) Ann. Rev. Biophys. Biomol. Struct. 24:405; Heim and Tsien, (1996) Curr. Biol. 6:178). High-content screens can be performed on either fixed cells, using fluorescently labeled antibodies, biological ligands, and/or nucleic acid hybridization probes, or live cells using multicolor fluorescent indicators and "biosensors." The choice of fixed or live cell screens depends on the specific cell-based assay required.

As understood by one of skill in the art, any of the methods and screens discussed above may be utilized with the compounds described herein.

Some aspects of the present invention utilize the high affinity and/or fluorescence or luminescence of the pyridinium and/or thiazolium molecules and analogs with respect to contacting specific cellular components, such as organelles. The affinity for specific components is governed by forces such as ionic interactions, covalent bonding (which includes chimeric fusion with protein-based chromophores, fluorophores, and lumiphores), as well as hydrophobic interactions, electrical potential, and, in some cases, simple entrapment within an organelle.

Aspects of the present invention may include utilizing the pyridinium and/or thiazolium compounds as staining agentsfor cell components in their isolated state or for isolated cells and organelles, including utilizing the compounds as staining agents for cell based assays. Further aspects may include utilizing the compounds to identify and/or stain tissues and/or organs in whole animals, excised tissue and/or organs, as well as embryos, larvae, nematodes, insects and other parasites. In some embodiments of the present invention, these compounds may be complexed with specific agents or antibodies for imaging of specific organ tissues in the whole animal model systems. The term "antibody" or "antibody molecule" in the various grammatical forms as used herein refers to an immunoglobulin molecule (including IgG, IgE, IgA, IgM, IgD) and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope and can bind antigen. An "antibody combining site" or "antigen binding site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable (CDR) regions that specifically binds antigen. As is known in the art, particular properties of antibodies relate to immunoglobulin isotype. In representative embodiments, the antibody or antigen-binding fragment is an IgG2a, an IgGl or an IgG2b isotype molecule. The antibody or fragment can further be from any species of origin including avian (e.g., chicken, turkey, duck, geese, quail, etc.) and mammalian (e.g., human, non-human primate, mouse, rat, rabbit, cattle, goat, sheep, horse, pig, dog, cat, etc.) species.

Accordingly, in some embodiments, the invention features a technique for determining, directly or indirectly, the presence, location, or quantity of a cell, and thus, cellular organelles such as the nucleus, smooth and/or rough endoplasmic reticulum, centrosome, cytoskeleton, cell wall, cell membrane, flagella, cilia, chloroplast, mitochondria, golgi apparatus, ribosome, lysosome, centriole, acrosome, glyoxysome, secretory vesicle, peroxisome, vacuole, melanosome, myofibril and parenthesome. In other embodiments, the invention features a technique for determining, directly or indirectly, the presence, location, quantity and/or health of analytes and microorganisms.

Embodiments of the invention also include assays which may determine the presence or absence of a cell, analyte, nucleic acid or microorganism in a sample, such as a fluid or aqueous sample, suspected of containing a microorganism, said assay comprising combining the sample with a labeling reagent to form a labeled cell, nucleic acid or microorganism, said labeling reagent comprising a dye which directly stains the cell, analyte, nucleic acid or microorganism to provide a stained sample comprising a stained cell, analyte, nucleic acid or microorganism, wherein said dye is a compound represented by the following structure:

or a solvate thereof wherein the compound is substantially in the E, E configuration, or the compound can also be in the E,Z or Z,Z configuration. The amino moieties can be in either the ortho, meta or para positions. The anion X^" can be an anionic salt. The anion X^" can be fluoride, chloride, bromide, iodide, halide, methanesulfonate (mesylate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), napthylate, m- nitrobenzenesulfonate (nosylate), para-aminobenzoate, lauryl sulfate, 2,4-dihydroxy benzophenone, or 2-(2-hydroxy-5'-methylphenyl) benzotriazole. Rj and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, C _MO alkyl (linear or branched) and alkenes (linear or branched), or wherein Ri and R₂ may be taken together with the nitrogen atom to which they are attached form substituted or unsubstituted pyrrolidino or piperidino rings. R₃ can be selected from the group consisting of methyl, ethyl, C_MO alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties. R₃ may also be an organometallic compound such as organotin, organosilicon, or organogermanium. Additionally, R₃ may be (CH₂)_H-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound. In some embodiments, R₃ is selected from the group consisting of methyl, ethyl, C_MO alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i- propyl, n-butyl, i-butyl, an organometallic compound, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties. R₃ can also be a polyalkylene glycol moiety including a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol. The end-terminals of these polyethylene glycol moieties can be hydroxy, methoxy, ethoxy and acetyloxy. R₄ through R₈ are the same or different and may be selected from the group consisting of hydrogen, C_{M O} alkyl (linear or branched), representative examples of alkyl including, but not limited to, n-propyl, i-propyl, n-butyl, i- butyl, alkenes (linear or branched), alkynes, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties, hydroxy, alkoxy, SCH₃, (Ci-C₃) alkylthio, SH, (Ci -C₃) haloalkoxy, (Ci-C₃) perhaloalkoxy, NH₂, NH(lower alkyl), N(Io wer alkyl)₂, halogen, (Ci-C₃) haloalkyl, (Ci-C₃) perhaloalkyl, -CF₃, -CH₂CF₃, -CF₂CF₃, -CN, -NC, -OCN, -SCN, -NO, -NO₂, -N₃, -S(O) (lower alkyl), -S(O) (aryl), -S(O)₂ (lower alkyl), -S(O)₂ (aryl), S(O)₂ (alkoxyl) , -S(O)₂(aryloxy), -S(O)NH₂; -S(O)₂NH-lower alkyl, -S(O)₂NH-aryl, -S(O)₂N- (lower alkyl)₂, -S(O)₂N-(aryl)₂, -C(O)R₃, -C(O)OR₃, -C(0)NR_aR_b, -C(NH)NR₃R,,, -OC(O)R₃, -SC(O)R₃, -OC(O)OR₃, -SC(O)OR₃, -0C(0)NR_aR_b, -SC(O)NR₃R_b, -OC(NH)NR₃R,,, -SC(NH)NR₃R_b, -[NHC(O)J_nR₃, -[NHC(O)J_nOR₃, -[NHC(O)J_nNR₃R_b and -[NHC(NH)J_nNR₃R_b, wherein n is an integer from 1 to 5, and wherein R₃ and R_b can be the same or different and are independently selected from the group consisting of hydrogen, halogen, trifluoromethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamide, and any of R₄ through R₈ together can form a fused ring. In some embodiments, the compound is thiazolium or a salt thereof.

The dye can be encapsulated. In some embodiments, the dye can be loaded into a microcapsule or a lipid vesicle such as a liposome, micelle and/or bicelle, to form a microencapsulated formulation or a lipid vesicle formulation, respectively. The dye can also be pegylated. In various embodiments of the invention, a cell may be bound by one of the compounds according to Formula VIII. In some embodiments, the cell is a prokaryotic cell, such as a gram-negative or gram-positive bacterial cell. In other embodiments, the cell is a eukaryotic cell. For example, the cell may be a yeast, Caenorhabditis, Xenopus, Drosophila, zebrafish, squid, plant, mammalian, embryonic, or human cell. In yet other embodiments, the cell or the sample is contacted with the fluorophore by incubating the cell or the sample with the fluorophore. In still other embodiments, the fluorophore is injected into the cell or administered to a plant, embryo, mammal, transgenic animal, or human including the cell.

By "fluorophore" is meant a compound that is capable of emitting a fluorescent signal. As described herein, fluorophores of use in the invention have a higher fluorescence intensity when bound to a nucleic acid, protein, or cell component than when unbound in solution. The fluorescence intensity of the bound fluorophore can be at least about 1, 5, 10, 50, 100, 500, or 1000 times that of the unbound fluorophore in an aqueous solution. Examples of conditions that may enhance the fluorescence of the bound fluorophore include rigidification, conformational restriction, and sequestration from solvent. In one embodiment, the fluorophore does not covalently bind the aptamer.

Other fluorophores have a lower fluorescence intensity when bound to a nucleic acid, protein, or cell component than when unbound in solution. The fluorescence intensity of the bound fluorophore can be at least about 2, 5, 10, 50, 100, 500, or 1000 less than that of the unbound fluorophore in an aqueous solution. An example of a condition that may decrease the fluorescence of the bound fluorophore is a change in the conformation of the fluorophore that decreases its fluorescence intensity. In one embodiment, the fluorophore does not covalently bind the aptamer.

Other fluorophores for use in multiplexing, such as calcium-sensing dyes, may adopt at least two different conformational states that result in different fluorescence intensities. An aptamer of the invention may modulate the fluorescence of the fluorophore by increasing the percentage of the fluorophore in a particular conformational state with increased or decreased fluorescence.

The fluorophore is soluble in an aqueous solution at a concentration of about 0.1 μM, lμM, lOμM, and 50μM. Incubating a cell with these concentrations of the fluorophore may not affect the viability of the cell. In another embodiment, incubating a cell with the fluorophore at these concentrations for as few as about 5 minutes to as many as about 1, 2, 4, 8, 12, 24, 36 or more hours does not require the presence of another compound to prevent toxic effects of the fluorophore, such as the inactivation of proteins in the cell; inhibition of replication, transcription, or translation; or the induction of cell death. By "cell permeable" is meant capable of migrating through a cell membrane or cell wall into the cytoplasm or periplasm of a cell by active or passive diffusion. The fluorophore can migrate through both the outer and inner membranes of gram-negative bacteria or both the cell wall and plasma membrane of plant cells. Additionally, the fluorophore can be used to visualize a cell, analyte and/or nucleic acids in an in vitro sample.

Embodiments of the present invention may include probes comprising a single- stranded oligonucleotide and a compound represented by Formula VIII:

or a solvate thereof wherein the compound is substantially in the E, E configuration, or the compound can also be in the E₅Z or Z,Z configuration. The amino moieties can be in either the ortho, meta or para positions. The anion X^" can be an anionic salt. The anion X^" can be fluoride, chloride, bromide, iodide, halide, methanesulfonate (mesylate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), napthylate, m- nitrobenzenesulfonate (nosylate), para-aminobenzoate, lauryl sulfate, 2,4-dihydroxy benzophenone, or 2-(2-hydroxy-5'-methylphenyl) benzotriazole. R₁ and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, C_1-10 alkyl (linear or branched) and alkenes (linear or branched), or wherein R₁ and R₂ may be taken together with the nitrogen atom to which they are attached form substituted or unsubstituted pyrrolidino or piperidino rings. R₃ can be selected from the group consisting of methyl, ethyl, C_MO alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties. R₃ may also be an organometallic compound such as organotin, organosilicon, or organogermanium. Additionally, R₃ may be (CHi)_n-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound. In some embodiments, R₃ is selected from the group consisting of methyl, ethyl, C_1-Io alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i- propyl, n-butyl, i-butyl, an organometallic compound, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties. R₃ can also be a polyalkylene glycol moiety including a C_1-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol. The end-terminals of these polyethylene glycol moieties can be hydroxy, methoxy, ethoxy and acetyloxy. R₄ through R₈ are the same or different and may be selected from the group consisting of hydrogen, C_1-I0 alkyl (linear or branched), representative examples of alkyl including, but not limited to, n-propyl, i-propyl, n-butyl, i- butyl, alkenes (linear or branched), alkynes, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties, hydroxy, alkoxy, SCH₃, (C₁-C₃) alkylthio, SH, (Ci-C₃) haloalkoxy, (Ci-C₃) perhaloalkoxy, NH₂, NHQower alkyl), N(lower alkyl)₂, halogen, (C₁-C₃) haloalkyl, (Ci-C₃) perhaloalkyl, -CF₃, -CH₂CF₃, -CF₂CF₃, -CN, -NC, -OCN, -SCN, -NO, -NO₂, -N₃, -S(O) (lower alkyl), -S(O) (aryl), -S(O)₂ (lower alkyl), -S(O)₂ (aryl), S(O)₂ (alkoxyl) , -S(O)₂(aryloxy), -S(O)NH₂; -S(O)₂NH-lower alkyl, -S(O)₂NH-aryl, -S(O)₂N- (lower alkyl)₂, -S(O)₂N-(aryl)₂, -C(O)R₃, -C(O)OR₃, -C(0)NR_aR_b, -C(NH)NR₃R_b, -OC(O)R₃, -SC(O)R₃, -OC(O)OR₃, -SC(O)OR₃, -0C(0)NR_aR_b, -SC(O)NR₃R_b, -OC(NH)NR_aR_b, -SC(NH)NR₃R_b, -[NHC(0)]_nR_a, -[NHC(0)]_n0R_a, -[NHC(0)]_ΛNR_aR_b and -[NHC(NH)]^NR₃R_I,, wherein n is an integer from 1 to 5, and wherein R₃ and R_b can be the same or different and are independently selected from the group consisting of hydrogen, halogen, trifluoromethyl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a heterocyclic group, a substituted heterocyclic group, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamide, and any of R₄ through R₈ together can form a fused ring. In some embodiments, the compound is thiazolium or a salt thereof. The single-stranded oligonucleotide may be a DNA oligomer. A phosphorus atom in the DNA oligomer can be linked by the chemical bond via a linker. The single-stranded oligonucleotide can have a sequence complementary to a specific sequence in a target nucleic acid containing the specific sequence. Embodiments of the present invention may include methods of selecting a nucleic acid molecule which binds to Formula VIII, wherein said binding increases the fluorescence intensity of said Formula VIII, said method comprising the steps of: (a) providing a population of candidate nucleic acid molecules; (b) selecting said candidate nucleic acid molecules which bind said Formula VIII; (c) contacting said candidate nucleic acid molecules which bind said Formula VIII with said Formula VIII; and (d) selecting said nucleic acid molecules which, upon binding said Formula VIII or, increase its fluorescence intensity. The nucleic acid can be a DNA or an RNA.

Another embodiment of the present invention includes methods of determining the presence, location, or quantity of a nucleic acid or component of interest in a cell or an in vitro sample, said method comprising the steps of: (a) expressing in said cell or said sample a nucleic acid or component of interest; (b) contacting said cell or said sample with Formula VIII; whereby said compound binds to said Formula VIII and increases its fluorescence intensity; and (c) visualizing or measuring the fluorescence of said Formula VIII, thereby determining the presence, location, or quantity of said nucleic acid or component of interest in said cell or said in vitro sample.

Embodiments of the present invention also include methods of determining whether Formula VIII is capable of modulating the transcription of a nucleic acid of interest, said method further comprising the steps of: (a) expressing in a cell or an in vitro sample a nucleic acid of interest; (b) contacting said cell or said sample with said Formula VIII or with said Formula VIII alone, whereby said compound binds to said Formula VIII and increases its fluorescence intensity; and (c) measuring said fluorescence intensity in the presence and absence of said compound, whereby said compound is determined to modulate said transcription if said compound effects a change in said fluorescence intensity.

Other embodiments include kits for staining nucleic or amino acids in a sample, comprising: (a) a staining mixture that contains one or more dyes to form a combined mixture; wherein each dye independently has the Formula VIII; b) instructions for combining said dye or dyes with a sample containing or thought to contain nucleic or amino acids, said instructions comprising i) combining a sample that is thought to contain nucleic or amino acids with a staining mixture that contains said dye or dyes to form a combined mixture; and ii) incubating the combined mixture for a time sufficient for the dye in the staining mixture to associate with the nucleic or amino acids in the sample mixture to form a dye-amino acid or dye-nucleic acid complex that gives a detectable optical response upon illumination.

Additional embodiments include methods of detecting a target analyte in a sample containing or suspected of containing one or more analytes, comprising the steps of: (a) providing the sample on a solid support wherein the analyte is a nucleic acid molecule or cell component; (b) combining with said sample a specific-binding molecule, wherein (i) said specific-binding molecule is a polymerase chain reaction amplification product comprising biotin as a detectable label, and (ii) said combining is performed under conditions that allow formation of a first complex comprising said specific-binding molecule and said analyte, when present; (c) removing any unbound specific-binding molecule; (d) providing a compound having the Formula VIII; and (e) detecting an optical response based upon the binding of the compound.

Embodiments of the present invention include methods for analyzing cells comprising providing an array of locations which contain multiple cells wherein the cells contain one or more fluorescent reporter molecules; scanning multiple cells in each of the locations containing cells to obtain fluorescent signals from the fluorescent reporter molecule in the cells; converting the fluorescent signals into digital data; and utilizing the digital data to determine the distribution, environment or activity of the fluorescent reporter molecule within the cells.

Embodiments of the present invention may also be utilized within cell arrays. Cell arrays are used for screening large numbers of compounds for activity with respect to a particular biological function and involves preparing arrays of cells for parallel handling of cells and reagents. Standard 96 well microtiter plates which are 86 mm by 129 mm, with 6 mm diameter wells on a 9 run pitch, are used for compatibility with current automated loading and robotic handling systems. The microplate is typically 20 mm by 30 mm, with cell locations that are 100-200 microns in dimension on a pitch of about 500 microns. Methods for making microplates are described in U.S. Patent No. 6,103,479. Microplates may include coplanar layers of materials to which cells adhere, patterned with materials to which cells will not adhere, or etched 3 -dimensional surfaces of similarly pattered materials. The terms "well" and "microwell" refer to a location in an array of any construction to which cells adhere and within which the cells are imaged. Microplates may also include fluid delivery channels in the spaces between the wells. The smaller format of a microplate increases the overall efficiency of the system by minimizing the quantities of the reagents, storage and handling during preparation and the overall movement required for the scanning operation. In addition, the whole area of the microplate can be imaged more efficiently, allowing a second mode of operation for the microplate reader as described later in this document.

According to the present invention, the compounds may be used to show a large fluorescent enhancement upon absorbed by a target cell component and may show a significant difference between the excitation wavelength and the emission wavelength (i.e., has a large Stokes shift).

Accordingly, the present invention provides for use of pyridinium and/or thiazolium compounds, homologs, analogs and derivatives thereof as reagents for use as a targeting vehicle, dye, stain or fluorescent stain to detect, visualize and quantify the presence of a substance in a sample including, but not limited to microorganisms, cells, organelles, DNA, RNA, proteins, lipids and carbohydrates and to understand the distribution and localization of biomarkers in tissues. Further uses include in vitro applications (fixed samples) for gram staining such as gram positive/gram negative bacterial detection/typing, histology such as tissue, cell and/or organelle visualization, protein detection such as electrophoresis and in situ detection, lipid detection and carbohydrate detection. Additionally, uses further include in vivo applications (living samples) such as cell tracking, sorting typing and selection and flow cytometry, cell viability and kinetic and endpoint experiments and real time detection of target cell components and/or microorganisms. Particular applications may include the use of pyridinium (for example, stilbazium) and/or thiazolium compounds, homologs, analogs and derivatives thereof as a fluorescent stain in automated nucleic acid sequencing, nucleic acid detection, nucleic acid microarray, i.e., DNA chip, fluorescent-activated cell sorting, ion detection, nucleic acid and protein structure determination such as fluorescence resonance energy transfer and characterization of an environment and immunohistochemistry where the principle of antibody binding specificity to antigens can be exploited, immunoblotting and immunocytochemistry. Particular aspects of the present invention may include the use of the compounds described herein for fluorescence microscopy, cell tracking experiments and flow cytometry analysis, including co-labeling applications.

Diagnostic Tools The activities of the compounds described above may confer utility of the pyridinium

(for example, stilbazium) and/or thiazolium compounds, homologs, analogs and derivatives thereof as diagnostic tools. More specifically, embodiments of the present invention may provide methods of using these compounds in analyses to identify, study, and/or diagnose diseases and disorders using further procedures known to those skilled in the art.

For example, the compounds may be used to identify, study, and/or diagnose Acquired Immune Deficiency Syndrome (AIDS) by detecting an anti-human immunodeficiency virus (HIV), bovine spongiform encephalopathy (BSE or mad cow disease), lyme disease, an autoimmune disease such as multiple sclerosis, rheumatoid arthritis, juvenile oligoarthritis, type I diabetes mellitus, inflammatory bowel disease, Crohn's disease, scleroderma, psoriasis, atherosclerosis, Hashimoto's thyroiditis, Addison's disease, and systemic lupus erythematosus (SLE); an allergic condition such as asthma, seasonal and perennial allergic rhinitis, sinusitus, conjunctivitis, drug allergy, food allergy and insect allergy, scombroid poisoning, psoriasis, urticaria, pruritus, eczema, rheumatoid arthritis, inflammatory bowel disease, chronic obstructive pulmonary disease, thrombotic disease, otitis media, atopic dermatitis, adolescent atopic dermatitis, hives, contact dermatitis, senile dermatosis, pollenosis, ischemic diseases, cardia anaphylaxis and endotoxic shock; inflammatory conditions, infections and immune disorders such as those of the lungs, throat, mouth, joints, eyes, nose, bowel, and skin; particularly those associated with the infiltration of leucocytes into inflamed tissue, including asthma, adult respiratory distress syndrome, bronchitis and cystic fibrosis (which may additionally or alternatively involve the bowel or other tissues, laryngitis and orophoryngeal mucositis, gingivitis and periodontitis, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, uveitis (including iritis) and conjunctivitis, rhinitis and chronic rhinosinusitis, Crohn's disease, ulcerative colitis and distal proctitis, psoriasis, eczema and dermatitis (whether or not of allergic origin), and allergic-induced pruritis, such as prurigo, tissue necrosis in chronic inflammation; tumors, cancers, and neoplastic conditions tissue such as breast cancers; osteosarcomas; angiosarcomas; fibrosarcomas and other sarcomas; leukemias; lymphomas; sinus tumors; ovarian, uretal, bladder, prostate and other genitourinary cancers; colon, esophageal and stomach cancers and other gastrointestinal cancers; lung cancers; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas; and bacterial, mycobacterial, spirochetal, rickettsial, chlamydial, mycoplasmal, algal, fungal, viral, and parasitic infections by detecting various biomarkers for the disorder, viewing abnormal cell structure under magnification or utilizing other techniques known to those skilled in the art. The invention also provides methods of using the compounds described herein in analyses to identify, study and/or diagnose diseases and disorders associated with the cellular organelles described herein. For example, the compounds described herein may be used to identify, study, and/or diagnose mitochondrial disorders including, but not limited to, Alpers Disease (Progressive Infantile Poliodystrophy), Barth Syndrome (Cardiomyopathy- Neutropenia Syndrome), Lethal Infantile Cardiomyopathy (LIC), Beta-oxidation defects, Carnitine Deficiency & Disorders, CPEO (Chronic Progressive External Ophthalmoplegia), Kearns-Sayre Syndrome (KSS), Lactic Acidosis, Leber Hereditary Optic Neuropathy (LHON), Leigh's Disease (Subacute Necrotizing Encephalomyelopathy), Long-Chain Acyl- CoA Dehydrongenase (LCAD) Deficiency, Luft Disease, Medium-Chain Acyl-CoA Dehydrongenase (MCAD) Deficiency, Mitochondrial Cytopathy, Mitochodnrial Encephalomyopathy Lactic Acidosis & Stroke-like Episodes (MELAS), Mitochondrial Encephalopathy, Mitochondrial Myopathy, Multiple Acyl-CoA Dehydrogenase (MAD) Deficiency, Glutaric Aciduria Type II, Myoclonic Epilepsy & Ragged Red Fiber Disease (MERRF), Myoneurogastrointestinal Disorder and Encephalopathy (MNGIE), Neuropathy Ataxia & Retinitis Pigmentosa (NARP), Pearson Syndrome, Pyruvate Carboxylase Deficiency, Pyruvate Dehydrogenase Deficiency (PHD), Short-Chain Acyl-CoA Dehydrogenase (SCAD) Deficiency, Respiratory Chain Disorders, Complex I - NADH Dehydrogenase (NADH-CoQ reductase) Deficiency, Complex II - Succinate dehydrogenase Deficiency, Complex III - Ubiquinone-cytochrome c oxidoreductase Deficiency, Complex IV - Cytochrome c oxidase (COX) Deficiency and Complex V - ATP Synthase Deficiency.

The compounds may also be used to evaluate physiological processes and function, for example, by illuminating target cells, cell components, and/or microorganisms in tissue and/or biological fluids including, but not limited to, blood, urine, semen, mucus and saliva and/or substances included therein.

Therapeutic Agents

Embodiments of the present invention further provide methods of using the pyridinium (for example, stilbazium) and/or thiazolium compounds, homologs, analogs and derivatives thereof as therapeutic agents. As noted above, these compounds may be used to target cells, cellular organelles, nucleic acids, and/or microorganisms. Embodiments of the present invention may provide these compounds conjugated to an antibody or a therapeutic moiety, such as a drug. Accordingly, these compounds can facilitate delivery of a substance of interest, antibody or therapeutic moiety to a target or specific site of interest. The target or site of interest may include, but is not limited to, an organelle, cell, tissue, organ, drug, antigen, antibody or microorganism. In some embodiments, the compounds may facilitate delivery of the substance of interest, antibody or therapeutic moiety in an amount effective to only identify and/or monitor a disorder or disease or other target of interest or by increasing the dosage to an amount effective to treat a disorder or disease afflicting the subject. In some embodiments of the present invention, the antibody is IgG, IgM, IgA, IgD or IgE. The disorder or disease may include Acquired Immune Deficiency Syndrome (AIDS), bovine spongiform encephalopathy (BSE or mad cow disease), lyme disease, an autoimmune disease such as multiple sclerosis, rheumatoid arthritis, juvenile oligoarthritis, type I diabetes mellitus, inflammatory bowel disease, Crohn's disease, scleroderma, psoriasis, atherosclerosis, Hashimoto's thyroiditis, Addison's disease, and systemic lupus erythematosus (SLE); an allergic condition such as asthma, seasonal and perennial allergic rhinitis, sinusitus, conjunctivitis, drug allergy, food allergy and insect allergy, scombroid poisoning, psoriasis, urticaria, pruritus, eczema, rheumatoid arthritis, inflammatory bowel disease, chronic obstructive pulmonary disease, thrombotic disease, otitis media, atopic dermatitis, adolescent atopic dermatitis, hives, contact dermatitis, senile dermatosis, pollenosis, ischemic diseases, cardia anaphylaxis and endotoxic shock; inflammatory conditions, infections and immune disorders such as those of the lungs, throat, mouth, joints, eyes, nose, bowel, and skin; particularly those associated with the infiltration of leucocytes into inflamed tissue, including asthma, adult respiratory distress syndrome, bronchitis and cystic fibrosis (which may additionally or alternatively involve the bowel or other tissues, laryngitis and orophoryngeal mucositis, gingivitis and periodontitis, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, uveitis (including iritis) and conjunctivitis, rhinitis and chronic rhinosinusitis, Crohn's disease, ulcerative colitis and distal proctitis, psoriasis, eczema and dermatitis (whether or not of allergic origin), and allergic- induced pruritis, such as prurigo, tissue necrosis in chronic inflammation; tumors, cancers, and neoplastic conditions affecting tissue such as breast cancers; osteosarcomas; angiosarcomas; fibrosarcomas and other sarcomas; leukemias; lymphomas; sinus tumors; ovarian, uretal, bladder, prostate and other genitourinary cancers; colon, esophageal and stomach cancers and other gastrointestinal cancers; lung cancers; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas and autoimmune disorders such as multiple sclerosis, rheumatoid arthritis, juvenile oligoarthritis, type I diabetes mellitus, inflammatory bowel disease, Crohn's disease, scleroderma, psoriasis, acne, atherosclerosis, Hashimoto's thyroiditis, Addison's disease, and systemic lupus erythematosus (SLE); and bacterial, mycobacterial, spirochetal, rickettsial, chlamydial, mycoplasmal, algal, fungal, viral, and parasitic infections. The diseases and disorders can also include those associated with the cellular organelles described herein such as the mitochondrial disorders described herein.

Accordingly, the therapeutic moiety may be any therapeutic agent known to treat the disorder or disease. Exemplary therapeutic moieties include, but are not limited to, ACE- inhibitors; anti-anginal drugs; anti-arrhythmias; anti-asthmatics; anti-cholesterolemics; anticonvulsants; anti-depressants; anti-diarrhea preparations; anti-histamines; anti-hypertensive drugs; anti-infectives; anti-inflammatory agents; anti-lipid agents; anti-manics; anti- nauseants; anti-stroke agents; anti-thyroid preparations; anti-tumor drugs; anti-tussives; anti- uricemic drugs; anti-viral agents; acne drugs; alkaloids; amino acid preparations; anabolic drugs; analgesics; angiogenesis inhibitors; antacids; anti-arthritics; antibiotics; anticoagulants; antiemetics; antiobesity drugs; antiparasitics; antipsychotics; antipyretics; antispasmodics; antithrombotic drugs; anxiolytic agents; appetite stimulants; appetite suppressants; beta blocking agents; bronchodilators; cardiovascular agents; cerebral dilators; chelating agents; cholecystokinin antagonists; chemotherapeutic agents; cognition activators; contraceptives; coronary dilators; cough suppressants; decongestants; dermatological agents; diabetes agents; diuretics; emollients; enzymes; erythropoietic drugs; expectorants; fertility agents; fungicides; gastrointestinal agents; growth regulators; hormone replacement agents; hyperglycemic agents; hypnotics; hypoglycemic agents; laxatives; migraine treatments; mineral supplements; mucolytics; narcotics; neuroleptics; neuromuscular drugs; NSAIDS; nutritional additives; peripheral vasodilators; prostaglandins; psychotropics; renin inhibitors; respiratory stimulants; steroids; stimulants; sympatholytics; thyroid preparations; tranquilizers; uterine relaxants; vaginal preparations; vasoconstrictors; vasodilators; vertigo agents; vitamins; and wound healing agents.

According to further aspects of the present invention, pyridinium and/or thiazolium compounds, homologs, analogs and derivatives thereof may serve as laser gain media in laser applications, for example, chemical and dye lasers. A laser can be composed of an active laser medium or gain medium and a resonant optical cavity. Many materials produce laser light by having the active laser medium or gain medium to power a laser. An optical cavity contains a sharp coherent beam of light between reflective surfaces, such that each light photon passes through the gain medium more than once before it is emitted from an output aperture. The laser gain medium can transfer external energy into the laser beam. The gain medium can be energized or pumped by an external energy source such as light and electricity. The pump energy can be absorbed by the laser medium, placing some of its particles into high-energy quantum states. When the number of particles from one excited state exceeds the number of particles of a lower excited state of the laser medium, population inversion can occur. In this condition, an optical beam passing through the laser medium can produce more stimulated emission than the stimulated absorption and this beam is thus amplified, and is amplified exponentially.

The compounds may be provided in an aqueous solution such as methanol, phenol or phenol derivatives and optionally an acid to adjust pH for such laser applications.

Methods of using the pyridinium (for example, stilbazium) and/or thiazolium compounds, homologs, analogs and derivatives thereof in laser applications include, but are not limited to, spectroscopy (light sources in Raman Spectroscopy Equipment that avoid light scattering "noise" and thereby produce sharp Raman spectra), in holography (creating three- dimensional holograms with the sharply focused dye laser light), and in a number of biomedical applications. The biomedical applications include, but are not limited to, functions in cardiology, oncology, ophthalmology, gastroenterology, dermatology and urology applications, retinal eye surgery, LASIK eye surgery for myopia, cauterizing surgeries that prevent bleeding as laser cutting occurs in blood vessels, laser thrombolysis in cardiology, blood-rich cardiac and liver tissues, removal of tattoos and birthmarks and isotope separation. See "Preparation of Multi-Layered Pyridinium as Non-Linear Optical Materials", Tang, J., et al., Faming Zhuanii Shenqing Gongkai Shuomingshu (Chinese Patent Application) CN 1394854, (filed 04 July 2002: published 5 February 2003), 7 pages; Chem. Abst. 141 :411081 (2004); "Synthesis and Optical Properties of Cross-Conjugated Bis(dimethylaminophenyl)-pyridylvinylene Derivatives", Wang, H., et al., J. Org. Chem.. 2000, 65 (18), 5862-5867; Chem. Abst. 133:310158 (2000); "Multiple Reflection Correction in Transmitted Second Harmonic Generation of Stilbazium Salt Langmuir-Blodgett Monolayers", Liu, L.-Y., et al., WuIi Xuebao. 1997, 46 (1), 78-86; Chem. Abst. 126:349258 (1997); "Angular Dependence of Second Harmonic Generation in a Langmuir-Blodgett Monolayer under a Full 360°", Liu, L.-Y., et al., J. Opt. Soc. Amer. B: Optical Physics. 1997. 14 (2), 382-390; Chem. Abst. 126:284743 (1997); "Study of Second-Order Nonlinear Optics for Stilbazium Salt Langmuir-Blodgett Monolayer at Different Surface Pressures", Li, J., et al., Guangxue Xuebao, 1995, 15 (10), 1346-1350; Chem. Abst. 124:159673 (1995). "SAXS on the Microstructure and Superstructure of Finite-Layer Langmuir-Blodgett Crystal-Like Films. II. The Mixed Film Systems of Stilbazium Iodide and Cadmium Arachidate", Cong, H. J., et al., Huaxue Xuebao. 1994, 52 (3), 248-256; Chem. Abst. 120:281322 (1994). "Study on the Second-Order Nonlinear Optical Property of Stilbazium Salt Polystyrene in Langmuir- Blodgett Films", Liu, L.- Y., et al., Guangxue Xuebao, 1993, 13 (14), 319-323; Chem. Abst. 120:192842 (1993); "Synthesis and Characterization of Mono-, Bis-, and Tris Substituted Pyridinium and Pyrylium Dyes", Matsui, M., et al., Bull. Chem. Soc. Japan. 1992, 65 (1), 71- 74; Chem. Abst. 116:131151 (1992); "Preparation of Multi-Layered Pyridinium as Non- Linear Optical Materials", Tang, J., et al., Faminfi Zhuanii Shenqing Gongkai Shuomingshu (Chinese Patent Application) CN 1394854, (filed 04 July 2002: published 5 February 2003), 7 pages; Chem. Abst. 141 :41 1081 (2004); and "On the Adsorption Behavior of Soluble, Surface-Chemically Pure Hemicyanine Dyes at the Air/Water Interface", Lunkenheimer, K., et al., J. Colloid & Interface Chem.. 2002, 248 (2), 260-267; Chem. Abst. 137:83893 (2002).

Methods of using the stilbazium and/or thiazolium compounds, homologs, analogs and derivatives thereof in laser applications may also include use in photodynamic therapy (PDT). In PDT, either a photosensitizer or the metabolic precursor of one is administered to the subject. The tissue to be treated is exposed to light suitable for exciting the photosensitizer. The photosensitizer can be excited from a ground singlet state to an excited singlet state. It can then undergo intersystem crossing to a longer-lived excited triplet state. One of the few chemical species present in tissue with a ground triplet state is molecular oxygen. When the photosensitizer and an oxygen molecule are in proximity, an energy transfer can take place that allows the photosensitizer to relax to its ground singlet state, and create an excited singlet state oxygen molecule. Singlet oxygen is an aggressive chemical species and will very rapidly react with any nearby biomolecules. (The specific targets depend heavily on the photosensitizer chosen.) Ultimately, these destructive reactions can result in cell killing through apoptosis or necrosis. PDT can be used in the treatment of tumors, cancers, and neoplastic conditions, dermatological disorders and ophthalmologic disorders.

Specificity of treatment can be achieved in several ways. Light may be delivered only to tissues that the clinician wishes to treat. In the absence of light, there may be no activation of the photosensitizer and no cell killing. Photosensitizers may be administered in ways that restrict their mobility. Photosensitizers may be chosen which are selectively absorbed at a greater rate by targeted cells. In some embodiments of the present invention, heat or ultrasonic energy could also potentially be directed at the target to trigger identification, alteration, or destruction (e.g., excite the compound to destroy the cell) or release the encapsulated/microencapsulated compound.

Accordingly, the invention further includes the following statements. Embodiments of the present invention provide a method of using a compound having the following structure:

or a solvate thereof, wherein X^" is an anionic salt; Ri_, R₂, R₃, or R₄ are the same or different and independently selected from the group consisting of methyl, ethyl, C_1-Io alkyl (linear or branched) and alkenes (linear or branched), or wherein when Ri and R₂ or when R₃ and R₄ are taken together with the nitrogen atom to which they are attached, they form substituted and unsubstituted pyrrolidino or piperidino rings; R₅ is selected from the group consisting of methyl, ethyl, C_1-10 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₅ is an organometallic; R₅ may be (CH₂)_U-MR₆, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₆ is a selected from the group consisting of propyl, butyl, or any alkyl compound; R₅ is a polyalkylene glycol moiety comprising a Cj_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol, or

or a solvate thereof wherein

X^" is an anionic salt;

Ri and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, C_1-I0 alkyl (linear or branched) and alkenes (linear or branched), or wherein R] and R₂ may be taken together with the nitrogen atom to which they are attached form substituted and unsubstituted pyrrolidino or piperidino rings;

R₃ is selected from the group consisting of methyl, ethyl, Ci-io alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₃ is an organometallic compound; R₃ is (CH₂)_n-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound; or R₃ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol, in a laser application. In further embodiments, the compound is stable in an organic solvent. In certain aspects of the invention, the laser is a chemical laser or a dye laser. The laser can tunable. In further embodiments, the laser application is a photodynamic therapy (PDT). Subjects suitable to be treated include, but are not limited to, plant, avian and mammalian subjects. Mammals of the present invention include, but are not limited to, canines, felines, bo vines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates, humans, and the like, and mammals in utero. Any mammalian subject in need of being treated according to the present invention is suitable. Human subjects are preferred. Human subjects of both genders and at any stage of development {i.e., neonate, infant, juvenile, adolescent, adult) may be treated according to the present invention.

Illustrative avians according to the present invention include chickens, ducks, turkeys, geese, quail, pheasant, ratites {e.g., ostrich) and domesticated birds (e.g., parrots and canaries), and birds in ovo. The invention can also be carried out on animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, livestock and horses for veterinary purposes (such as treatment of traumatic and surgical wounds, strains, musculoskeletal pain and dysfunction, rheumatoid and osteoarthritis, neurologic applications and sports injuries such as contusions and muscle tears), and for drug screening and drug development purposes.

In therapeutic use for treatment of conditions in mammals (i.e. humans or animals) for which the compounds of the present invention or an appropriate pharmaceutical composition thereof are effective, the compounds described herein may be administered in an effective amount. Since the activity of the compounds and the degree of the therapeutic effect vary, the actual dosage administered will be determined based upon generally recognized factors such as age, condition of the subject, route of delivery and body weight of the subject.

The present invention is explained in greater detail in the examples that follow. The examples are intended as illustrative of the invention and is not to be taken are limiting thereof.

EXAMPLE 1 Spectral Characterization

Nine compounds were screened for their staining behavior. Figure 1 shows the structures of the nine compounds employed in the experiments. Compounds A-C are exemplary pyridinium compounds, and Compounds D-I are exemplary thiazolium compounds.

The compounds were first analyzed to determine optimum excitation and emission parameters for imaging. 5μg/ml solutions in ethanol of the compounds were found to exhibit an absorption maxima around 500nm (data not shown). Using 500nm as the excitation wavelength, the compounds were then screened in a fluorescence spectrophotometer for their emission spectra. All nine compounds were found to have appreciable emission around 580nm indicating a stokes shift of about 80nm (data not shown). Excitation of the compounds at 315nm also produced marked emission peaks in the 580 to 630nm range (data not shown). EXAMPLE 2 Cell Staining Results

Based on the spectral characterization data of Example 1 , it was clear that all nine exemplary compounds were suitable for fluorescence microscopy and would likely be detectable under filters normally used in fluorescence microscopy. In order to determine the need for liposomes in effective staining, it was decided to first evaluate the behavior of the dyes as ethanol solutions. For these preliminary cell-staining experiments, 4Tl Mouse mammary carcinoma cells were grown on glass coverslips in 6 well plates till 60-80% confluent. Cells were then stained by adding lμl of ethanol stock solution directly into the 2 ml of culture medium in each well. After 5 min incubation, the cells were washed 3 times in Phosphate Buffered Saline, pH 7.4 (PBS) and mounted in Fluoromount medium on glass slides. Fluorescence micrographs were taken at 40 and 100 x objective magnifications using UV, green and red filter sets. The dyes were found to exhibit appreciable staining of the cells with a distinct staining pattern that was visible with all the filter sets although with varying intensities (data not shown). These observations indicated that although the compounds were generally poorly soluble in water, they were taken up by the cells tested and were able to stain the cells at readily detectable intensities.

To determine whether the concentration of stains used was sufficient to saturate the cells, staining was carried out as before, but the medium from each well was collected after the staining incubation. The fluorescence of the collected medium was measured to provide an estimate of the dye remaining in the medium after staining. For comparison, the amount of dye added to each well was diluted in 2 ml of fresh medium and assayed for fluorescence. Results show that a significant amount of dye is retained in the medium for all compounds tested (data not shown).

In order to determine if cell staining exhibits a dose dependent behavior, cells were stained with compound B at different concentrations (1-0.0001 μg/ml) in a 96 well plate. After staining, cells were washed and the fluorescence in each well measured with a plate reader at 485/590nm EX/EM. The results indicated a marked linear response over the concentrations tested (data not shown).

To demonstrate that compounds described herein could potentially be used as UV excitable dyes for simultaneous imaging with other UV excitable dyes, cells were co-stained with Hoechst and compound B. Figure 2 shows an epifluorescence micrograph of 4Tl cells excited with UV wavelength light. The nucleus can be seen stained in blue and the mitochondria stained red by compound B.

EXAMPLE 3 Liposomal Incorporation

Compounds B and G were formulated in liposomes at 0.05μg/ml in a total of

10mg/ml lipid (phosphatidyl choline: Cholesterol 70:30 molar ratio). At this concentration, the compound was stably incorporated with no evidence of precipitation after storage for several days. Attempts to incorporate 0.5μg/ml compound in the same amount of lipid resulted in an unstable preparation as evidenced by heavy precipitation of the compounds.

Liposomal incorporation did not, however, alter the staining behavior (staining time, subcellular localization, or staining intensity) of the compounds. Liposomal incorporation provides an alternative for in vitro applications; and further provides the potential for use in in vivo applications.

EXAMPLE 4 Cytotoxicity

The compounds were assayed for toxicity at the concentration used for staining and over the time period typically associated with staining protocols. For the purpose of evaluating cytotoxicity, the well-established (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) assay was used. Based on the metabolic conversion of a tetrazolium salt to a colored product, this assay primarily determines cell viability based on the measurement of metabolic activity of the cell. The established staining concentration of the compound (0.5μg/ml) was incubated with the cells for the indicated times followed by washing and a 4 hour recovery period in regular cell culture medium followed by the an MTS assay performed according to the manufacturer's instructions. All compounds had comparable toxicity to that of MitoTracker Red CMXRos (a commercially available cellular stain) under the conditions of the MTS assay (Figure 3). To determine possible long term cytotoxic effects from conditions typically encountered in cell tracking experiments, 4Tl mouse mammary carcinoma cells were exposed to various concentrations of the compound in culture medium for 24 hours. The MTS assay was then used to determine the cell viability after exposure to the compounds. Both free and liposomal forms of the compounds were used. Both compounds tested were found to have no detectable effect on cell viability over all concentrations tested (see Figure 4).

EXAMPLE 5

Cell Tracking Experiments

In view of the observation that the compounds appeared to be non-toxic and were well retained by cells after staining and washing, the applicability of these compounds as cell trackers was explored. As a preliminary experiment, 4Tl cells were stained with the compounds according to the standard incubation procedure (0.5μg/ml for 5min) and the stained cells were then washed and allowed to grow on coverslips for up to 3 days. Coverslips were removed from culture and examined by microscopy 1, 2 and 3 days after staining. Fluorescence from the compounds was detectable even at 3 days post staining. The intensity of staining was however reduced over the 3 day period in a manner consistent with the compound being redistributed among the cells as cell-division took place. Based on a visual estimation, the growth rates of the stained cells did not appear to be changed over the untreated control group.

To provide a more quantitative analysis of the staining, flow cytometry was explored. Cells (4Tl) were harvested from large-scale cultures and stained with compounds at 0.5, 0.05, and 0.005 μg/ml concentrations for 5 min. The cells were washed and approximately 250,000 cells were seeded into culture flasks and allowed to grow for up to 3 days post staining. Cells were collected at 1, 2, and 3 days post staining washed again and fixed with 800μl of 4% paraformaldehyde in PBS. The resulting preparations were then analyzed on a flow cytometer for detection of the red fluorescence of the compounds. Figure 5 shows a representative result of such an experiment with compound B. It can be clearly seen that the fluorescence intensity is directly proportional to the starting amount of compound used but also inversely proportional to the time of growth post staining. Similar data have been collected with all compounds tested so far. These data not only show the applicability of the compounds as cell trackers, but also show the applicability of the compounds described herein to flow cytometry-based applications. EXAMPLE 6 Fluorescence Microscopic Imaging

Selected compounds were tested on a 2-photon fluorescence microscope. First, a slide prepared from cells stained with compound B (M340) was scanned from 930nm to730nm in lOnm steps with a green collection filter.

910 run was found to be optimal for imaging. Figure 6 shows images from the relevant part of the scan showing that 910nm excitation produced the brightest image.

Compound B thus showed a strong 2-Photon signal and was found to be reasonably resistant to photobleaching. Figures 7 and 8 show representative images with some representative compounds at various magnifications.

This investigation confirmed that the compounds described herein can be applied to 2- photon based fluorescence microscopic imaging as well.

EXAMPLE 7 Determination of Subcellular Distribution of Exemplary Compounds

The use of compounds described herein as mitochondria specific stains was explored, wherein three compounds (B, D and G) were selected for confocal imaging analysis. Cells were co-stained with the compounds and the green mitochondrial dye Mitofluor Green followed by confocal analysis of the subcellular distribution of the dye in comparison with the known mitochondrial stain. All 3 compounds showed excellent co-localization with the mitochondria through 20micron z-stacks. Figure 9 shows representative slices taken from acquired z-stacks of treated cells. All images show overlaid red and green channels with regions in yellow indicating co-localization. MitoTracker Red, a well known mitochondrial stain, was used to indicate the validity of such an approach for proving mitochondria specificity.

EXAMPLE 8

Effect of Mitochondrial Membrane Potential on Staining Behavior

In an effort to evaluate the effect of mitochondrial membrane potential on the staining behavior of the compounds described herein, staining was carried out on cells pretreated for 30min with the known mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone

(CCCP). Treatment with CCCP effectively abolishes the mitochondrial proton gradient and hence lowers the magnitude of the negative membrane potential. MitoTracker Red CMXRos, which has been documented to exhibit mitochondrial membrane potential sensitive staining, was used as control. Of the compounds tested, some seemed to exhibit a change in intensity of staining in the presence of CCCP suggesting an influence of the mitochondrial membrane potential on the subcellular accumulation of these compounds (Figure 10). In particular compounds B and G appear potential sensitive, similar to MitoTracker Red. In an attempt to provide a quantitative basis for this comparison, cell associated fluorescence was measured in extracts prepared by lysis of the contents of treated wells with buffer containing l%Triton X- 100. The cell associated fluorescence in the presence or absence of CCCP is shown in Figure 11. There appeared to be no detectable difference in cell-associated fluorescence in the presence of CCCP. This observation in combination with microscopic analysis in Figure 10 suggests that the mitochondrial membrane potential might only influence the subcellular localization of the dyes and not the total uptake into cells.

The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. Although exemplary embodiments of this invention have been described, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the claims. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

What is claimed is:

1. A method of diagnosing a disease or disorder, comprising: contacting a cell with a compound having the following structure:

or a solvate thereof, wherein

X^" is an anionic salt;

Ri, R₂, R₃, or R₄ are the same or different and independently selected from the group consisting of methyl, ethyl, Ci_-I0 alkyl (linear or branched) and alkenes (linear or branched), or wherein when Ri and R₂ or when R₃ and R₄ are taken together with the nitrogen atom to which they are attached, they form substituted and unsubstituted pyrrolidino or piperidino rings;

R₅ is an organometallic compound; R₅ may be (CH₂)_n-MR₆, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₆ is an alkyl; R₅ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol, or

or a solvate thereof wherein X^" is an anionic salt;

Ri and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, C_M0 alkyl (linear or branched) and alkenes (linear or branched), or wherein Ri and R₂ may be taken together with the nitrogen atom to which they are attached form substituted and unsubstituted pyrrolidino or piperidino rings;

R₃ is selected from the group consisting of methyl, ethyl, C_M0 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₃ is an organometallic compound; R₃ is (CH₂)H-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is an alkyl; or R₃ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol, wherein contact with the cell allows (a) detection of a biomarker, or (b) viewing of abnormal cellular structure, wherein (a) and/or (b) are associated with characteristics of the disease or disorder.

2. The method of claim 1, wherein the method is carried out in vitro.

3. The method of claim 1, wherein the method is carried out in vivo.

4. The method of claim 1, wherein the compound is coupled to an antibody.

5. The method of claims 1 through 4, wherein the compound is a stain.

6. The method of claims 1 through 4, wherein the compound is a fluorescent stain.

7. The method of claims 1 through 6, wherein the method further comprises immunohistochemical techniques.

8. The method of claims 1 through 7, wherein the disease or disorder is Acquired Immune Deficiency Syndrome (AIDS), bovine spongiform encephalopathy (BSE or mad cow disease), lyme disease, an autoimmune disease, an allergic condition, an inflammatory condition, tumor, cancer, neoplastic condition, a bacterial infection, a mycobacterial infection, a spirochetal infection, a rickettsial infection, a chlamydial infection, a mycoplasmal infection, an algal infection, a fungal infection, a viral infection or a parasitic infection.

9. The method of claim 8, wherein the disease or disorder is a tumor or cancer and the method further comprises detecting (a) an antigen directed to, or (b) a biomarker for the tumor or cancer.

10. A method of evaluating physiological processes and/or functions comprising, administering a compound having the following structure:

or a solvate thereof, wherein

X^" is an anionic salt;

Ri₁ R₂, R₃, or R₄ are the same or different and independently selected from the group consisting of methyl, ethyl, Ci_-I0 alkyl (linear or branched) and alkenes (linear or branched), or wherein when Ri and R₂ or when R₃ and R₄ are taken together with the nitrogen atom to which they are attached, they form substituted and unsubstituted pyrrolidino or piperidino rings;

R₅ is selected from the group consisting of methyl, ethyl, Ci_-I0 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₅ is an organometallic; R₅ may be (CI^)_n-MRe, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₆ is a selected from the group consisting of propyl, butyl, or any alkyl compound; R₅ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol, or

or a solvate thereof wherein

X^" is an anionic salt;

Ri and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, C_MO alkyl (linear or branched) and alkenes (linear or branched), or wherein Ri and R₂ may be taken together with the nitrogen atom to which they are attached form substituted and unsubstituted pyrrolidino or piperidino rings;

R₃ is selected from the group consisting of methyl, ethyl, Ci_-I0 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₃ is an organometallic compound; R₃ is (CH₂)_n-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound; or R₃ is a polyalkylene glycol moiety comprising a Cj_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol, wherein administration of the compound provides illumination of biological fluids, cells or tissues that can be monitored to evaluate physiological processes and/or function.

11. The method of claim 10, wherein the compound is a stain.

12. The method of claim 10, wherein the compound is a fluorescent stain.

13. The method of claims 10 through 12, wherein the biological fluid is blood, urine, semen, mucus and/or saliva.

14. The method of claims 10 through 12, wherein the cell is an isolated cell.

15. A method of facilitating delivery of an antibody or therapeutic moiety to a target of interest, the method comprising coupling said antibody or therapeutic moiety to a compound of formula I or formula VIII.

16. The method of claim 15, wherein the antibody is IgG, IgM, IgA, IgD or IgE.

17. The method of claim 15, wherein the therapeutic moiety is a therapeutic agent for the treatment of Acquired Immune Deficiency Syndrome (AIDS), bovine spongiform encephalopathy (BSE or mad cow disease), lyme disease, an autoimmune disease, an allergic condition, an inflammatory condition, tumor, cancer, neoplastic condition, a bacterial infection, a mycobacterial infection, a spirochetal infection, a rickettsial infection, a chlamydial infection, a mycoplasmal infection, an algal infection, a fungal infection, a viral infection or a parasitic infection.

18. The method of claims 1 through 17, wherein the compound is encapsulated.

19. The method of claims 1 through 18, wherein the compound is in a liposomal formulation.

20. An assay for determining the presence, absence or health of a cell, analyte, nucleic acid or microorganism in a sample, said assay comprising combining the sample with a labeling reagent to form a labeled cell, analyte, nucleic acid or microorganism, said labeling reagent comprising a dye that stains the cell, analyte, nucleic acid or microorganism to provide a stained sample comprising a stained cell, analyte, nucleic acid or microorganism, wherein said dye is a compound having the following structure:

or a solvate thereof wherein X^" is an anionic salt;

Ri and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, Ci_-I0 alkyl (linear or branched) and alkenes (linear or branched), or wherein Rj and R₂ may be taken together with the nitrogen atom to which they are attached form substituted and unsubstituted pyrrolidino or piperidino rings;

R₃ is selected from the group consisting of methyl, ethyl, Cj-io alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₃ is an organometallic compound; R₃ is (CH₂)_n-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound; or R₃ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol, and observing the accumulation of the stained cell, analyte, nucleic acid or microorganism.

21. The assay of claim 20, wherein the dye is encapsulated.

22. The assay of claim 20, wherein R₅ is (CH^_n-MR^ and n is a number from 1 to 6, M is an organometallic compound selected from the group consisting of tin, silicon, and germanium, and wherein R₆ is selected from the group consisting of propyl, butyl, and alkyl, substituted or unsubstituted.

23. A probe comprising a ligand or antibody and a compound having the following structure:

or a solvate thereof wherein X^" is an anionic salt;

Ri and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, Cj.io alkyl (linear or branched) and alkenes (linear or branched), or wherein Ri and R₂ may be taken together with the nitrogen atom to which they are attached form substituted and unsubstituted pyrrolidino or piperidino rings;

R₃ is selected from the group consisting of methyl, ethyl, C_MO alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₃ is an organometallic compound; R₃ is (CH₂)_n-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound; or R₃ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol, and wherein said compound is linked to the ligand or antibody by a chemical bond.

24. The probe of claim 23, wherein the compound is encapsulated.

25. The probe of claim 23, wherein the compound is in a liposomal formulation.

26. A method of selecting an analyte that binds to a compound having the following structure:

or a solvate thereof wherein

X^" is an anionic salt;

Ri and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, C_MO alkyl (linear or branched) and alkenes (linear or branched), or wherein Ri and R₂ may be taken together with the nitrogen atom to which they are attached form substituted and unsubstituted pyrrolidino or piperidino rings; R₃ is selected from the group consisting of methyl, ethyl, Ci-io alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₃ is an organometallic compound; R₃ is (CH₂)_n-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound; or R₃ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol; and wherein said binding increases the fluorescent intensity of the analyte compound, said method comprising the steps of:

(a) providing a population of analytes;

(b) contacting said analytes with said compound; and

(d) selecting said analytes that, upon binding said compound, show an increased fluorescent intensity.

27. The method of claim 26, wherein said analyte is a small molecule.

28. The method according to claim 26, wherein said analyte is a microorganism.

29. The method according to claim 26, wherein said analyte is a cell.

30. A method for determining the presence or absence of one or more target compounds in a sample, wherein said compound is a fluorescent molecule having the following structure:

or a solvate thereof wherein X^" is an anionic salt; Ri and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, C_MO alkyl (linear or branched) and alkenes (linear or branched), or wherein Ri and R₂ may be taken together with the nitrogen atom to which they are attached form substituted and unsubstituted pyrrolidino or piperidino rings;

R₃ is selected from the group consisting of methyl, ethyl, C_{M O} alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₃ is an organometallic compound; R₃ is (CH₂)_n-MR9, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound; or R₃ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol, and the method comprising the steps of: providing a plurality of electrophoretic probes specific for the one or more target compounds, each electrophoretic probe having a target-binding moiety; combining with the sample the plurality of electrophoretic probes such that in the presence of a target compound a complex is formed between each target compound and one or more electrophoretic probes specific therefor; and separating and identifying the compounds to determine the presence or absence of the one or more target compounds.

31. The method of claim 30, wherein the compound is encapsulated.

32. A kit for staining cells, analytes, nucleic acids or microorganisms in a sample, comprising:

(a) a staining mixture comprising one or more dyes to form a combined mixture; wherein at least one dye having the following structure:

or a solvate thereof wherein

X^" is an anionic salt;

R₁ and R₂ are the same or different and are independently selected from the group consisting of methyl, ethyl, C_1-10 alkyl (linear or branched) and alkenes (linear or branched), or wherein Ri and R₂ may be taken together with the nitrogen atom to which they are attached form substituted and unsubstituted pyrrolidino or piperidino rings;

R₃ is selected from the group consisting of methyl, ethyl, Ci_-I0 alkyl (linear or branched), alkenes (linear or branched), alkynes, n-propyl, i-propyl, n-butyl, i-butyl, substituted and unsubstituted aryl moieties and substituted and unsubstituted benzyl moieties; R₃ is an organometallic compound; R₃ is (CH₂)_n-MR₉, wherein n is a number from 1 to 6, M is an organometallic compound such as tin, silicon, or germanium, and wherein R₉ is a selected from the group consisting of propyl, butyl, or any alkyl compound; or R₃ is a polyalkylene glycol moiety comprising a Ci_-5 alkyl (linear or branched) substituted polyethylene glycol, a C_2-5 alkene (linear or branched) substituted polyethylene glycol or a C_2-5 alkyne substituted polyethylene glycol; and b) instructions for combining said dye or dyes with a sample comprising cells, analytes, nucleic acids and/or microorganisms, said instructions comprising i) combining a sample of cells, analytes, nucleic acids and/or microorganisms with a staining mixture comprising said at least one dye or dyes to form a combined mixture; and ii) incubating the combined mixture for a time sufficient for the dye in the staining mixture to associate with the cells, analytes, nucleic acids or microorganisms in the sample mixture to form stained cells, analytes, nucleic acids or microorganisms complex that gives a detectable optical response upon illumination.

33. The kit of claim 32, wherein said kit is used for cell differentiation.

34. The kit of claim 32, wherein said kit is used for cell sorting.