Patent Application
20080181965
The present invention relates to the use of fluorescent particles for biological assays, particularly as fluorescent probes for Optical Molecular Imaging.
Dyes have been incorporated into silica particles. (Ow, H.; Larson, D. R.; Srivastava, M.; Baird, B. A.; Webb, W. W.; Wiesner, U. “Bright and Stable Core-Shell Fluorescent Nanoparticles” Nano Letters 2005, 5, 113-117/Verhaegh, N. A. M.; Blaaderen, A. v. “Dispersions of Rhodamine-Labeled Silica Spheres: Synthesis, Characterization, and Fluorescence Confocal Scanning Laser Microscopy” Langmuir 1994, 10, 1427-1438./Imhof, A.; Megens, M.; Engelberts, J. J.; Lang, D. T. N. d.; Sprik, R.; Vos, W. L. “Spectroscopy of Fluorescein (FITC) Dyed Colloidal Silica Spheres” J. Phys. Chem. B 1999, 103, 1408-1415.).
Loaded latexes with IR dyes are known for imaging and photographic applications (US 2002/0113854, U.S. Pat. No. 6,706,460). Latexes loaded with non-IR dyes are known for biological and diagnostic applications.
US 2005/0244976 relates to methods of detecting anionic proteins in a sample with fluorescent carbocyanine dye compounds. The invention is of use in a variety of fields including immunology, diagnostics, proteomics, molecular biology and fluorescence based assays. Anionic proteins are detected in a sample with fluorescent carbocyanine dye compounds. The invention also describes methods of simultaneously detecting anionic and non-anionic proteins in a sample with discrete fluorescent signals produced by carbocyanine dye compounds.
U.S. Pat. No. 6,964,844 relates generally to the synthesis of novel dyes and labels and methods for the detection or visualization of analytes and more specifically to fluorescent latex particles which incorporate the novel fluorescent dyes and utilize, in certain aspects, fluorescence energy transfer and intramolecular energy transfer, for the detection of analytes in immunoassays or in nucleic acid assays. These dyes are water soluble hybrid phthalocyanine derivatives useful in competitive and noncompetitive assays immunoassays, nucleic acid and assays are disclosed and claimed having (1) at least one donor subunit with a desired excitation peak; and (2) at least one acceptor subunit with a desired emission peak, wherein said derivative(s) is/are capable of intramolecular energy transfer from said donor subunit to said acceptor subunit. Such derivatives also may contain an electron transfer subunit. Axial ligands may be covalently bound to the metals contained in the water soluble hybrid phthalocyanine derivatives. Ligands, ligand analogues, polypeptides, proteins and nucleic acids can be linked to the axial ligands of the dyes to form dye conjugates useful in immunoassays and nucleic acid assays.
U.S. Pat. No. 4,997,772 relates to a core/shell polymer particle containing a detectable tracer material in the core only. It also relates to an immunoreactive reagent and the use of that reagent in analytical elements and methods. A water-insoluble polymeric particle has an inner core comprising a detectable tracer material distributed in a first polymer for which the tracer material has a high affinity. This first polymer has a glass transition temperature (Tg1) less than about 100 degree C. The particle also has an outer shell comprising a second polymer for which the tracer material has substantially less affinity relative to said first polymer. This second polymer has a glass transtion temperature (Tg2) which is greater than or equal to the term [Tg1-10 degree C.]. It also contains groups which are either reactive with free amino or sulfhydryl groups of an immunoreactive species or which can be activated for reaction with such groups. Such a species can be covalently attached to this particle to form an immunoreactive reagent which is useful in analytical elements and various analytical methods including immunological methods, for example, agglutination assays.
US 2004/0038318 relates to a reagent set, as well as to a method, for carrying out simultaneous analyses of multiple isoenzymes in a test sample, particularly a bodily fluid. The invention is especially useful for measuring creatine kinase isoenzymes in particle, or bead, based multiplexed assay systems.
U.S. Pat. No. 4,891,324 relates to methods for performing an assay for determining an analyte by use of a conjugate of a member of a specific binding pair consisting of ligands and receptors, for example, antigens and antibodies, with a particle. The method of the invention has particular application to heterogeneous immunoassays of biological fluids, for example, serum or urine. The method is carried out using a composition that includes a conjugate of a first specific binding pair member with a particle. A luminescer is reversibly associated with a nonaqueous phase of the particle. Where the first specific binding pair member is not complementary to the analyte, a second specific binding pair member that is capable of binding to the first specific binding pair member is employed. Unbound conjugate is separated from conjugate that is bound to the analyte or to the second specific binding pair member. A reagent for enhancing the detectability of the luminescer is added and the light emission of the luminescer acted on by the reagent is measured.
WO 2006/016166 relates to polymeric materials suitable for medical materials. This invention discloses a polymer containing an alkoxyethyl acrylate monomer, a monomer containing a primary, secondary, tertiary or quaternary amine group and a monomer containing an acid group. The polymer composition forms fibers with the preferred size of 0.5 to 2.0 um, which is still not sufficient to provide nanoparticles less than 100 nm in size which are colloidally stable and can be loaded with non-water soluble fluorescent dye for the purposes of diagnostic imaging.
U.S. Pat. No. 5,326,692 relates to polymeric materials incorporating multiple fluorescent dyes to allow for controlled enhancement of the Stokes shift. In particular, the invention describes microparticles incorporating a series of two or more fluorescent compounds having overlapping excitation and emission spectra, resulting in fluorescent microparticles with a desired effective Stokes shift. The novel fluorescent microparticles are useful in applications such as the detection and analysis of biomolecules, such as DNA and RNA, that require a very high sensitivity and in flow cytometric and microscopy analytical techniques. The invention relates to microparticles incorporating a series of two or more fluorescent dyes having overlapping excitation and emission spectra allowing efficient energy transfer from the excitation wavelength of the first dye in the series, transfer through the dyes in the series and re-emitted as an optical signal at the emission wavelength of last dye in the series, resulting in a desired effective Stokes shift which is controlled through selection of appropriate dyes.
IR-emissive nanoparticulate assemblies for physiological imaging suffer from several problems. First, the dyes are often highly aggregated and hence nonemissive. Second, the fluorescence for the dye-nanoparticle assemblies is often inefficient in an aqueous environment. Third, the dyes used in such assemblies are unstable to light and oxygen and bleach readily, which makes handling and administration difficult. Fourth, such assemblies are often colloidally unstable and cytotoxic.
The present invention relates to a loaded latex particle comprising a latex material made from a mixture represented by formula (X)m-(Y)n-(Z)o-(W)p, wherein Y is at least one monomer with at least two ethylenically unsaturated chemical functionalities; Z is at least one polyethylene glycol macromonomer with an average molecular weight of between 300 and 10,000; W is an ethylenic monomer different from X, Y, or Z; X is at least one water insoluble, alkoxethyl containing monomer; and m, n, o, and p, are the respective weight percentages of each component monomer. The particle may be loaded with a fluorescent dye.
The present invention includes several advantages, not all of which are incorporated in a single embodiment.
The loaded latexes of this invention give an advantageous combination of properties that make them well suited for specific biological applications. In addition to giving enhanced fluorescence efficiencies, they are highly biocompatible, are resistant to adhesion of serum proteins, and remain well dispersed over as wide range of conditions.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
This invention relates to hydrophobic infrared dyes non-covalently loaded into heavily PEGylated nanolatex particle, which when preferably used in IR-active assemblies, show highly efficient fluorescence, low dye aggregation, and high photostability, that is, are less subject to bleaching. These assemblies are also non-cytotoxic and are very colloidally stable, that is, are less prone to aggregation. The present inventive particle demonstrates an increase in the quantum yield of fluorescence. For purposes of the present invention, a nanolatex is a crosslinked polymer, which is less than 100 nm in size, is composed of alkoxyethyl methacrylate or alkoxyethyl acrylate monomers and is heavily PEGylated.
IR-emissive nanoparticulate assemblies for physiological imaging suffer from several problems. First, the dyes are often highly aggregated and hence nonemissive. Second, the fluorescence for the dye-nanoparticle assemblies is often inefficient in an aqueous environment. Third, the dyes used in such assemblies are unstable to light and oxygen and bleach readily, which makes handling and administration difficult. Fourth, such assemblies are often colloidally unstable and cytotoxic.
For purposes of the present invention: “PEGylated” refers to nanolatex compositions which are composed of at least 5 weight percent covalently bound poly(ethylene glycol). “Pegylation” typically refers to the reaction by which a PEG-protein/peptide conjugate is obtained starting from the activated PEG and the corresponding protein/peptide. This may also apply to PEG-Therapeutic Agent, PEG-Dye, PEG-bioligand, PEG-(MRI Contrast Agent), PEG-(X-Ray Contrast Agent), PEG-Antibody, PEG-(Enzyme Inhibitor) PEG-(radioactive isotope), PEG-(quantum dot), PEG-oligosaccharide, PEG-polygosaccharide, PEG-hormone, PEG-dextran, PEG-oligonucleotide, PEG-carbohydrate, PEG-neurotransmitter, PEG-hapten, PEG-carotinoid.
“Nanolatex” refers to a hydrophobic polymer particle which has a hydrodynamic diameter of less than 100 nm.
A “water dispersible crosslinked polymer particle” refers to a polymer particle which is a contiguous, crosslinked polymer network through which a through-bond path can be traced between any two atoms (not including counterions) in the particle. The particle is capable of existing in water in such a state of division that that each individual particle network is separated from every other by the aqueous continuous phase.
A “hydrophobic crosslinked polymer” refers to a polymer consisting of at least 45 weight percent of water-insoluble monomers. The polymer is a contiguous network through which a through-bond path can be traced between any two atoms (not including counter ions).
“Biocompatible” means that a composition does not disrupt the normal function of the bio-system into which it is introduced. Typically, a biocompatible composition will be compatible with blood and does not otherwise cause an adverse reaction in the body. For example, to be biocompatible, the material should not be toxic, immunogenic or thrombogenic.
“Colloidally stable” refers to the state in which the particle is capable of existing in aqueous phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 at pH 7.4.) in such a state of division that that each individual particle is separated from every other by the aqueous continuous phase without the formation of agglomerates (entities comprising multiple individual particles in intimate contact) or without bulk flocculation occurring.
“Loaded” or “embedded” refers to a non-covalent association between the dye and the polymer particle such that when the latex is dispersed in water at a concentration of less than 10%, less than 1% of the total dye in the system can be extracted into the water continuous phase.
The latex of this invention is composed of repetitive crosslinked ethylenically unsaturated monomers. The latex may have a volume-average hydrodynamic diameter of 5 and 100 nm, preferably 8 to 50 nm as determined by quasi-elastic light scattering in phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 at pH 7.4.).
One loaded latex particle of the invention comprises a latex material made from a mixture represented by the following Formula I:
(X)m-(Y)n-(Z)o-(W)p FORMULA I
wherein X is at least one water insoluble, alkoxethyl containing monomer; Y is at least one monomer with at least two ethylenically unsaturated chemical functionalities; Z is at least one polyethylene glycol macromonomer with an average molecular weight of between 300 and 10,000; W is an ethylenic monomer different from X, Y, or Z. The weight percent range of each component monomer is represented by m, n, o, and p: m ranges between 40-90 wt %, preferably from 45-60 wt %.; n ranges between 1-10 wt %, preferably 2-6 wt %; o ranges between 20-60 wt %, preferably between 40-50 wt %; and p is up to 10 wt %.
In Formula 1, X is a water-insoluble, alkoxyethyl-containing monomer described below by Formula 2. In Formula 2, R1 is methyl or hydrogen. R2 is an alkyl or aryl group containing up to 10 carbons. Preferably, X is methoxyethyl methacrylate or alkoxyethyl acrylate.
In Formula 1, Y is a water-insoluble or water-soluble monomer containing at least two ethylenically unsaturated chemical functionalities. These functionalities may be vinyl groups, acrylates, methacrylates, acrylamides, methacrylamides, allyl groups, vinyl ethers and vinyl esters. Y monomers include, but are not necessarily limited to aromatic divinyl compounds such as divinylbenzene, divinylnaphthalene or derivatives thereof, diethylene carboxylate esters and amides such as ethylene glycol dimethacrylate, diethylene glycol diacrylate, 1,4 butanediol diacrylate, 1,4 butanediol dimethacrylate, 1,3 butylene glycol diacrylate, 1,3 butylene glycol dimethacrylate, cyclohexane dimethanol diacrylate, cyclohexane dimethanol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, dipropylene glycol diacrylate, dipropylene glycol dimethacrylate, ethylene glycol diacrylate, ethylene glycol dimethacrylate, 1,6 hexanediol diacrylate, 1,6 hexanediol dimethacrylate, neopentyl glycol diacrylate, neopentyl glycol dimethacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, triethylene glycol diacrylate, triethylene glycol dimethacrylate, tripropylene glycol diacrylate, tripropylene glycol dimethacrylate, pentaerythritol triacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, dipentaerythritol pentaacrylate, di-trimethylolpropane tetraacrylate, pentaerythritol tetraacrylate, divinyl esters such as divinyl adipate, and other divinyl compounds such as divinyl sulfide or divinyl sulfone compounds of allyl methacrylate, allyl acrylate, cyclohexanedimethanol divinyl ether diallylphthalate, diallyl maleate, dienes such as butadiene and isoprene and mixtures thereof.
The W monomer basically consists of any other inert monomers which are added to somehow modify the properties. W is a non-chemically reactive monomer which can be added in small amounts to impart desired properties to the latex, such as water dispersibility, charge, more facile dye loading, or to make the latex more hydrophobic. W may be a water-soluble monomer such as 2-phosphatoethyl acrylate potassium salt, 3-phosphatopropyl methacrylate ammonium salt, vinylphosphonic acid, and their salts, vinylcarbazole, vinylimidazole, vinylpyrrolidone, vinylpyridines, acrylamide, methacrylamide, maleic acid and salts thereof, sulfopropyl acrylate and methacrylate, acrylic and methacrylic acids and salts thereof, N-vinylpyrrolidone, acrylic and methacrylic esters of alkylphosphonates, styrenics, acrylic and methacrylic monomers containing amine or ammonium functionalities, styrenesulfonic acid and salts thereof, acrylic and methacrylic esters of alkylsulfonates, vinylsulfonic acid and salts thereof, vinylpyridines, hydroxyethyl acrylate, glycerol acrylate and methacrylate esters, (meth)acrylamide, and N-vinylpyrrolidone. W may alternately be a water-insoluble monomer such as methyl methacrylate, ethyl methacrylate, isobutyl methacrylate, 2-ethylhexyl methacrylate, benzyl methacrylate, cyclohexyl methacrylate and glycidyl methacrylate, acrylic/acrylate esters such as methyl acrylate, ethyl acrylate, isobutyl acrylate, 2-ethylhexyl acrylate, benzyl methacrylate, phenoxyethyl acrylate, cyclohexyl acrylate, and glycidyl acrylate, styrenics such as styrene, a-methylstyrene, ethylstyrene, 3- and 4-chloromethylstyrene, halogen-substituted styrenes, and alkyl-substituted styrenes, vinyl halides and vinylidene halides, N-alkylated acrylamides and methacrylamides, vinyl esters such as vinyl acetate and vinyl benzoate, vinyl ether, allyl alcohol and its ethers and esters, and unsaturated ketones and aldehydes such as acrolein and methyl vinyl ketone, isoprene, butadiene and acrylonitrile.
Z is a polyethylene glycol macromonomer with a molecular weight of between 300 and 10,000, preferably between 500 and 5000. Preferably, the linking polymer is a polyethylene glycol backbone chain with specific functional end groups at each end, which allows the polyethylene glycol to act as a linking group between two materials through the two functional end groups.
The polyethylene glycol macromonomer contains a radical polymerizeable group at one end. This group can be, but is not necessarily limited to a methacrylate, acrylate, acrylamide, methacrylamide, styrenic, allyl, vinyl, maleimide, or maleate ester. Preferably, the polyethylene glycol macromonomer additionally contains a reactive chemical functionality at the other end which can serve as an attachment point for other chemical units, such as quenchers or antibodies. This chemical functionality may be, but is not limited to alcohols, thiols, carboxylic acids, primary or secondary amines, vinylsulfonyls, aldehydes, epoxides, hydrazides, succinimidyl esters, maleimides, a-halo carbonyl moieties (such as iodoacetyls), isocyanates, isothiocyanates, and aziridines. Preferably, these functionalities will be carboxylic acids, primary amines, maleimides, vinylsulfonyls, or secondary amines.
A class of polyethylene glycol macromonomers with a reactive functional group at one end can be described by Formula 3.
In Formula 3, R1 is hydrogen or methyl, q is 10-200, r is 0-10, and RG is a hydrogen or reactive chemical functionality which can be a alcohol, thiol, carboxylic acid, primary or secondary amine, vinylsulfonyl, aldehyde, epoxy, hydrazide, succinimidyl ester, maleimide, a substituted or unsubstituted acetate, or substituted carbamyl, substituted phosphate, substituted or unsubstituted sulfonate a-halo carbonyl moiety (such as iodoacetyl), isocyanate, isothiocyanate, or aziridine.
The linking polymer is typically utilized in two ways. First, a single linking polymer may be used to attach one functional compound of interest to another, thereby producing a single compound with two different desired functions. Multiple linking polymers may also be attached to a single large particle or bead at one end and a compound of interest on the other, thereby producing a single carrier particle for a large payload of functional compound of interest.
The linking polymer may be used in both the acylation and alkylation approaches and is compatible with aqueous and organic solvent systems, so that there is more flexibility in reacting with useful groups and the desired products are more stable in an aqueous environment, such as a physiological environment. The linking polymer has at least two reactive groups, one of which is an acrylate which is useful for forming nanogels and latexes and reacting with thiols through Michael addition, the other reactive groups is useful for conjugation to contrast agents, dyes, proteins, amino acids, peptides, antibodies, bioligands, therapeutic agents and enzyme inhibitors. The linking polymer may be branched or unbranched. Preferably, for therapeutic use of the end-product preparation, the linking polymer will be pharmaceutically acceptable.
RG in Formula 3 is hydrogen or a reactive chemical functionality. A reactive chemical functionality allows the loaded latex to be covalently bonded to a biomolecule and the location of the biomolecule can be determined by fluorescent imaging. The covalent attachment provides a link that is stable to handling, changes in solvent, pH, and ionic strength, and temperature. This stable association between the loaded latex particle and the biomolecule is important to insure that the fluorescent signal that is detected relates to the presence of the biomolecule. If a loaded latex is not covalently attached and associated with the biomolecule through ionic attraction, or Van der Waals forces, then the dye may become detached and the desired biomolecule signal will decrease and false signals may be obtained from the separated loaded latex such that the fluorescence image does not indicate the location of the biomolecule. The reactive chemical functionality (RG) should allow covalent bonding to occur in organic solvents such as N,N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, and non-organic solvents such as water.
The reactive chemical functionality (RG) will allow the use of linkers that are designed to form covalent bonds between the reactive chemical functionality (RG) on the loaded latex and an attachment group on a bio molecule such as an amine, alcohol, carboxylic acid or thiol from amino acids, peptides, protein, cells, RNA, DNA or other linkers which have been added to the biomolecule to allow for greater flexibility in the methods used to attach biomolecules to other materials. Such linkers would include but not be limited to hetero-bifunctional or homo-bifunctional linkers such as bis-sulfosuccinylsuberate,3-[2-(aminoethyl)dithio]propionic acid, p-azidobenzoylhydrazide, bis-maleimidohexane, N-succinimidyl-S-acetylthioacetate, N-Sulfosuccinimidyl-4-azidophenyl-1-3′-dithiopropionatte, Succinimidyl 4-[p-maleimidophenyl]butyrate, N-Succinimidyl[4-iodoacetyl]aminobenzoate, Sulfosuccinimidyl-[perfluoroazidobenzamido]ethyl-1,3′-dithiopropionate, Succinimidyl 3-[bromoacetamido]propionate, Sulfosuccinimidyl 2-[7-amino-4-methylcoumarin-3-acetamido]ethyl-1,3′dithiopropionate, 3-(2-Pyridyldithio)propionyl hydrazide), N-e-Maleimidocaproyloxy]succinimide ester, N-[4-(p-Azidosalicylamido) butyl]-3′-(2′-pyridyldithio)propionamide, Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate, 4-(4-N-Maleimidophenyl)butyric acid hydrazide hydrochloride, Disuccinimidyl suberate, Lomant's Reagent, Sulfosuccinimidyl[2-6-(biotinanido)-2-(p-azidobenzamido)-hexanoamido]-ethyl-1,3′-dithiopropionate, B-[Tris(hydroxymethyl) phosphino]propionic acid (betaine), (Sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate), Bis-Maleimidoethane, Bis-[b-(4-Azidosalicylamido)ethyl]disulfide, Succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate], N-[p-Maleimidophenyl]isocyanate, m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester, Sulfosuccinimidyl 4-N-maleimidomethyl cyclohexane-1-carboxylate, Bis[sulfosuccinimidyl]suberate, N-[g-Maleimidobutyryloxy]sulfosuccinimide ester, N-succinimidyl 4-pentynoate, and N-succinimidyl 4-azidoylbutanoate.
The reactive chemical functionality may also serve as an attachment point for a metal chelating group used to chelate metals such as radioisotopes (for PET imaging) and Gadolinium (for MRI) imaging. In one embodiment, the metal chelating group is S-2-(4-Isothiocyanatobenzyl)-1,4,7,10 tetraazododecane-tetraacetic acid or 2-(4-Isothiocyanatobenzyl)diethylenetriaminepentaacetic acid. The metal chelating group may be bound to a radioisotope or heavy metal.
By the proper use of reactive chemical functionality (RG), the loaded latex can be covalently attached to any drug or biomolecule in such a way to optimize the fluorescent signal and not interfere with the normal function of the biomolecule. The carboxylic acid attachment group can be converted to an active ester to enable the covalent bond formation. An N-hydroxysuccinimide ester is a preferred method of activating the carboxylic acid group. The carboxylic acid attachment group can also be activated for covalent bond formation with carbodiimide reageants such as dicylcohexylcarbodiimide. A hydroxyl attachment group can be activated for covalent bond formation by forming a chloroformate such as p-nitrophenyl chloroformate. An amine attachment group can be activated for covalent bond formation by forming using the carbodiimide activating agent to react with carboxylic acid functions of the biomolecule, or forming isocyanates or isothiocyanates or using an amine reactive linking group from the list above. The maleimide linking group can react with thiol groups typically available from cysteine residues in biomolecules or a thiol linking group from the list above, an isocyanate or isothiocyanate can be used directly to react with amine groups of a biomolecule. The trialkoxysilane can be use to react with other trialkoxysilanes or siloxide modified molecules or particles. The alkyne and azidoyl group can be used to form a stable triazole link often catalyzed by copper (I); such that if the dye contains an alkynyl attachment group, then an azidoyl attachment group is placed on the biomolecule or the opposite where an azidoyl group is the attachment group on the dye and an alkynyl group is added to the biomolecule.
A particularly preferred water-soluble linking polymer for use herein is a polyethylene glycol derivative of Formula 4. Polyethylene glycol (PEG) is a hydrophilic, biocompatible and non-toxic polymer of general formula H(OCH2CH2)nOH, wherein n>4.
wherein X═CH3 or H, Y═O, NR, or S, L is a linking group or spacer, FG is a functional group, n is greater than 4 and less than 1000. Most preferably, X═CH3, Y═O, NR, L is alkyl or aryl, and FG is NH2 or COOH, and n is between 6 and 500 or between 10 and 200. Most preferably, n=16.
The linking polymer may be used by attaching to biologically important materials, dyes and contrast agents for detection and of disease and the study of metabolic activity, therapeutic agents for the treatment of disease, agents for making thickener agents, pharmaceuticals, and cosmetics. The preferred biologically important materials for attachment of the linking polymer include targeting agents, diagnostic agents, and therapeutic agents, which can be greatly improved in effectiveness when linked.
Targeting agents are compounds with useful groups that will identify and associate with a specific site, such as a disease site, such that the particle or conjugated material will be concentrated in this site for greater effect. Also of particular interest are PEG-antibodies. Antibodies, also known as immunoglobulins (Igs), are proteins that help identify foreign substances to the immune system, such as a bacteria or a virus or any substance bearing an antigen, and are useful for identification and association of specific biological targets. Bioligands are useful groups that will associate with receptor sites expressed in or on cells or with enzymes. Examples of bioligands are growth factors such as biotin and folic acid, specific proteins, and peptide sequences of amino acids or molecules which have strong binding ability to the active sites of enzymes or help the material penetrate or concentrate on or in cells of interest.
Diagnostic agents are materials which enhance the signal of detection when a material is scanned with light, sound, magnetic, electronic and radioactive sources of energy. Examples would be dyes such as UV, visible or infrared absorbing dyes especially fluorescent dyes such as indocarbocyanines and fluorescein, MR contrast agents such as gadallinium and iron oxide complexes, and X-ray constrast agents such as a polyiodoaromatic compound. The loaded latex particles can be functionalized with chelating groups such as diethylenteriamepenatacetic acid (DTPA) or 1,4,7,10-tetra-azacyclododecane-N,N′,N″,N′″-tetra acetic acid (DOTA). These chelating groups can allow the chelating of metals such as Gadolinium used in magnetic resonance imaging and X-ray imaging. Tetra- and pentaacetic acid chelating groups also allow the loaded latex to be labeled with radioisotopes for radioscintigraphy, single photon emission and positive emission tomography.
The component being labeled can be in a mixture including other materials. The mixture, in which the labeling reaction occurs, can be a liquid mixture, particularly a water mixture. The detection step can occur with the mixture in a liquid or dry condition, such as a microscope slide.
“Labeling” refers to the attachment of the loaded latex or loaded latex conjugate to a material to aid in the identification of the material. Preferably, the material is identified by optical detection methods.
“Biocompatible” means that a composition does not disrupt the normal function of the bio-system into which it is introduced. Typically, a biocompatible composition will be compatible with blood and does not otherwise cause an adverse reaction in the body. For example, to be biocompatible, the material should not be toxic, immunogenic or thrombogenic.
“Biodegradable” means that the material can be degraded either enzymatically or hydrolytically under physiological conditions to smaller molecules that can be eliminated from the body through normal processes.
The term “diagnostic agent” includes components that can act as contrast agents and thereby produce a detectable indicating signal in the host or test sample. The detectable indicating signal may be gamma-emitting, radioactive, echogenic, fluoroscopic or physiological signals, or the like.
The term “biomedical agent” as used herein includes biologically active substances which are effective in the treatment of a physiological disorder, pharmaceuticals, enzymes, hormones, steroids, recombinant products and the like. Exemplary therapeutic agents are antibiotics, thrombolytic enzymes such as urokinase or streptokinase, insulin, growth hormone, chemotherapeutics such as adriamycin and antiviral agents such as interferon and acyclovir.
In one preferred embodiment, the loaded latex is associated with a material that is selective for a target material to be labeled and optionally detected. For example, nucleic acid detection generally involves probing a sample thought to contain target nucleic acids using a nucleic acid probe that contains a nucleic acid sequence that specifically recognizes the sequence of the target nucleic acids, such that the nucleic acid probe and the target nucleic acids in combination create a hybridization pair. The nucleic acid probe typically contains from greater than about 4 bases to as many as tens of thousands of bases, although probing entire chromosomes may involve millions of bases. Any of the dye-conjugates described below may be used to label the corresponding target materials.
The component or conjugate to which the loaded latex is attached, also referred to as the labeled component, can be antibodies, proteins, peptides, enzyme substrates, hormones, lymphokines, metabolites, receptors, antigens, haptens, lectins, toxins, carbohydrates, sugars, oligosaccharides, polysaccharides, nucleic acids, deoxy nucleic acids, derivatized nucleic acids, derivatized deoxy nucleic acids, DNA fragments, RNA fragments, derivatized DNA fragments, derivatized RNA fragments, natural drugs, virus particles, bacterial particles, virus components, yeast components, blood cells, blood cell components, biological cells, noncellular blood components, bacteria, bacterial components, natural and synthetic lipid vesicles, synthetic drugs, poisons, environmental pollutants, polymers, polymer particles, glass particles, glass surfaces, plastic particles and plastic surfaces.
A variety of loaded latex-conjugates may be prepared using the loaded latexes of the invention, including conjugates of antigens, steroids, vitamins, drugs, haptens, metabolites, toxins, environmental pollutants, amino acids, peptides, proteins, nucleic acids, nucleic acid polymers, carbohydrates, lipids, and polymers. In another embodiment, the conjugated substance is an amino acid, peptide, protein, polysaccharide, nucleotide, oligonucleotide, nucleic acid, hapten, drug, lipid, phospholipid, lipoprotein, lipopolysaccharide, liposome, lipophilic polymer, polymer, polymeric microparticle, biological cell or virus. In one aspect of the invention, the conjugated substance is labeled with a plurality of loaded latexes of the present invention, which may be the same or different.
The loaded latexes are useful as labels for probes and in immunoassays and also as labels for in-vivo imaging and in-vivo tumor therapy. When so used, these loaded latexes may be linked to one member of a specific binding pair (“labeled binding partner”) or an analog of such a member to form a loaded latex-conjugate.
These loaded latexes may be used as agents for in-vivo imaging. When used as imaging agents, these loaded latexes are conjugated to one member of a specific binding pair to give a labeled conjugate/binding complement. The loaded latex-conjugate is introduced into an animal. If the other member of the specific binding pair is present, the loaded latex-conjugate will bind thereto and the signal produced by the dye may be measured and its localization identified.
These loaded latexes may also be used in in-vivo tumor therapy. For example, photodynamic therapy involves using an additional dye component attached to the surface of the nanoparticle as a photosensitizing agent. The loaded latex with photosensitizing agent is further conjugated to a binding partner which may specifically recognize and bind to a component of a tumor cell. The localized triplet emission from the bound dye-loaded latex conjugate after excitation by light, causes chemical reactions and selective damage and/or destruction to the tumor cells.
In one embodiment, the loaded latex or loaded latex-conjugates are used to probe a sample solution for the presence or absence of a target analyte. By “target analyte” or “analyte” or grammatical equivalents herein is meant any atom, molecule, ion, molecular ion, compound or particle to be either detected or evaluated for binding partners. As will be appreciated by those in the art, a large number of analytes may be used in the present invention; basically, any target analyte can be used which binds a bioactive agent or for which a binding partner (i.e. drug candidate) is sought.
The target material is optionally a material of biological or synthetic origin that is present as a molecule or as a group of molecules, including, but not limited to, antibodies, amino acids, proteins, peptides, polypeptides, enzymes, enzyme substrates, hormones, lymphokines, metabolites, antigens, haptens, lectins, avidin, streptavidin, toxins, poisons, environmental pollutants, carbohydrates, oligosaccarides, polysaccharides, glycoproteins, glycolipids, nucleotides, oligonucleotides, nucleic acids and derivatized nucleic acids (including deoxyribo- and ribonucleic acids), DNA and RNA fragments and derivatized fragments (including single and multi-stranded fragments), natural and synthetic drugs, receptors, virus particles, bacterial particles, virus components, biological cells, spores, cellular components (including cellular membranes and organelles), natural and synthetic lipid vesicles, polymer membranes, polymer surfaces and particles, and glass and plastic surfaces and particles. Typically the target material is present as a component or contaminant of a sample taken from a biological or environmental system. Particularly preferred analytes are nucleic acids and proteins.
In one aspect of the invention, the conjugate is a bioreactive substance. The target material is optionally a bioreactive substance also. Bioreactive substances are substances that react with or bind to molecules that are derived from a biological system, whether such molecules are naturally occurring or result from some external disturbance of the system (e.g. disease, poisoning, genetic manipulation). By way of illustration, bioreactive substances include biomolecules (i.e. molecules of biological origin including, without limitation, polymeric biomolecules such as peptides, proteins, polysaccharides, oligonucleotides, avidin, streptavidin, DNA and RNA, as well as non-polymeric biomolecules such as biotin and digoxigenin and other haptens typically having a MW less than 1000), microscopic organisms such as viruses and bacteria, and synthetic haptens (such as hormones, vitamins, or drugs). Typically the target complement or the target material or both are amino acids, peptides (including polypeptides), or proteins (larger MW than polypeptides); or are nucleotides, oligonucleotides (less than 20 bases), or nucleic acids (i.e. polymers larger than oligonucleotides, including RNA and single- and multi-stranded DNA and fragments and derivitized fragments thereof); or are carbohydrates or carbohydrate derivatives, including monosaccharides, polysaccharides, oligosaccharides, glycolipids, and glycoproteins; or are haptens (a chemical compound that is unable to elicit an immunological response unless conjugated to a larger carrier molecule), which haptens are optionally conjugated to other biomolecules; or a microscopic organisms or components of microscopic organisms. For such bioreactive substances, there are a variety of known methods for selecting useful pairs of corresponding conjugates complementary to the target materials.
Where more than one material is targeted simultaneously, multiple conjugates which are target complements (one for each corresponding target material) are optionally included. Target complements are selected to have the desired degree of specificity or selectivity for the intended target materials.
In one embodiment, the target analyte is a protein. As will be appreciated by those in the art, there are a large number of possible proteinaceous target analytes that may be detected or evaluated for binding partners using the present invention. Suitable protein target analytes include, but are not limited to, (1) immunoglobulins; (2) enzymes (and other proteins); (3) hormones and cytokines (many of which serve as ligands for cellular receptors); and (4) other proteins. In a preferred embodiment, the target analyte is a nucleic acid. In a preferred embodiment, the probes are used in genetic diagnosis. For example, probes can be made using the techniques disclosed herein to detect target sequences such as the gene for nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which is a gene associated with a variety of cancers, the Apo E4 gene that indicates a greater risk of Alzheimer's disease, allowing for easy presymptomatic screening of patients, mutations in the cystic fibrosis gene, or any of the others well known in the art.
In an additional embodiment, viral and bacterial detection is done using the complexes of the invention. In this embodiment, probes are designed to detect target sequences from a variety of bacteria and viruses. For example, current blood-screening techniques rely on the detection of anti-HIV antibodies. The methods disclosed herein allow for direct screening of clinical samples to detect HIV nucleic acid sequences, particularly highly conserved HIV sequences. In addition, this allows direct monitoring of circulating virus within a patient as an improved method of assessing the efficacy of anti-viral therapies. Similarly, viruses associated with leukemia, HTLV-I and HTLV-II, may be detected in this way. Bacterial infections such as tuberculosis, clymidia and other sexually transmitted diseases, may also be detected.
In another embodiment, the nucleic acids of the invention find use as probes for toxic bacteria in the screening of water and food samples. For example, samples may be treated to lyse the bacteria to release its nucleic acid, and then probes designed to recognize bacterial strains, including, but not limited to, such pathogenic strains as, Salmonella, Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E. coli, and Legionnaire's disease bacteria.
The described composition can further comprise a biological, pharmaceutical or diagnostic component that includes a targeting moiety that recognizes a specific target cell. Recognition and binding of a cell surface receptor through a targeting moiety associated with loaded latexes can be a feature of the described compositions. This feature takes advantage of the understanding that a cell surface binding event is often the initiating step in a cellular cascade leading to a range of events, notably receptor-mediated endocytosis. The term “receptor mediated endocytosis” (“RME”) generally describes a mechanism by which, catalyzed by the binding of a ligand to a receptor disposed on the surface of a cell, a receptor-bound ligand is internalized within a cell. Many proteins and other structures enter cells via receptor mediated endocytosis, including insulin, epidermal growth factor, growth hormone, thyroid stimulating hormone, nerve growth factor, calcitonin, glucagon and many others.
Receptor Mediated Endocytosis (hereinafter “RME”) affords a convenient mechanism for transporting a dye-conjugate, possibly in combination with other biological, pharmaceutical or diagnostic components, to the interior of a cell.
In RME, the binding of a ligand by a receptor disposed on the surface of a cell can initiate an intracellular signal, which can include an endocytosis response. Thus, a loaded latex with a targeting moiety associated to form a loaded latex-conjugate, can bind on the surface of a cell and subsequently be invaginated and internalized within the cell. A representative, but non-limiting, list of moieties that can be employed as targeting agents useful with the present compositions is selected from the group consisting of proteins, peptides, aptomers, small organic molecules, toxins, diptheria toxin, pseudomonas toxin, cholera toxin, ricin, concanavalin A, Rous sarcoma virus, Semliki forest virus, vesicular stomatitis virus, adenovirus, transferrin, low density lipoprotein, transcobalamin, yolk proteins, epidermal growth factor, growth hormone, thyroid stimulating hormone, nerve growth factor, calcitonin, glucagon, prolactin, luteinizing hormone, thyroid hormone, platelet derived growth factor, interferon, catecholamines, peptidomimetrics, glycolipids, glycoproteins and polysacchlorides. Homologs or fragments of the presented moieties can also be employed. These targeting moieties can be associated with loaded latex and be used to direct the loaded latex-conjugate to a target cell, where it can subsequently be internalized. There is no requirement that the entire moiety be used as a targeting moiety. Smaller fragments of these moieties known to interact with a specific receptor or other structure can also be used as a targeting moiety.
An antibody or an antibody fragment represents a class of most universally used targeting moiety that can be utilized to enhance the uptake of loaded latex or loaded latex-conjugate into a cell. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988. Antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). A superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, “Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion.” Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.
Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides may be used in the purification process in, for example, an affinity chromatography step.
A number of “humanized” antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86:4220-4224. These “humanized” molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules that limits the duration and effectiveness of therapeutic applications of those moieties in human recipients.
Vitamins and other essential minerals and nutrients can be utilized as targeting moiety to enhance the uptake of loaded latex or loaded latex-conjugate by a cell. In particular, a vitamin ligand can be selected from the group consisting of folate, folate receptor-binding analogs of folate, and other folate receptor-binding ligands, biotin, biotin receptor-binding analogs of biotin and other biotin receptor-binding ligands, riboflavin, riboflavin receptor-binding analogs of riboflavin and other riboflavin receptor-binding ligands, and thiamin, thiamin receptor-binding analogs of thiamin and other thiamin receptor-binding ligands. Additional nutrients believed to trigger receptor mediated endocytosis, and thus also having application in accordance with the presently disclosed method, are carnitine, inositol, lipoic acid, niacin, pantothenic acid, pyridoxal, and ascorbic acid, and the lipid soluble vitamins A, D, E and K. Furthermore, any of the “immunoliposomes” (liposomes having an antibody linked to the surface of the liposome) described in the prior art are suitable for use with the described loaded latex or loaded latex-conjugates.
Since not all natural cell membranes possess biologically active biotin or folate receptors, use of the described compositions in-vitro on a particular cell line can involve altering or otherwise modifying that cell line first to ensure the presence of biologically active biotin or folate receptors. Thus, the number of biotin or folate receptors on a cell membrane can be increased by growing a cell line on biotin or folate deficient substrates to promote biotin and folate receptor production, or by expression of an inserted foreign gene for the protein or apoprotein corresponding to the biotin or folate receptor.
RME is not the exclusive method by which the loaded latex or loaded latex-conjugates can be translocated into a cell. Other methods of uptake that can be exploited by attaching the appropriate entity to a, loaded latex or loaded latex-conjugate include the advantageous use of membrane pores. Phagocytotic and pinocytotic mechanisms also offer advantageous mechanisms by which a loaded latex or loaded latex-conjugate can be internalized inside a cell.
The recognition moiety can further comprise a sequence that is subject to enzymatic or electrochemical cleavage. The recognition moiety can thus comprise a sequence that is susceptible to cleavage by enzymes present at various locations inside a cell, such as proteases or restriction endonucleases (e.g. DNAse or RNAse).
The water dispersible, loaded latex may also be useful in other biomedical applications, including, but not limited to, tomographic imaging of organs, monitoring of organ functions, coronary angiography, fluorescence endoscopy, detection, imaging, determining efficacy of drug delivery, and therapy of tumors, laser assisted guided surgery, photoacoustic methods, and sonofluorescent methods.
The compositions of the invention can be formulated into diagnostic compositions for enteral or parenteral administration. These compositions contain an effective amount of the dye along with conventional pharmaceutical carriers and excipients appropriate for the type of administration contemplated. For example, parenteral formulations advantageously contain a sterile aqueous solution or suspension of dye according to this invention. Parenteral compositions may be injected directly or mixed with a large volume parenteral composition for systemic administration. Such solutions also may contain pharmaceutically acceptable buffers and, optionally, electrolytes such as sodium chloride.
Formulations for enteral administration may vary widely, as is well known in the art. In general, such formulations are liquids which include an effective amount of the dye in aqueous solution or suspension. Such enteral compositions may optionally include buffers, surfactants, thixotropic agents, and the like. Compositions for oral administration may also contain flavoring agents and other ingredients for enhancing their organoleptic qualities.
The diagnostic compositions are administered in doses effective to achieve the desired enhancement. Such doses may vary widely, depending upon the particular dye employed, the organs or tissues which are the subject of the imaging procedure, the imaging equipment being used, and the like.
The diagnostic compositions of the invention are used in the conventional manner. The compositions may be administered to a patient, typically a warm-blooded animal, either systemically or locally to the organ or tissue to be imaged, and the patient then subjected to the imaging procedure.
Administration techniques include parenteral administration, intravenous administration and infusion directly into any desired target tissue, including but not limited to a solid tumor or other neoplastic tissue. Purification can be achieved by employing a final purification step, which disposes the loaded latex or loaded latex-conjugate composition in a medium comprising a suitable pharmaceutical composition. Suitable pharmaceutical compositions generally comprise an amount of the desired loaded latex or loaded latex conjugate with active agent in accordance with the dosage information (which is determined on a case-by-case basis). The described particles are admixed with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give an appropriate final concentration. Such formulations can typically include buffers such as phosphate buffered saline (PBS), or additional additives such as pharmaceutical excipients, stabilizing agents such as BSA or HSA, or salts such as sodium chloride.
For parenteral administration it is generally desirable to further render such compositions pharmaceutically acceptable by insuring their sterility, non-immunogenicity and non-pyrogenicity. Such techniques are generally well known in the art. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biological Standards. When the described loaded latex or loaded latex-conjugate composition is being introduced into cells suspended in a cell culture, it is sufficient to incubate the cells together with the nanoparticle in an appropriate growth media, for example Luria broth (LB) or a suitable cell culture medium. Although other introduction methods are possible, these introduction treatments are preferable and can be performed without regard for the entities present on the surface of a loaded latex carrier.
The loaded latex-conjugates described above, whether for single or multicolor detection systems, are combined with a sample thought to contain target materials. Typically the sample is incubated with an aqueous suspension of the loaded latex-conjugates. Where a single color detection system is used, the aqueous suspension contains substantially identical, loaded latex-conjugates. Where a multicolor detection system is used, the aqueous suspension contains a number of detectably different loaded latex-conjugates. In each case, the loaded latex-conjugates are specific for a particular target or combination of targets.
Prior to combination with the loaded latex-conjugates, the sample is prepared in a way that makes the target materials in the sample accessible to the probes. The target materials may require purification or separation prior to labeling or detection. For example, the sample may contain purified nucleic acids, proteins, or carbohydrates, either in mixtures or individual nucleic acid, protein, or carbohydrate species; the sample may contain nucleic acids, proteins, or carbohydrates in lysed cells along with other cellular components; or the sample may contain nucleic acids, proteins, or carbohydrates in substantially whole, permeabilized cells. Preparation of the sample will depend on the way the target materials are contained in the sample. When the sample contains cellular nucleic acids (such as chromosomal or plasmid borne genes within cells, RNA or DNA viruses or mycoplasma infecting cells, or intracellular RNA) or proteins, preparation of the sample involves lysing or permeabilizing the cell, in addition to the denaturation and neutralization already described.
Following the labeling of the sample with the loaded latex-conjugates, unbound loaded latex-conjugates are optionally removed from the sample by conventional methods such as washing.
For detection of the target materials, the sample is illuminated with means for exciting fluorescence in the loaded latex-conjugates. Typically a source of excitation energy emitting within the range of the excitation peak of the loaded latex-conjugates is used. Fluorescence resulting from the illuminated, loaded latex-conjugates that have formed a complex with the target materials can be used to detect the presence, location, or quantity of target materials.
Fluorescence from the loaded latex-conjugates can be visualized with a variety of imaging techniques, including ordinary light or fluorescence microscopy and laser scanning confocal microscopy and CCD cameras. Three-dimensional imaging resolution techniques in confocal microscopy utilize knowledge of the microscope's point spread function (image of a point source) to place out-of-focus light in its proper perspective. Multiple labeled target materials are optionally resolved spatially, chronologically, by size, or using detectably different spectral characteristics (including excitation and emission maxima, fluorescence intensity, or combinations thereof). Typically, multiple labeled target materials are resolved using different loaded latex conjugates with distinct spectral characteristics for each target material. Alternatively, the loaded latex-conjugates are the same but the samples are labeled and viewed sequentially or spatially separated. If there is no need or desire to resolve multiple targets, as in wide scale screening (e.g. pan-viral or bacterial contamination screening), loaded latex-conjugates containing multiple target complements need not be separately applied to samples
Therapeutic agents are materials which effect enhance or inhibit cellular function, blood flow, or biodistribution, or bioabsorbtion. Examples would be pharmaceutical drugs for cancer, heart disease, genetic disorders, bacterial and viral infection and many other disorders.
Other useful materials to conjugate would be: PEG-peptide, PEG-protein, PEG-enzyme inhibitor PEG-oligosaccharide, PEG-polygosaccharide, PEG-hormone, PEG-dextran, PEG-oligonucleotide, PEG-carbohydrate, PEG-neurotransmitter, PEG-hapten, PEG-carotinoid.
The PEG could be functionalized with mixtures of these materials to improve effectiveness.
The following is a list of preferred linking polymers, but is not intended to an exhaustive and complete list of all linking polymers according to the present invention:
In one method of use, multiple linking polymers are attached to a nanogel. For example, a first mixture of monomer(s) of interest, the linking polymer, and initiator is prepared in water. The first mixture was added to the second mixture of additional initiator and reacted, after which, additional initial may be added to produce a nanogel composition. In another preferred method of use, multiple linking polymers are attached to a nanolatex. A mixture of monomers, linking polymer, initiator, surfactant, and buffer was prepared in water. The mixture is added to an aqueous solution of initiator, surfactant and buffer and reacted to produce a nanolatex particle according to the present invention.
In general, the derivatization may be performed under any suitable condition to react a biologically active substance with an activated water soluble linking polymer molecule. In general, the optimal reaction conditions for the acylation reactions will be determined case-by-case based on known parameters and the desired result. For example, the larger the ratio of PEG: protein, the greater the percentage of polypegylated product. One may choose to prepare a mixture of linking polymer/polypeptide conjugate molecules by acylation and/or alkylation methods, and the advantage provided herein is that one may select the proportion of monopolymer/polypeptide conjugate to include in the mixture.
The latexes useful in this invention may be prepared by any method known in the art for preparing particles of 5-100 nm in mean diameter. Especially useful methods include emulsion and miniemulsion polymerization. Such techniques are reviewed in “Suspension, Emulsion, and Dispersion Polymerization: a Methodological Survey” Colloid. Polym. Sci. vol. 270, p. 717-732, 1992 and in Lovell, P. A.; El-Aaser, M. S. “Emulsion Polymerization and Emulsion Polymers”, Wiley: Chichester, 1997. An alternate method involves intramolecularly crosslinking individual polymer chains to form very small particles. This method is described in U.S. Pat. No. 6,890,703.
Dyes useful for this invention are fluorescent, hydrophobic dyes which fluoresce at 400-1000 nm. Classes of dyes include, but are not necessarily limited to oxonol, pyrylium, Squaric, croconic, rodizonic, polyazaindacenes or coumarins, scintillation dyes (usually oxazoles and oxadiazoles), aryl- and heteroaryl-substituted polyolefins (C2-C8 olefin portion), merocyanines, carbocyanines, phthalocyanines, oxazines, carbostyryl, porphyrin dyes, dipyrrometheneboron difluoride dyes aza-dipyrrometheneboron difluoride dyes and oxazine dyes. Commercially available fluorescent dyes are listed in Table 1 and generic structures are shown in Table 2. Preferred dyes are carbocyanine, phthalocyanine, or aza-dipyrrometheneboron difluoride.
The fluorescent dyes utilized with the latex particle are solvent soluble and demonstrate insolubility in water. When the dyes are loaded into the water soluble latex particle, a boost is observed in quantum yield of fluorescence as compared to the quantum yield of the dye in aqueous solvent.
The fluorescent dye can be loaded into the latex by a variety of known methods. For example, a solution of the dye in a water-miscible organic solvent can be mixed with the latex, and then the solvent can be removed by evaporation, dilution with water, or dialysis, as described in U.S. Pat. No. 6,706,460, U.S. Pat. No. 4,368,258, U.S. Pat. No. 4,199,363 and U.S. Pat. No. 6,964,844. A solution of the dye in a water-immiscible organic solvent can be combined with the aqueous latex and the mixture subjected to high shear mixing, as described in U.S. Pat. No. 5,594,047. Alternately, the dye can be incorporated during the preparation of the latex. Such a method is described in Journal of Polymer Science Part A: Polymer Chemistry, Vol. 33, p. 2961-2968, 1995 and in Colloid and Polymer Science, vol. 282, p. 119-126, 2003.
The loaded latex particle may be used as an imaging probe for use in animals, as well as other physiological systems. The particle may be used as a diagnostic contrast element or in other in vitro/in vivo, physiological imaging applications. Preferably, the particle is provided in an aqueous, biocompatible dispersion.
The described composition can further comprise a biological, pharmaceutical or diagnostic component that includes a targeting moiety that recognizes the specific target cell or other target biological molecules. As used herein “target cells” refers to healthy cells, disease cells, mammalian cell, or plant cells. “Target biological molecules” include, but not limited to, proteins, protein fragments, nucleic acids, or any essential metabolites.
A representative, but non-limiting, list of moieties that can be employed as targeting agents useful with the present compositions is selected from the group consisting of proteins, peptides, aptomers, small organic molecules, toxins, diptheria toxin, pseudomonas toxin, cholera toxin, ricin, concanavalin A, Rous sarcoma virus, Semliki forest virus, vesicular stomatitis virus, adenovirus, transferrin, low density lipoprotein, transcobalamin, yolk proteins, epidermal growth factor, growth hormone, thyroid stimulating hormone, nerve growth factor, calcitonin, glucagon, prolactin, luteinizing hormone, thyroid hormone, platelet derived growth factor, interferon, catecholamines, peptidomimetrics, glycolipids, glycoproteins and polysacchlorides. Homologs or fragments of the presented moieties can also be employed. These targeting moieties can be associated with a nanoparticulate and be used to direct the nanoparticle to bind a chosen target. There is no requirement that the entire moiety be used as a targeting moiety. Smaller fragments of these moieties known to interact with a specific receptor or other structure can also be used as a targeting moiety.
An antibody or an antibody fragment represents a class of most universally used targeting moiety that can be linked to a nanolatex. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988. Antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). A superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol, 6:511-519, 1976, and improvements thereto.
Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.
A number of “humanized” antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described (Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86:4220-4224. These “humanized” molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules that limits the duration and effectiveness of therapeutic applications of those moieties in human recipients.
The recognition moiety can further comprise a sequence of peptides or nucleic acids that can be recognized by a select target. The peptides and nucleic acids can be selected from a sequence known in the art for their ability to bind to a chosen target, or to be selected from combinatorial peptide or nucleic acid libraries for their ability to bind a chosen target.
Vitamins and other essential minerals and nutrients can be utilized as targeting moiety to enhance the binding of nanolatex particle to a target. In particular, a vitamin ligand can be selected from the group consisting of folate, folate receptor-binding analogs of folate, and other folate receptor-binding ligands, biotin, biotin receptor-binding analogs of biotin and other biotin receptor-binding ligands, riboflavin, riboflavin receptor-binding analogs of riboflavin and other riboflavin receptor-binding ligands, and thiamin, thiamin receptor-binding analogs of thiamin and other thiamin receptor-binding ligands.
Since not all natural cell membranes possess biologically active biotin or folate receptors, use of the described compositions in-vitro on a particular cell line can involve altering or otherwise modifying that cell line first to ensure the presence of biologically active biotin or folate receptors. Thus, the number of biotin or folate receptors on a cell membrane can be increased by growing a cell line on biotin or folate deficient substrates to promote biotin and folate receptor production, or by expression of an inserted foreign gene for the protein or apoprotein corresponding to the biotin or folate receptor.
The recognition moiety can further comprise a sequence that is subject to enzymatic or electrochemical cleavage. The recognition moiety can thus comprise a sequence that is susceptible to cleavage by enzymes present at various locations inside a cell, such as proteases or restriction endonucleases (e.g. DNAse or RNAse).
For cell targeting, a cell surface recognition sequence is not a must-have requirement. Thus, although a cell surface receptor targeting moiety can be useful for targeting a given cell type, or for inducing the association of a described nanoparticle with a cell surface, there is no requirement that a cell surface receptor targeting moiety be present on the surface of a nanolatex particle.
To assemble the biological, pharmaceutical or diagnostic components to a described nanoparticulate carrier, the components can be associated with the nanoparticle carrier through a linkage. By “associated with”, it is meant that the component is carried by the nanoparticle, for example the surface of the nanoparticle. The component can be dissolved and incorporated in the particle non-covalently. A preferred method of associating the component is by covalent bonding through the amine function on the surface.
Generally, any manner of forming a linkage between a targeting moiety of interest and a nanolatex particulate carrier can be utilized. This can include covalent, ionic, or hydrogen bonding of the ligand to the exogenous molecule, either directly or indirectly via a linking group. The linkage is typically formed by covalent bonding of the targeting moiety, biological, pharmaceutical or diagnostic component to the nanoparticle carrier through the formation of amide, ester or imino bonds between acid, aldehyde, hydroxy, amino, or hydrazo groups on the respective components of the complex. Art-recognized biologically labile covalent linkages such as imino bonds and so-called “active” esters having the linkage —COOCH, —O—O— or —COOCH are preferred. Hydrogen bonding, e.g., that occurring between complementary strands of nucleic acids, can also be used for linkage formation.
In a preferred embodiment of this invention, the targeting moiety is covalently attached to the reactive group at then end of the polyethylene glycol macromonomer. The covalent linkage used will be dependent on the reactive group at the end of the polyethylene glycol. For example, if the reactive group is an amine, it can react with an activated carboxylic acid derivative on the targeting moiety (such as an N-hydroxysuccinimidyl ester) to form an amide bond. If the reactive group is a vinylsulfone, it can react with a primary amine on the targeting moiety to afford a secondary amine linkage.
The following examples are provided to illustrate the invention.
To a mixture of 3H-Indolium salt (2 eqv.) and N-(5-(phenylamino)-2,4-pentadienylidene)-benzenamine monohydrochloride (the dianil)(1 eqv.) in acetonitrile was added acetic anhydride and triethylamine (1.5 eqv.). The mixture was heated at reflux for 5˜25 min. The resulting mixture was then cooled to room temperature and poured to either water or ether to obtain the crude product, which was further purified either by silica-gel chromatography, or by recrystallization or by reverse phase HPLC.
This dye was prepared following the general procedure described above, using 2,3,3-trimethyl-1-octadecyl-3H-Indolium perchlorate (4.28 g, 10 mmol) and the dianil (1.4 g, 5 mmol) in 40 mL of acetic anhydride containing triethylamine (1.5 g, 15 mmoles). The reaction time was 5 minutes. The reaction was cooled to 25 degrees and poured into 2 liters of ice water with vigorous stirring. The water was decanted and the oil was dissolved in 100 mL of 80/20 dichlomethane-methanol. The material was chromatographed on a silica gel column eluting with 80/20 dichlomethane-methanol. Evaporation of the solvent after drying with anhydrous magnesium sulfate afforded pure dye (4 g, 32% yield), λmax=747 nm in methanol, extinction coefficient=220,020.
This dye was prepared following the general procedure described above, using 2,3,3-trimethyl-1-butyl-3H-Indolium perchlorate (12 g, 38 mmoles) and the dianil (5.4 g, 19 moles) in 100 mL of acetic anhydride containing tributylamine (10.5 g, 57 mmoles). The reaction was carried out for 15 minutes, cooled to 25 degrees and poured into 2000 mL of ice water with vigorous stirring. The water was decanted from the oily product then chromatographed on silica gel eluting with 90/10 methylene chloride-methanol. Evaporation of the solvent after drying with anhydrous magnesium sulfate afforded pure dye (8 g, 71% yield). λmax=746 nm in methanol with extinction coefficient of 259,500.
This dye was prepared following the general procedure described above using 1,2,3,3-tetramethyl-3H-Indolium borontetrabromide (5.22 g, 20 mmol) and the dianil (2.84 g, 10 mmol) in 25 mL of isopropyl alcohol containing acetic anhydride (3 ml) and triethylamine (5.6 ml) for 2 hours. The reaction was cooled to 25° C. and poured into 1 liter of ice water with vigorous stirring. The crude product was collected by filtration and washed again with water. The crude product was purified by recrystallization from hot ethyl alcohol. 3.4 g pure product was obtained. The 1H NMR spectrum is consistent with the structure. □max=739 nm in methanol, extinction coefficient=294,000
This dye was prepared following the general procedure described above, using 2,3,3-trimethyl-1-(4-sulfobutyl)-3H-Indolium inner salt (2.3 g, 6.7 mmoles) and the dianil (0.95 g, 3.3 moles) in 20 mL of acetic anhydride. Triethylamine (2 g, 20 mmoles) was added with vigorous stirring and the reaction heated to reflux for 5 minutes. The reaction was cooled and diluted to 300 mL with diethyl ether and stirred for 10 minutes. The ether was decanted from the oil and 25 mL of absolute ethanol was added. The mixture was heated to reflux then 1.5 g (0.01 moles) of sodium iodide was added. Heating was continued for 3 minutes and the mixture was cooled to 25 degrees with stirring. The solid was filtered, washed with absolute ethanol and dried. Wt=2.4 g (94% yield, 85% purity). The product was purified by reverse phase HPLC to yield 1 g of desired dye (39% yield, 99% purity by HPLC. λmax=784 nm methanol, extinction coefficient=221,700.
Dye 2 was synthesized using the synthetic scheme described in the literature (e.g., Zhao, W. et al. Chem. Int. Ed. 2005, 44, 1677). □max=739 nm in methanol, extinction coefficient=129945.
Isostearyl alcohol (1.8 g, 6.6 mmol) was mixed with N,N-dimethylformamide (50 ml), treated with sodium hydride (0.31 g of 50% oil mixture, 6.6 mmol) and stirred at ambient conditions under nitrogen atmosphere for 2 hrs. Silicon phthalocyanine dichloride was added and the reaction was heated at reflux overnight. The reaction was portioned between water and ethyl acetate and the organic layer was washed 3 times with water. The organic layer was dried over magnesium sulfate, filtered and concentrated. The residue was chromatographed on silica to yield 0.8 g of product. The 1H NMR spectrum was consistent with the structure. □max in toluene (672 nm)
A mixture of 3H-Indolium, 2,3,3-trimethyl-1-octadecyl-, perchlorate salt (2.14 g, 5 mmol), N-((2-chloro-3-((phenylamino)methylene)-1-cyclohexen-1-yl)methylene)-benzenamine monohydrochloride (0.9 g, 2.5 mmol) and 1-methyl-2-pyrrolidinone (30 ml) was heated at 60° C. overnight. The mixture was cooled to room temperature and poured into water. The dye was collected by filtration and washed with water and dried in a vacuum oven to afford the crude product (1.8 g). The crude dye was used in the next step without further purification.
To a solution of phenol (130 mg, 1.4 mmol) in anhydrous THF (20 ml) was added sodium hydride (60 mg, 1.4 mmol, 60% in mineral oil) at room temperature. The mixture was stirred for 30 minutes, then the dye (0.8 g, 0.9 mmol) was added and the mixture was heated at 60° C. for overnight. The solvent was then removed and residue was purified chromatographically (silica-gel column; dichloromethane with 2% Methanol) to give the pure dye product (560 mg). □max=771 nm in acetone. Both the mass spectrum and H1NMR are consistent with the structure.
To a round bottom flask charged with 3H-Indolium, 2,3,3-trimethyl-1-octadecyl-, p-toluenesulfonate (PTS) salt (5.75 g, 12 mmoles), N-(3-(phenylamino)-2-propenylidene)-benzenamine, monohydrochloride (1.55 g, 6 mmoles), and acetonitrile (15 ml) was added acetic anhydride (1.3 ml) and triethylamine (3.4 ml). The mixture was heated to reflux and a second portion of triethylamine (2.0 ml) was added. The resulting mixture was refluxed for two hours. After the mixture was cooled to room temperature, water was added while stirring. The crude dye with PTS as a counter ion (6.2 g) was collected by filtration and air-dried.
The PTS counter ion of the dye was next exchanged to perchlorate. To a solution of the dye (6.1 g) in methanol (55 ml) was added a solution of sodium perchlorate (1.3 g) in methanol. The mixture was stirred at room temperature for 1 hour and the dye was precipitated out and collected by filtration. The dye was further purified by recrystallization from methanol. 3.2 g of dye 6 was obtained, □max=682 nm in methanol, extinction coefficient=2.33×105.
This dye was prepared following the procedure of Dye Synthesis Example 7 with an additional ion exchange step. The condensation step used 3H-Indolium, 2,3,3-trimethyl-1-butyl, p-toluenesulfonate salt (10.76 g, 20 mmoles), N-(3-(phenylamino)-2-propenylidene)-benzenamine, monohydrochloride (2.6 g, 10 mmoles), acetonitrile (30 ml), acetic anhydride (2.5 ml) and triethylamine (6.5 ml). 10.2 gram of crude dye was obtained, the dye was ion exchanged to the perchlorate as described in Dye Synthesis example 7 using the crude dye (10 g, 18.6 mmoles), MeOH (70 ml), and sodium perchlorate (2.6 g, 21.2 mmoles). 8.2 g of the perchlorate dye was obtained. The crude perchlorate dye (5 g, 9.3 mmoles) was stirred in methanol (300 ml), with amberlite IRA-400 (Cl) resins (40 g) for several hours. The resin was filtered off and this was repeated with a second portion of resin. The solvent was removed on a rotary evaporator and the dye was dried overnight in a vacuum oven at 60° C. The final dye 7 was obtained (4.7 g) with □max=641 nm in methanol, extinction coefficient=2.56×105.
Dye 11 was prepared according to the procedures described by Lou Kai-yan, Qian Xu-huong, Song Gong-hua, Journal of East China University of Science and Technology, 28, (2), 212-5, 2002. □max=751 nm in methanol. extinction coefficient=2.31×105.
Polyethyleneglycol dimethacrylate (Aldrich, Mn=875, 335 g) was mixed with 100 ml of methanol and treated with cysteamine (Aldrich, 5.8 g) and diisopropylethylamine (Hunigs base) and was stirred at RT for 2 days and concentrated using a rotary evaporator. The residue was taken up in 1 L of ethyl acetate and extracted with aqueous 10% HCl. The aqueous layer was collected and made basic by the addition of 50% aqueous sodium hydroxide followed by extraction with ethyl acetate. The organic layer was dried over MgSO4, filtered and concentrated. The residue was taken up in anhydrous diethyl ether and treated with gaseous HCl and allowed to stand. The ether was decanted to leave a dark blue oil. This material was washed with fresh diethyl ether, which was decanted. The dark blue oil was concentrated using a rotary evaporator to give 37 g of the desired product as the hydrochloride salt.
1H-NMR (300 MHZ, CDCl3): D 1.18 (d, 3H), 1.93 (bs, 3H), 2.04 (bs, 2H), 2.43-2.77 (bm, 7H), 3.6-3.7 (vbs, —CH2CH2O—), 3.73 (bt, 2H), 3.29 (bt, 2H), 5.56 (bs, 1H), 6.12 (bs, 1H).
A 500 ml 3-neck round bottomed flask was modified with Ace #15 glass threads at the bottom and a series of adapters allowing connection of 1/16 inch ID Teflon tubing. The flask (hereafter referred to as the “header” flask) was outfitted with a mechanical stirrer, rubber septum with syringe needle nitrogen inlet. The header flask was charged with methoxyethyl methacrylate (5.63 g), divinylbenzene (0.63 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (6.25 g, Mn=1100), cetylpyridinium chloride (0.31 g), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.06 g), sodium bicarbonate (0.06 g) and distilled water (78.38 g). A IL 3-neck round bottomed flask outfitted with a mechanical stirrer, reflux condenser, nitrogen inlet, and rubber septum (hereafter referred to as the “reactor”) was charged with 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.06 g), sodium bicarbonate (0.06 g), and distilled water (159.13 g). Both the header and reactor contents were stirred until homogeneous and were bubble degassed with nitrogen for 20 minutes. The reactor flask was placed in a thermostatted water bath at 60° C. and the header contents were added to the reactor over two hours using a model QG6 lab pump (Fluid Metering Inc. Syossett, N.Y.). The reaction mixture was then allowed to stir at 60° C. for 16 hours. The reaction mixture was then dialyzed for 48 hours using a 3.5K cutoff membrane in a bath with continual water replenishment. 286.0 g of a colorless dispersion of 2.64% solids was obtained. The volume average diameter was found to be 10.8 nm with a coefficient of variation of 0.25 by quasi-elastic light scattering (QELS). QELS was performed using a Nanotrac Ultrafine Particle Analyzer (Microtrac Inc. Montgomeryville, Pa.) at 3-5% solids.
This nanolatex was prepared using the same method as described in Example 2. The header contained methoxyyethyl methacrylate (22.50 g), divinylbenzene (2.50 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (22.50 g, Mn=1100), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.25 g), sodium bicarbonate (0.25 g), and distilled water (313.50 g). The reactor contents were composed of distilled water (636.50 g), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.25 g), and sodium bicarbonate (0.25 g). 771 g of a clear dispersion of 5.59% solids was obtained. The volume average diameter was found to be 10.4 nm with a coefficient of variation of 0.27 by quasi-elastic light scattering. 200 g of this latex was dialyzed for 48 hours using a 3.5K cutoff membrane. 50 g of the dialyzed latex was then stirred over 15 cc Dowex 50Wx4 ion exchange resin (converted to the sodium form and washed 3× with distilled water) to afford an ion exchanged dispersion of 3.85% solids.
This nanolatex was prepared using the same method as described in Example 2. The header contained methoxyyethyl methacrylate (45.00 g), divinylbenzene (5.00 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (50.00 g, Mn=1100), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.50 g), sodium bicarbonate (0.50 g), and distilled water (627.00 g). The reactor contents were composed of distilled water (1273.00 g), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.50 g), and sodium bicarbonate (0.50 g). The latex was subjected to ultrafiltration using an Amicon LP1 diafiltrations system (Millipore Inc) with a 30K cutoff spiral wound cartridge. After 8 turnovers against distilled water, the latex was treated with 600 cc Dowex 50Wx4 ion exchange resin (converted to the sodium form and washed 3× with distilled water) to afford an ion exchanged dispersion of 5.76% solids. The volume average diameter was found to be 12.0 nm with a coefficient of variation of 0.28 by quasi-elastic light scattering.
This nanolatex was prepared using the same method as described in Example 2. The header contained methoxyyethyl methacrylate (22.50 g), divinylbenzene (2.50 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (22.50 g, Mn=1100), sodium persulfate (0.50 g), sodium bicarbonate (0.25 g), and distilled water (313.50 g). The reactor contents were composed of distilled water (636.50 g), sodium metabisulfite (0.36 g), and sodium bicarbonate (0.25 g). 759 g of a clear dispersion of 4.94% solids was obtained. The volume average diameter was found to be 22.2 nm with a coefficient of variation of 0.25 by quasi-elastic light scattering. 200 g of this latex was dialyzed for 48 hours using a 3.5K cutoff membrane to afford 292 g of a dispersion of 3.24% solids.
This nanolatex was prepared using the same method as described in Example 2. The header contained methoxyyethyl methacrylate (112.5 g), divinylbenzene (1.25 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (11.25 g, Mn=1100), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.13 g), cetylpyridinium chloride(0.63), and distilled water (156.75 g). The reactor contents were composed of distilled water (318.25 g), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.13 g), and cetylpyridinium chloride (1.88 g). The latex was twice stirred for 1 hour with 200 cc Dowex 88 ion exchange resin and dialyzed for 48 hours using a 3.5K cutoff membrane to afford clear latex of 4.39% solids. The volume average diameter was found to be 10.98 nm with a coefficient of variation of 0.29 by quasi-elastic light scattering.
This nanolatex was prepared using the same method as described in Example 2. The header contained methoxyethyl methacrylate (5.63 g), divinylbenzene (0.63 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (6.25 g, Mn=1100), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.06 g), cetylpyridinium chloride (0.31), sodium bicarbonate (0.06 g) and distilled water (78.38 g). The reactor contents were composed of distilled water (159.13 g), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.06 g), sodium bicarbonate (0.06 g) and cetylpyridinium chloride (0.94 g). The latex was treated twice with 100 cc Dowex 88 ion exchange resin and dialyzed for 48 hours using a 14K cutoff membrane to afford to afford 312 g of a clear latex of 3.26% solids. The volume average diameter was found to be 20.89 nm with a coefficient of variation of 0.24 by quasi-elastic light scattering.
This nanolatex was prepared using the same method as described in Example 2. The header contained styrene (8.75 g), divinylbenzene (0.63 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (3.13 g, Mn=1100), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.06 g), cetylpyridinium chloride (0.31), and distilled water (78.38 g). The reactor contents were composed of distilled water (159.13 g), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.63 g), and cetylpyridinium chloride (0.94 g). The latex was dialyzed for 48 hours using a 3.5K cutoff membrane to afford 251 g of a clear latex of 3.38% solids. The volume average diameter was found to be 12.82 nm with a coefficient of variation of 0.36 by quasi-elastic light scattering.
This nanolatex was prepared using the same method as described in Example 2. The header contained styrene (11.25 g), divinylbenzene (1.25 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (12.50 g, Mn=1100), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.13 g), cetylpyridinium chloride (0.31), sodium bicarbonate (0.13 g) and distilled water (156.75 g). The reactor contents were composed of distilled water (318.25 g), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.13 g), sodium bicarbonate (0.13 g) and cetylpyridinium chloride (0.94 g). The latex was dialyzed for 48 hours using a 3.5K cutoff membrane and treated with 250 cc Dowex 88 ion exchange resin to afford to afford 561.23 g of a clear latex of 4.08% solids. The volume average diameter was found to be 13.22 nm with a coefficient of variation of 0.19 by quasi-elastic light scattering.
This nanolatex was prepared using the same method as described in Example 2. The header contained methyl methacrylate (11.25 g), divinylbenzene (1.25 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (12.50 g, Mn=1100), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.13 g), cetylpyridinium chloride (0.31), sodium bicarbonate (0.13 g) and distilled water (156.75 g). The reactor contents were composed of distilled water (318.25 g), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.13 g), sodium bicarbonate (0.13 g) and cetylpyridinium chloride (0.94 g). The latex was dialyzed for 48 hours using a 3.5K cutoff membrane and treated with 250 cc Dowex 88 ion exchange resin to afford to afford 610.25 g of a clear latex of 3.74% solids. The volume average diameter was found to be 13.30 nm with a coefficient of variation of 0.18 by quasi-elastic light scattering.
This nanolatex was prepared using the same method as described in Example 2. The header contained butyl acrylate (5.63 g), divinylbenzene (0.63 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (6.25 g, Mn=1100), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.06 g), cetylpyridinium chloride (0.31), sodium bicarbonate (0.06 g) and distilled water (78.38 g). The reactor contents were composed of distilled water (159.13 g), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.06 g), sodium bicarbonate (0.06 g) and cetylpyridinium chloride (0.94 g). The latex was dialyzed for 48 hours using a 14K cutoff membrane to afford to afford 263 g of a clear latex of 4.32% solids. The volume average diameter was found to be 24.56 nm with a coefficient of variation of 0.29 by quasi-elastic light scattering.
Under dim lighting, a dye stock solution of 0.2784% w/w was prepared by dissolving 0.0280 g of Dye 1 in sufficient tetrahydrofuran to afford a final solution weight of 24.5244 g. A 1.4365 g portion of the dye solution was added to a glass vial and was diluted to a final weight of 10.0 g with tetrahydrofuran. 9.9960 g of Nanolatex 1 was added to the vial and the solution was stripped to approximately 40-50% volume on a rotary evaporator. Residual tetrahydrofuran was further removed by twice adding 3-5 ml distilled water and again stripping ˜1/4 to 1/3 of the volatiles. 9.4069 g of a loaded latex (LL-1A) of 4.27% solids containing 3.94×10−3 mol dye per gram of solid latex. Three additional samples with higher loading levels (LL-1B, 1C, 1D) were prepared in an identical manner using the reagent quantities in the table below.
Under dim lighting, a dye stock solution of 0.0919% w/w was prepared by dissolving 0.0138 g of Dye 2 in sufficient tetrahydrofuran to afford a final solution weight of 15.0239 g. Loaded latexes LL-2A and 2B were prepared in brown glass vials from Nanolatex 1 using the procedure as described in Example 12 and the reagent quantities in the table below. In order to convert the latex serums to phosphate buffered saline, portions of a salt mixture (137 parts NaCl, 2.7 parts KCl, 10 parts Na2HPO4, 2 parts KH2PO4) were added to each sample in the quantities listed below.
Loaded latexes LL-3A and 3B were prepared from Nanolatex 4 using the procedure described in Example 13 and the reagent quantities in the table below. A dye stock solution of 0.0903% w/w was prepared by dissolving 0.0296 g of Dye 3 in sufficient tetrahydrofuran to afford a final solution weight of 29.8012 g.
Loaded latexes LL-4A, 4B, and 4C were prepared from Nanolatex 2 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0402% w/w) was prepared by dissolving 0.0101 g of Dye 1 in sufficient tetrahydrofuran to afford a final solution weight of 25.1201 g.
Loaded latex LL-5A was prepared from Nanolatex 3 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0358% w/w) was prepared by dissolving 0.0087 g of Dye 6 in sufficient tetrahydrofuran to afford a final solution weight of 24.3270 g.
Loaded latex LL-6A was prepared from Nanolatex 3 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0385% w/w) was prepared by dissolving 0.0096 g of Dye 7 in sufficient tetrahydrofuran to afford a final solution weight of 24.9403 g.
Loaded latexes LL-7A, 7B, and 7C were prepared from Nanolatex 6 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0693% w/w) was prepared by dissolving 0.0197 g of Dye 1 in sufficient tetrahydrofuran to afford a final solution weight of 28.4269 g.
Loaded latexes LL-8A, and 8B were prepared from Nanolatex 6 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0385% w/w) was prepared by dissolving 0.0096 g of Dye 9 in sufficient tetrahydrofuran to afford a final solution weight of 24.9403 g.
Loaded latexes LL-9A, and 9B were prepared from Nanolatex 6 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0912% w/w) was prepared by dissolving 0.0200 g of Dye 4 in sufficient tetrahydrofuran to afford a final solution weight of 21.9372 g.
Loaded latexes LL-11A was prepared from Nanolatex 3 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0250% w/w) was prepared by dissolving 0.0075 g of Dye 5 in sufficient tetrahydrofuran to afford a final solution weight of 30.0000 g.
Loaded latexes LL-12A was prepared from Nanolatex 3 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0693% w/w) was prepared by dissolving 0.0197 g of Dye 1 in sufficient tetrahydrofuran to afford a final solution weight of 28.4269 g.
To 1 mL of dye loaded nanolatex sample LL-7A, 5 mL of PBS buffer was added followed by the addition of 5 mg of NHS-PEG-Biotin (MW5000, Nektar). The reaction, mixture was stirred and protected from light for 3 hours. At the end of the reaction, the solution was filtered through YM-30 (Millipore) filters and washed 4 times with PBS buffer. A control sample without dye loaded nanolatex was included.
To determine the amount of biotin attachment to the surface of dye loaded nanolatex, a HABA/Avidin replacement binding assay was performed as follows: in a 1 cm black walled cuvette, a HABA/Avidin assay premix (Pierce Biotechnology) was dissolved in 800 μL of PBS buffer. The absorbance at 500 nm was recorded. Then, 100 μL of biotin attached dye loaded nanolatex sample was added to the cuvette, and after waiting for 15 min, the absorbance at 500 nm was re-measured absorbance at 500 nm. The differential absorbance after the volume adjustment corrections was used to determine the amount of biotin attachment. It was found that ca. 80 μg of biotin was attached for every mg of dye loaded nanolatex.
Comparative loaded latex CLL-1A was prepared from Nanolatex 7 using the procedure as described in Example 12 and the reagent quantities in the table below. The dye stock solution (0.2784% w/w) was prepared by dissolving 0.0795 g of Dye 1 in sufficient tetrahydrofuran to afford a final solution weight of 28.5549 g.
Comparative Loaded latex CLL-2A, was prepared from Nanolatex 8 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0350% w/w) was prepared by dissolving 0.0100 g of Dye 1 in sufficient tetrahydrofuran to afford a final solution weight of 28.5743 g.
Comparative Loaded latex CLL-3A, was prepared from Nanolatex 9 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0350% w/w) was prepared by dissolving 0.0100 g of Dye 1 in sufficient tetrahydrofuran to afford a final solution weight of 28.5743 g.
Comparative Loaded latex CLL-4A, was prepared from Nanolatex 10 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0350% w/w) was prepared by dissolving 0.0100 g of Dye 1 in sufficient tetrahydrofuran to afford a final solution weight of 28.5743 g.
Comparative Loaded latex CLL-5A was prepared from Nanolatex 3 using the procedure described in Example 13 and the reagent quantities in the table below. The dye stock solution (0.0307% w/w) was prepared by dissolving 0.0075 g of Dye 8 in sufficient tetrahydrofuran to afford a final solution weight of 24.4668 g.
Fluorescence RQY determinations were performed as detailed in the HORIBA Jobin Yvon Application Note, “A Guide to Recording Fluorescence Quantum Yields” (available from Horiba Jobin Yvon, Middlesex, UK).
Aqueous dyed nanolatex dispersions were diluted volumetrically with phosphate-buffered saline (PBS, pH 7.4) to produce a dye concentration series in the range 10−7 to 10−8 moles/L, such that their absorbance values at peak maximum did not exceed 0.1 in a 1 cm path length cell. A concentration series of the standard (reference) dye was prepared by first dissolving the solid dye in spectroscopic-grade methanol at room temperature and then diluting the methanolic dye solution with PBS buffer to produce a dye concentration series in the range 10−7 to 10−8 moles/L. The final dye solutions typically contained less than 1% methanol.
All dilutions were performed under low-light conditions and the samples were stored in amber Nalgene bottles to minimize photodecomposition. All samples were measured within hours of dilution.
Absorbance measurements were made using a Perkin Elmer Lambda 900 UV/VIS/NIR spectrometer with dye solutions contained in 5 cm path length stoppered cuvettes. The absorbance intensity of the dye was measured at the specific excitation wavelength used for the fluorescence measurement from the solvent-subtracted and baseline-zeroed absorbance spectrum. These measured absorbance values were then converted to 1 cm path length cuvette-equivalent values for the RQY determinations.
The fluorescence spectrum of the dye solution contained in a 1 cm path length cuvette was recorded in right-angle detection mode using a SPEX fluorolog 1680 0.22 m double spectrometer. The instrumental parameter settings and the excitation wavelength used for the inventive and reference dyes were co-optimized and were identical for each experiment. The baseline-resolved fluorescence spectrum of each dye sample was corrected for solvent contributions and instrumental response characteristics as a function of emission wavelength and the integrated fluorescence intensity was measured.
The integrated fluorescence intensity for each dye was plotted as a function of absorbance (1 cm path length equivalent) measured at the excitation wavelength of interest. The resultant F/A plots were linear with zero intercepts and regression coefficients better than 0.97. The slope of the F/A plot is proportional to the dye's fluorescence quantum yield. The slope value for the inventive dye was then normalized to the slope value for the reference dye to yield a “normalized RQY” metric for the inventive dye. The data are presented in Table 4.
It is evident from the data tabulated above that the fluorescence quantum yield of latex nanoparticles loaded with a hydrophobic near-infrared dye and dispersed in aqueous PBS buffer is significantly improved compared to a hydrophilic dye structural analogue directly dissolved in aqueous PBS buffer (Example 17, LL-6A versus dye 12, Example 18, LL-7A versus dye 11 and Example 22, LL-11A versus dye 11).
It is also evident from the data tabulated above that the fluorescence quantum yield of latex nanoparticles loaded with a near-infrared hydrophobic dye and dispersed in aqueous PBS buffer is significantly improved compared to the exact same hydrophobic dye dissolved in an organic solvent, such as methanol (Example 16, LL-5A versus dye 6 and Example 17, LL-6A versus dye 7).
It is also evident from the data tabulated above that the fluorescence quantum yields of latex nanoparticles loaded with a variety of structurally disparate near-infrared hydrophobic cyanine, aza-bodipy and phthalocyanine dyes and dispersed in aqueous PBS buffer are significantly improved compared to the hydrophilic near-infrared fluorophore, Indocyanine Green (Example 13, LL-2A versus dye 10, Example 14, LL-3A versus dye 10, Example 15, LL-4A versus dye 10, Example 19, LL-8A versus dye 10).
It is also evident from the data tabulated above that the fluorescence quantum yield of latex nanoparticles loaded with a near-infrared hydrophobic cyanine dye can be improved by increasing the size of the bis N-alkyl hydrophobic moieties (Comparative example 5, CLL-5A loaded with dye 8 versus LL-11A loaded with dye 1).
It is also evident from the data tabulated above that fluorescence quantum yield of latex nanoparticles loaded with a near-infrared hydrophobic dye is significantly improved for lattices derived from methoxyethyl methacrylate monomer as opposed to styrene (comparative examples 1 and 2), methyl methacrylate (comparative example 3) or butyl acrylate (comparative example 4) monomer.
In the example 33 the R2 of General formula 2 is varied for polymers prepared from methoxyethyl methacrylate (R2=methyl group for nanolatex X, and X1), ethoxyethylmethacrylate (R2=ethyl group for nanolatex X2), butoxyethylmethacrylate (R2=butyl group for nanolatex X3) and phenoxyethylmethacrylate (R2=phenyl group for nanolatex X4). The table shows structure variation produces nanoparticles similar in size and polydispersibility. The table shows that fluorescent quantum yield is similar for the different R2 groups
The nanolatex X for this study was comprised of alkoxyethyl methacrylate (45% w/w), divinylbenzene (4%), ethylstyrene (1%), and amine-terminated polyethylene glycol macromonomer of Example 1 (amino—PEGMA)(50%)
This nanolatex X1-X4 was prepared using the same method as described in Example 2. The header contained alkoxyethyl methacrylate (5.63 g), divinylbenzene (0.63 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), poly(ethylene glycol) monomethyl ether methacrylate (PEGMA)(6.25 g, Mn=1100), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.06 g), cetylpyridinium chloride (0.31), sodium bicarbonate (0.06 g) and distilled water (78.38 g). The reactor contents were composed of distilled water (159.13 g), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride (0.06 g), sodium bicarbonate (0.06 g) and cetylpyridinium chloride (0.94 g). The latex was treated twice with 100 cc Dowex 88 ion exchange resin and dialyzed for 48 hours using a 14K cutoff membrane and was treated with ˜50 cc Dowex 50Wx4 ion exchange resin (converted to the sodium form and washed 3× with distilled water) to afford to afford clear latexes.
Under dim lighting, a dye stock solution of 0.0440% w/w was prepared by dissolving 0.0132 g of Dye 1 in sufficient tetrahydrofuran to afford a final solution weight of 30.00 g. The loaded latexes were prepared in brown glass vials from Nanolatexes X-X4 using the procedure as described in Example 12 and the reagent quantities in the table below. In order to convert the latex serums to phosphate buffered saline, portions of a salt mixture (137 parts NaCl, 2.7 parts KCl, 10 parts Na2HPO4, 2 parts KH2PO4) were added to each sample in the quantities listed below.
Equal volumes of the dye 1-loaded nanolatex dispersion LL-1A in PBS buffer and dye 11 solution in PBS buffer (both prepared at 0.45 micromoles/liter dye) were placed in translucent Nalgene HDPE bottles. The samples were irradiated continuously over a period of approximately 30 hours at a light intensity of approximately 2 kLux using a bench top fluorescent light (manufactured by the Dazor Manufacturing Corporation and fitted with two Sylvania 15-Watt fluorescent “Cool White” 4100K, F15T8/CW bulbs). Decreases in dye spectral absorbance at λ-max were measured as a function of irradiation time using a Perkin Elmer Lambda 900 UV/VIS/NIR spectrometer for samples contained in 5 cm path length stoppered cuvettes.
The first-order rate constant for dye photobleaching (loss of absorbance) was 2.8 times slower for the dye-loaded latex dispersion LL-1A in PBS buffer relative to the comparative dye 11 solution in PBS buffer.
Samples of the dye 1-loaded nanolatex dispersion LL-1A in PBS buffer and dye 11 solution in PBS buffer (both prepared at equal “time zero” absorbance values of 0.37 in 5 cm path length cuvettes) were stored in amber Nalgene bottles at room temperature. Periodically, aliquots were removed and the dye's optical density at λ-max was measured using a Perkin Elmer Lambda 900 UV/VIS/NIR spectrometer.
Decreases in dye spectral absorbance at λ-max were measured as a function of “dark storage” time. The first-order rate constant for dye decomposition (loss of absorbance) was 1.7 times slower for the dye-loaded latex dispersion LL-1A in PBS buffer relative to the comparative dye 11 solution in PBS buffer.
sulfo-SMCC
1) Take 2.2 ml of latex solution LL1A, and add 1.1 ml of PBS buffer (7.4).
2) Dissolve 7.3 mg of sulfo-SMCC (Pierce Biotechnology) in 73 μl of DMSO and add into above latex solution.
3) Let the reaction go under vortex or stirring for 1 hrs.
4) Pour the reaction solution into a YM-30centriprep tube (Millipore) and add large quantity of PBS buffer (around 15 ml), then centrifuge to remove excess linker. After centrifugation, latex particle solution should be around 700 μl. Usually two-three cycles of centrifugation is needed (Starting from 15 ml, and ending by 700%1)
5) Weigh out 12 mg of DTT (Dithiothreitol), dissolve in 100 uL of (sodium phosphate buffer saline (PBS) with 1 mM EDTA (ethylenediaminetretraacetic acid), pH 7.4.
6) Dissolve 1 mg of Secondary Antibody (Goat anti-rabbit) in 500 μl (concentration of 2 mg/mL) in PBS buffer with 1 mM EDTA, pH 7.4.
7) Add 5 uL of DTT solution to the Antibody solution, let react with agitation for one hour.
8) Filter excess DTT from sample by filtering through YM-30 filter 3× with PBS buffer with 1 mM EDTA, pH 7.4. In the final wash, bring volume of sample to less than 1 mL.
9) Combine the linker-functionalized loaded latex solution with the DTT-modified Antibody solution and stir at room temperature overnight at 4 C
A small piece of nitrocellulose was spotted with target rabbit protein and allowed to stand for 15 minutes. The prepared slide was treated with 5% blotto in PBS solution for 30 minutes and washed with fresh PBS solution.
Loaded latex-conjugated antibody (100 ul of solution from Step 9) was diluted to 1 0 ml in PBS buffer (pH7.4) with 5% blotto milk and this solution was rocked with the test nitrocellulose for 30 minutes and then washed with fresh PBS buffer. The nitrocellulose strip was placed in a Kodak Image Station MM4000 and exposed for 1 minute to record fluorescence (720 nm excitation 40 nm bandpass filter/790 nm emission 40 nm bandpass filter). As shown in
While the invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to adapt to particular situations without departing from the scope of the invention. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope and spirit of the appended claims.
This is a 111A application of Provisional Application Ser. No. 60/790,643, filed Apr. 10, 2006. Reference is made to commonly assigned, co-pending U.S. patent Applications: Ser. No. 11/401,343 by Leon et al. filed on Apr. 10, 2006 entitled “NANOGEL-BASED CONTRAST AGENTS FOR OPTICAL MOLECULAR IMAGING”; and Ser. No. 11/400,935 by Harder et al. filed on Apr. 10, 2006 entitled “FUNCTIONALIZED POLY(ETHYLENE GLYCOL)”, the disclosures of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60790643 | Apr 2006 | US |
