WO2002000265A1 - Intramolecularly-quenched near infrared fluorescent probes - Google Patents
Intramolecularly-quenched near infrared fluorescent probes Download PDFInfo
- Publication number
- WO2002000265A1 WO2002000265A1 PCT/US2001/019941 US0119941W WO0200265A1 WO 2002000265 A1 WO2002000265 A1 WO 2002000265A1 US 0119941 W US0119941 W US 0119941W WO 0200265 A1 WO0200265 A1 WO 0200265A1
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- WO
- WIPO (PCT)
- Prior art keywords
- probe
- fluorescence
- fluorochromes
- fluorochrome
- near infrared
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
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- A—HUMAN NECESSITIES
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0058—Antibodies
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0062—Arrangements for scanning
- A61B5/0068—Confocal scanning
Definitions
- the invention relates to biochemistry, cell biology, and in vivo optical imaging.
- Advantages of near infrared imaging over other currently used clinical imaging techniques include the following: potential for simultaneous use of multiple, distinguishable probes (important in molecular imaging) ; high temporal resolution (important in functional imaging) ; high spatial resolution (important in in vivo microscopy) ; and safety (no ionizing radiation) .
- filtered light or a laser with a defined bandwidth is used as a source of excitation light.
- the excitation light travels through body tissues. When it encounters a near infrared fluorescent molecule ("contrast agent”) , the excitation light is absorbed. The fluorescent molecule then emits light (fluorescence) spectrally distinguishable (slightly longer wavelength) from the excitation light.
- contrast agent a near infrared fluorescent molecule
- fluorescent molecule then emits light (fluorescence) spectrally distinguishable (slightly longer wavelength) from the excitation light.
- conventional near infrared fluorescence probes are subject to many of the same limitations encountered with other contrast agents, including low target/background ratios.
- the invention features an intramolecularly-quenched fluorescence probe comprising a polymeric backbone and a plurality of near infrared fluorochro es covalently linked to the backbone at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites.
- the backbone can be any biocompatible polymer.
- it can be a polypeptide, a polysaccharide, a nucleic acid, or a synthetic polymer.
- Polypeptides useful as a backbone include, for example, polylysine, albumins, and antibodies.
- Poly-L-lysine is a preferred polypeptide backbone.
- the backbone also can be a synthetic polymer such as polyglycolic acid, polylactic acid, poly(glycolic- colactic) acid, polydioxanone, polyvalerolactone, poly- -caprolactone, poly(3-hydroxybutyrate, poly (3-hydroxyvalerate) polytartronic acid, and poly( ⁇ - malonic acid) .
- the probe can include one or more protective chains covalently linked to the backbone.
- Suitable protective chains include polyethylene glycol, methoxypolyethylene glycol, methoxypolypropylene glycol, copolymers of polyethylene glycol and methoxypolypropylene glycol, dextran, and polylactic-polyglycolic acid.
- the backbone is polylysine and the protective chains are methoxypolyethylene glycol.
- Fluorescence activation sites can be located in the backbone, e.g., when the fluorochromes linked directly to ⁇ - amino groups of polylysine.
- each fluorochrome can be linked to the backbone by a spacer containing a fluorescence activation site.
- the spacers can be oligopeptides. Oligopeptide sequences useful as spacers include: Arg-Arg; Arg-Arg-Gly; Gly-Pro-Ile-Cys-Phe-Phe-Arg- Leu-Gly (SEQ ID NO:l); and His-Ser-Ser-Lys-Leu-Gln-Gly (SEQ ID NO: 2) .
- Near infrared fluorochromes useful in this invention include Cy5.5, Cy5, Cy7, IRD41, IRD700, NIR-1, LaJolla Blue, indocyanine green (ICG) and analogs thereof, indotricarbocyanine (ITC) , and chelated lanthanide compounds that display near infrared fluorescence.
- the fluorochromes can be covalently linked to the backbone, or spacers, using any suitable reactive group on the fluorochrome and a compatible functional group on the backbone or spacer.
- a probe according to this invention also can include a targeting moiety such as an antibody, antigen-binding antibody fragment, a receptor-binding polypeptide, or a receptor-binding polysaccharide.
- the invention also features an in vivo optical imaging method.
- the method includes: (a) administering to a living animal or human an intramolecularly-quenched fluorescence probe that accumulates preferentially in a target tissue, and comprises a fluorochrome attachment moiety and a plurality of near infrared fluorochromes covalently linked to the fluorochrome attachment moiety at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites; (b) allowing time for (1) the probe to accumulate preferentially in the target tissue, and (2) enzymes in the target tissue to activate the probe by enzymatic cleavage at fluorescence activation sites, if the target tissue is present; (c) illuminating the target tissue with near infrared light of a wavelength absorbable by the fluorochromes; and (d) detecting fluorescence emitted by the fluorochromes .
- the fluorochrome attachment moiety is a poly
- the invention also features an in vivo optical imaging method comprising: (a) administering to a living animal or human an intramolecularly-quenched fluorescence probe comprising a fluorochrome attachment moiety and a plurality of near infrared fluorochromes covalently linked to the fluorochrome attachment moiety at fluorescence- quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites, which enzymatic cleavage occurs preferentially in a target tissue; (b) allowing time for enzymes in the target tissue to activate the probe by enzymatic cleavage at fluorescence activation sites, if the target tissue is present; (c) illuminating the target tissue with near infrared light of a wavelength absorbable by the fluorochromes; and (d) detecting fluorescence emitted by the fluorochromes.
- the fluorochrome attachment moiety is a polymeric backbone. Alternatively, it can be a
- the above methods can be used, e.g., for in vivo imaging of a tumor in a human patient, or in vivo detection or evaluation of arthritis in a joint of a human patient.
- the invention also features an in vivo method for selectively imaging two different cell or tissue types simultaneously.
- the method includes administering to an animal or human patient two different intramolecularly- quenched fluorescence probes, each of which accumulates preferentially in a target tissue.
- Each of the two probes includes a fluorochrome attachment moiety and a plurality of near infrared fluorochromes covalently linked to the fluorochrome attachment moiety at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites.
- Each of the two probes comprises a fluorochrome whose fluorescence wavelength is distinguishable from that of the other fluorochrome, and each of the two probes contains a different activation site.
- backbone means a biocompatible polymer to which near infrared fluorochromes are covalently linked in fluorescence-quenching interaction-permissive positions.
- fluorescence activation site means a covalent bond within a probe, which bond is: (1) cleavable by an enzyme present in a target tissue, and (2) located so that its cleavage liberates a fluorochrome from being held in a fluorescence-quenching interaction-permissive position.
- fluorescence-quenching interaction- permissive positions means the positions of two or more atoms (in a single polymer) to which fluorochromes can be covalently linked (directly or indirectly through a spacer) so that the fluorochromes are maintained in a position relative to each other that permits them to interact photochemically and quench each other's fluorescence.
- fluorochrome attachment moiety means a molecule to which two or more fluorochromes are covalently linked (directly or through a spacer) and maintained in fluorescence-quenching interaction-permissive positions relative to one another.
- protective chain means a biocompatible polymeric moiety covalently linked to the backbone of a probe to inhibit undesired biodegradation, clearance, or immunogenicity of the backbone.
- targeting moiety means a moiety bound covalently or noncovalently to a self-quenched probe, which moiety enhances the concentration of the probe in a target tissue relative to surrounding tissue.
- Figs. 1A and IB are schematic diagrams indicating the chemical components, and their structural arrangement, in probes representing two embodiments of the invention.
- Figs. 2A and 2B are the chemical structures of six near infrared fluorochromes.
- Fig. 2A includes the structures of Cy5.5, Cy5, IRD41, and IRD700.
- Fig. 2B includes the structures of NIR-1 and LaJolla Blue.
- Figs. 3A and 3B are spectrophotometer scans of the near infrared fluorochrome, Cy5.5, before (Fig. 3A) and after (Fig. 3B) covalent linkage to PL-MPEG.
- Fig. 4 is a bar graph summarizing data on intramolecular quenching and probe activation. The data were obtained using Cy-PL-MPEG probes with different levels of fluorochrome loading.
- Fig. 5A is a schematic diagram illustrating the location of the murine flank tumor shown in Figs. 5B and 5C.
- Fig. 5B is a visible light photograph of the skin covering a human flank tumor in a 9L-bearing nude mouse.
- Fig. 5C is a near infrared fluorescence image of the tumor in Fig. 5B.
- Fig. 6 is a schematic diagram illustrating the use of an endoscope in the invention. Detailed Description Probe Design and Synthesis
- Probe architecture i.e., the particular arrangement of probe components, can vary as long as the probe retains a polymeric backbone and a plurality of near infrared fluorochromes covalently linked to the backbone at fluorescence-quenching interaction-permissive positions separable by enzymatic cleavage at fluorescence activation sites.
- the activation sites points of enzymatic cleavage
- a pair of fluorochromes in fluorescence-quenching interaction-permissive positions can be in a single side chain.
- a fluorescence activation site is placed in the side chain between the pair of fluorochromes.
- the probe comprises a polypeptide backbone containing only a small number of amino acids, e.g., 5 to 20 amino acids, with fluorochromes attached to amino acids on opposite sides of a protease cleavage (activation) site.
- a polypeptide backbone containing only a small number of amino acids, e.g., 5 to 20 amino acids, with fluorochromes attached to amino acids on opposite sides of a protease cleavage (activation) site.
- Guidance concerning various probe components, including backbone, protective side chains, fluorochromes, fluorochrome attachment moieties, spacers, activation sites and targeting moieties is provided in the paragraphs below.
- Probe backbone design will depend on considerations such as biocompatibility (e.g., toxicity and immunogenicity) , serum half-life, useful functional groups (for conjugating fluorochromes, spacers, and protective groups) , and cost.
- Useful types of backbone include polypeptides (polya ino acids) , polyethylenea ines, polysaccharides, aminated polysaccharides, aminated oligosaccharides, polyamidoamines, polyacrylic acids and polyalcohols.
- the backbone consists of a polypeptide formed from L-amino acids, D-amino acids, or a combination thereof.
- Such a polypeptide can be, e.g., a polypeptide identical or similar to a naturally occurring protein such as albumin, a homopolymer such as polylysine, or a copolymer such as a D-tyr-D-lys copolymer.
- a polypeptide identical or similar to a naturally occurring protein such as albumin, a homopolymer such as polylysine, or a copolymer such as a D-tyr-D-lys copolymer.
- the ⁇ -amino groups on the side chains of the lysine residues can serve as convenient reactive groups for covalent linkage of fluorochromes and spacers (Figs. 1A and IB) .
- the backbone is a polypeptide, preferably the molecular weight of the probe is from 2 kD to 1000 kD. More preferably, its molecular weight is from 4 kD to 500 kD.
- a backbone may be chosen or designed so as to have a suitably long in vivo persistence (half-life) inherently. Therefore, protective chains are not necessary in some embodiments of the invention.
- a rapidly- biodegradable backbone such as polylysine can be used in combination with covalently-1inked protective chains.
- useful protective chains include polyethylene glycol (PEG) , methoxypolyethylene glycol (MPEG) , methoxypolypropylene glycol, polyethylene glycol-diacid, polyethylene glycol monoamine, MPEG monoamine, MPEG hydrazide, and MPEG imidazole.
- the protective chain can also be a block-copolymer of PEG and a different polymer such as a polypeptide, polysaccharide, polyamidoamine, polyethyleneamine or polynucleotide.
- a useful backbone-protective chain combination is methoxypoly (ethylene) glycol-succinyl-JNT- ⁇ -poly-L-lysyine (PL-MPEG). The synthesis of this material, and other polylysine backbones with protective chains, is described in Bogdanov et al., U.S. Patent No. 5,593,658 and Bogdanov et al., 1995, Advanced Drug Delivery Reviews 16:335-348.
- fluorochromes are commercially available and can be used to construct probes according to this invention.
- exemplary fluorochromes include the following: Cy5.5, Cy5 and Cy7 (Amersham, Arlington Hts.,
- IRD41 and IRD700 (LI-COR, Lincoln, NE) ; NIR-1, (Dejindo, Kumamoto, Japan) ; LaJolla Blue (Diatron, Miami, FL) ; indocyanine green (ICG) and its analogs (Licha et al., 1996, SPIE 2927:192-198; Ito et al. , U.S. Patent No. 5,968,479); indotricarbocya ine (ITC; WO 98/47538) ; and chelated lanthanide compounds.
- Fluorescent lanthanide metals include europium and terbium. Fluorescence properties of lanthanides are described in Lackowicz, 1999, Principles of Fluorescence Spectroscopy , 2 Ed. , Kluwar Academic, New York.
- Fluorescent probes with excitation and emission wavelengths in the near infrared spectrum are used, i.e., 650-1300 n . Use of this portion of the electromagnetic spectrum maximizes tissue penetration and minimizes absorption by physiologically abundant absorbers such as hemoglobin ( ⁇ 650 nm) and water (>1200 nm) .
- Ideal near infrared fluorochromes for in vivo use exhibit: (1) narrow spectral characteristics, (2) high sensitivity (quantum yield) , (3) biocompatibility, and (4) decoupled absorption and excitation spectra. Table 1 summarizes information on the properties of six commercially-available near infrared fluorochromes, whose structures are shown in Figs. 2A and 2B. Table 1 Exemplary Near Infrared Fluorochromes
- Intramolecular fluorescence quenching by non- activated probes can occur by any of various quenching mechanisms.
- Several mechanisms are known, including resonance energy transfer between two fluorochromes.
- the emission spectrum of a first fluorochrome should be very similar to the excitation of a second fluorochrome, which is in close proximity to the first fluorochrome.
- Efficiency of energy transfer is inversely proportional to r 6 , where r is the distance between the quenched chromophore and excited chromophore.
- Self-quenching can also result from fluorochrome aggregation or exci er formation. This effect is strictly concentration dependent. Quenching also can result from a non-polar-to- polar environmental change.
- the fluorochrome can be covalently linked to a fluorochrome attachment moiety, backbone, or spacer using any suitable reactive group on the fluorochrome and a compatible functional group on the fluorochrome attachment moiety, backbone, or spacer.
- a carboxyl group (or activated ester) on a fluorochrome can be used to form an amide linkage with a primary amine such as the ⁇ -amino group of the lysyl side chain on polylysine.
- the fluorochromes are linked directly to the backbone or linked to the backbone through nonbiodegradable spacers. In such embodiments, the fluorescence activation sites are in the backbone.
- Some probes of this type accumulate in tumor interstitium and inside tumor cells, e.g., by fluid phase endocytosis. By virtue of this preferential accumulation, such probes can be used to image tumor tissues, even if the enzyme (s) activating the probe are not tumor specific.
- fluorochromes are linked to the backbone through spacers containing fluorescence activation sites.
- Oligopeptide spacers can be designed to contain amino acid sequences recognized by specific proteases associated with target tissues.
- two paired fluorochromes in fluorescence-quenching positions are in a single polypeptide side chain containing an activation site between the two fluorochromes.
- a side chain can be synthesized as an activatable fluorescence module that can be used as a probe per se , or covalently attached to a backbone (carrier) or targeting molecule, e.g., an albumin, antibody, receptor binding molecule, synthetic polymer or polysaccharide.
- a useful conjugation strategy is to place a cysteine residue at the N-terminus or C-terminus of the module and then employ SPDP for covalent linkage between the side chain of the terminal cysteine residue and a free amino group of the carrier or targeting molecule.
- Prostate Specific Antigen is a 33 kD chymotrypsin-like serine protease is secreted exclusively by prostatic epithelial cells. Normally, this enzyme is primarily involved in post-ejaculation degradation of the major human seminal protein. Normally, serum concentrations of PSA are proportional to the volume of prostatic epithelium. The release of PSA from prostate tumor cells, however, is about 30-fold higher than that from normal prostate epithelium cells. Damages basal membrane and deranged tissue architecture allow PSA to be secreted directly into the extracellular space and into the blood.
- PSA protein-binding protein
- PSA activity can be used as a marker for prostate tumor tissue
- prostate tumor tissue is highly enriched in PSA.
- spacers containing the amino acid sequence recognized by PSA can be used to produce a near infrared probe that undergoes fluorescence activation specifically in prostate tumor tissue.
- An example of a PSA-sensitive spacer is His-Ser-Ser-Lys-Leu- Gln-Gly (SEQ ID NO: 2) .
- Other PSA-sensitive spacers can be designed using information known in the art regarding the substrate specificity of PSA. See, e.g., 1997, Denmeade et al., Cancer Res . 57:4924-4930.
- Cathepsin D is an abundant lysosomal aspartic protease distributed in various mammalian tissues. In most breast cancer tumors, cathepsin D is found at levels from 2-fold to 50-fold greater than levels found in fibroblasts or normal mammary gland cells. Thus, cathepsin D can be a useful marker for breast cancer. Spacers containing the amino acid sequence recognized by cathepsin D can be used to produce a near infrared probe that undergoes fluorescence activation specifically in breast cancer tissue.
- An example of a cathepsin D-sensitive spacer is the oligopeptide: Gly- Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly (SEQ ID NO:l).
- cathepsin D-sensitive spacers can be designed using information known in the art regarding the substrate specificity of cathepsin D. See, e.g., Gulnik et al., 1997, FEBS Let . 413:379-384.
- Table 2 provides information on 5 exemplary enzymes (including substrate sequence recognized and cleavage point) and associated diseases.
- probe activation is by cleavage of the backbone.
- High fluorochrome loading of the backbone can interfere with backbone cleavage by activating enzymes such as trypsin. Therefore, a balance between fluorescence quenching and accessibility of the backbone by probe-activating enzymes.
- probes representing a range of fluorochrome loading densities can be produced and tested in vitro to determine the optimal fluorochrome loading percentage.
- Preferential accumulation of a probe in a target tissue can be achieved or enhanced by binding a tissue-specific targeting moiety (targeting ligand) to the probe.
- the binding can be covalent or non-covalent.
- targeting moieties include a monoclonal antibody (or antigen-binding antibody fragment) directed against a target-specific marker, a receptor-binding polypeptide directed to a target-specific receptor, and a receptor- binding polysaccharide directed against a target-specific receptor.
- Antibodies or antibody fragments can be produced and conjugated to probes of this invention using conventional antibody technology (see, e.g., Folli et al., 1994, "Antibody-Indocyanin Conjugates for
- receptor-binding polypeptides and receptor-binding polysaccharides can be produced and conjugated to probes of this invention using known techniques.
- a probe After a probe is designed and synthesized, it can be tested routinely in vitro to verify a requisite level of intramolecular fluorescence quenching before activation. Preferably, this is done by obtaining a fluorescence value for the intramolecularly quenched, fluorochrome-containing probe in a dilute, physiological buffer. This value is then compared to the fluorescence value obtained from an equimolar concentration of free fluorochrome in the same buffer, under the same fluorescence-measuring conditions. Preferably, this comparison will be done at a series of dilutions, to verify that the measurements are taking place on a linear portion of the fluorescence vs. fluorochrome concentration curve.
- the molar amount of an intramolecularly-quenched fluorochrome on a probe can be determined by one of ordinary skill in the art using any suitable technique.
- the molar amount can be determined readily by near infrared absorption measurements.
- it can be determined readily by measuring the loss of reactive linking groups on the backbone (or spacers), e.g., decrease in ninhydrin reactivity due to loss of amino groups.
- "de-quenching" i.e., fluorescence, upon exposure to an activating enzyme also can be verified in vitro .
- fluorescence of an intramolecularly-quenched probe is measured before and after treatment with an activating enzyme.
- de-quenching should be tested at various levels of fluorochrome loading, where "loading” refers to the percentage of possible fluorochrome linkage sites on the backbone actually occupied by fluorochromes.
- cells grown in culture can be used routinely to test intramolecularly-quenched near infrared fluorescence probes.
- Probe molecules free in cell culture medium should be non-detectable by fluorescence microscopy.
- Cellular uptake should result in probe activation and a fluorescence signal from probe-containing cells.
- Microscopy of cultured cells thus can be used to verify that activation takes place upon cellular uptake of a probe being tested.
- Microscopy of cells in culture is also a convenient means for determining whether activation occurs in one or more subcellular compartments.
- An imaging system useful in the practice of this invention typically includes three basic components: (1) a near infrared light source, (2) a means for separating or distinguishing fluorescence emissions from light used for fluorochrome excitation, and (3) a detection system.
- the light source provides monochromatic (or substantially monochromatic) near infrared light.
- the light source can be a suitably filtered white light, i.e., bandpass light from a broadband source.
- bandpass light i.e., bandpass light from a broadband source.
- light from a 150-watt halogen lamp can be passed through a suitable bandpass filter commercially available from Omega Optical (Brattleboro, VT) .
- the light source is a laser. See, e.g., Boas et al., 1994, Proc. Natl . Acad . Sci . USA 91:4887-4891; Ntziachristos et al., 2000, Proc . Natl . Acad . Sci .
- a high pass filter 700 nm can be used to separate fluorescence emissions from excitation light.
- a suitable high pass filter is commercially available from Omega Optical.
- the light detection system can be viewed as including a light gathering/image forming component and a light detection/image recording component. Although the light detection system may be a single integrated device that incorporates both components, the light gathering/image forming component and light detection/image recording component will be discussed separately.
- a particularly useful light gathering/image forming component is an endoscope.
- Endoscopic devices and techniques that have been used for in vivo optical imaging of numerous tissues and organs, including peritoneum (Gahlen et al., 1999, J. Photochem . Photobiol . B 52:131-135), ovarian cancer (Major et al., 1997, Gynecol . Oncol . 66:122- 132), colon (Mycek et al., 1998, Gastrointest . Endosc .
- catheter-based devices including fiber optics devices.
- fiber optics devices are particularly suitable for intravascular imaging. See, e.g., Tearney et al., 1997, Science 276:2037-2039; Proc. Natl . Acad . Sci . USA 94:4256- 4261.
- Still other imaging technologies including phased array technology (Boas et al., 1994, Proc . Natl . Acad . Sci . USA 91:4887-4891; Chance, 1998, Ann. NY Acad . Sci . 838:29- 45), diffuse optical tomography (Cheng et al., 1998, Optics Express 3:118-123; Siegel et al., 1999, Optics Express 4:287-298), intravital microscopy (Dellian et al., 2000, Br. J. Cancer 82:1513-1518; Monsky et al, 1999, Cancer Res .
- phased array technology Boas et al., 1994, Proc . Natl . Acad . Sci . USA 91:4887-4891; Chance, 1998, Ann. NY Acad . Sci . 838:29- 45
- diffuse optical tomography Choeng et al., 1998, Optics Express 3:118-123; Siegel et al.
- Any suitable light detection/image recording component e.g., charge coupled device (CCD) systems or photographic film, can be used in the invention.
- CCD charge coupled device
- the choice of light detection/image recording will depend on factors including type of light gathering/image forming component being used. Selecting suitable components, assembling them into a near infrared imaging system, and operating the system is within ordinary skill in the art.
- two (or more) probes containing: (1) fluorochromes that emit fluorescence at different near infrared wavelengths, and (2) activation sites recognized by different enzymes, e.g., cathepsin D and MMP2, are used simultaneously. This allows simultaneous evaluation of two (or more) biological phenomena.
- Cy-RRG-PL- MPEG the Cy5.5 fluorochrome was linked to the polylysine by a spacer consisting of Arg-Arg-Gly.
- Cy-GPICFFRLG-PL-MPEG the Cy 5.5 fluorochrome was linked to the polylysine by a spacer consisting of Gly- Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly (SEQ ID NO:l). Trypsin and trypsin-like proteases are capable of cleaving the polylysine backbone of Cy-PL-MPEG, when it is only partially derivatized.
- Probes Cy-RRG-PL-MPEG and Cy-GPICFFRLG-PL-MPEG were designed to allow fluorochrome cleavage of the spacer, but not necessarily the backbone.
- the peptide spacer RRG sensitive to trypsin cleavage, was used to derivatize the PL-MPEG, and then Cy5.5 was linked to the N- terminus of the RRG spacers.
- the cathepsin D sensitive peptide spacer, GPICFFRLG (SEQ ID NO:l) was similarly used to derivatize the PL-MPEG.
- Cy5.5 commercially available as a monofunctional NHS-ester (Amersham, Arlington Heights, IL) , was used according to the vendor's instructions, to label free ⁇ -amino groups of the polylysine backbone in PL-MPEG. Cy5.5 was added to a pre-mixed MPEG-PL solution (0.2 mg PL-MPEG in 1 ml 80 mM sodium bicarbonate solution) to a final concentration of 17 ⁇ M. After three hours, the reaction mixture was applied to a SephadexTM G-25 (Pharmacia) column (12 cm) for separation of the reaction product (Cy-PL-MPEG) from the unreacted fluorochrome and other low-molecular weight components of the reaction mixture. Average fluorochrome loading was about 20%, i.e., 11 out of 55 free amino groups on the PL-MPEG labeled with Cy5.5 (based on TNBS assay and absorption measurement) .
- Fig. 3A shows the excitation and emission spectra of Cy5.5 free in solution.
- Fig. 3B shows the excitation and emission spectra of Cy5.5 fluorochromes of Cy-PL-MPEG.
- the excitation and emission wavelengths of Cy5.5 are 675 nm and 694 nm, respectively.
- the fluorescence level of the Cy-MPEG-PL was approximately 30-fold lower than that of the unbound Cy5.5.
- Fig. 4 shows the relative fluorescent signal of Cy(n) -MPEG-PL (white bars) as a function of percentage of ⁇ -amino groups on the polylysine backbone occupied by fluorochrome.
- 20% loading 11 of 55 groups
- intramolecular quenching was observed, and the fluorescence signal was lowered in comparison to probes with lower fluorochrome loading.
- fluorescence signal was recovered, as shown by the black bars in Fig. 4.
- In vivo mouse imaging was carried out using a system composed of three main parts: light source, platform/holder, and image recording device.
- a fiber optic light bundle with a 150 W halogen bulb (Fiberlite high intensity illuminator series 180, Dolan-Jennen Industries) provided broad spectrum white light.
- a sharp cut off band pass optical filter (Omega Filter Corp. , Brattleboro, VT) was mounted at the end of the fiber optic bundle to create a uniform excitation source in the 610-650 nm range.
- the light was placed approximately 15 cm above the imaging platform to provide homogenous illumination of the entire mouse.
- the platform itself was a matte black surface that decreased the number of excitation photons reflected (and possibly detected) by the recording device.
- Fluorescent (emission) photons were selected using a low pass filter with a sharp cut off at 700 nm (Omega Filter Corp.). Cy5.5 dye has an excitation peak at approximately 670 nm, with a broad shoulder extending below 610 nm. Peak emission is at 694 nm. Sharp cut-off filters with more than 5 OD attenuation combined with widely spaced frequencies for the filter set markedly decreased "cross talk" of incident excitation photons recorded as fluorescent emission signal. The narrow angle between light source and recording device ensured that only fluorescent emission photons or scattered photons that interacted with the mouse tissue reached the low pass filter.
- the low-pass filter was mounted on a low power microscope (Leica StereoZoom 6 photo, Leica microscope systems, Heerbrugg, Switzerland).
- a low light CCD (SenSys 1400, 12 bit cooled CCD, Photometries, Tuscon, AZ) recorded the fluorescent emission images. Images were transferred to a PowerMac 7600/120 PC (Apple Computer, Cupertino, CA) and processed using IPLab Spectrum 3.1 software (Signal Analytics Corp., Vienna, VA) .
- Post processing included standard routines to exclude bad CCD pixels, and superimposition routines to overlay emission images with localization images of the entire mouse obtained using a second white light source. Typical acquisition time was 30 seconds for the near infrared emission images, and 1 second for the white light (non-selective images) .
- Fig. 5A is a schematic diagram of the imaged mouse, illustrating the location of tumor shown in Figs. 5B and 5C.
- Fig. 5B is visible light photograph of the skin covering a tumor on the side of a nude mouse into which the
- Fig. 5C is a corresponding near infrared fluorescence image. The tumor is visible as an area of intense fluorescence, in contrast to the surrounding tissue.
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Also Published As
Publication number | Publication date |
---|---|
AU2001271380A1 (en) | 2002-01-08 |
US8486373B2 (en) | 2013-07-16 |
US20030219383A1 (en) | 2003-11-27 |
US6592847B1 (en) | 2003-07-15 |
US20060275775A1 (en) | 2006-12-07 |
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