EP1414989A1 - Sonde organometallique - Google Patents

Sonde organometallique

Info

Publication number
EP1414989A1
EP1414989A1 EP02750835A EP02750835A EP1414989A1 EP 1414989 A1 EP1414989 A1 EP 1414989A1 EP 02750835 A EP02750835 A EP 02750835A EP 02750835 A EP02750835 A EP 02750835A EP 1414989 A1 EP1414989 A1 EP 1414989A1
Authority
EP
European Patent Office
Prior art keywords
sulphide
selenide
mts
probe
organometallic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02750835A
Other languages
German (de)
English (en)
Inventor
Gorm Danscher
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Berlock ApS
Original Assignee
Berlock ApS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Berlock ApS filed Critical Berlock ApS
Publication of EP1414989A1 publication Critical patent/EP1414989A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles

Definitions

  • the present invention relates to an organometallic probe for biological specimens comprising a colloidal core with a membrane translocating signal (MTS) molecule attached thereto.
  • the invention further relates to a method for staining a biological entity, and to a method for identifying a biological entity in a surrounding substance.
  • MTS membrane translocating signal
  • colloidal gold particles had been introduced a few years before as a marker of, for example, antibodies to be traced in the electron microscope, which is references in the article by M. Horisberger and J. Rosset: "Colloidal gold, a useful marker for transmission and scanning electron microscopy.” which has been published in J. Histochem. Cytochem. 1977; 25: 295.
  • AMG for colloidal gold visualisation as published by G. Danscher and J.O.R. N ⁇ rgaard: "Light microscopic visualisation of colloidal gold on resin-embedded tissue.” in J. Histochem. Cytochem. 1983; 31/12: 1394-1398; and by C.S. Holgate, P. Jackson, P.N. Cowen, and C.C.
  • an organothiol metal cluster compound for detecting the histological localisation of biological substances, for example specific molecules, lipids or proteins.
  • the organometallic probe comprises a metal core surrounded by a polymer shell to which biological substances as proteins, peptides, antibodies, lipids, carbohydrates, nucleic acids, drugs or hormones are covalently attached to impart desirable physical and chemical properties to the probe.
  • This metal cluster was introduced as an alternative to colloidal gold particles because of, among other things, its small size that allowed the tagged substances to penetrate a little further into embedded tissues. This metal cluster suffers from having a strongly reduced catalytic power for inducing the binding to silver.
  • the particles were bound to a new and for this purpose very effective HIV-Tat peptide.
  • Detection by magnetic resonance suffers from low spatial resolution, which is also demonstrated by the MR images presented in the publications.
  • This method is rather a scientific tool for demonstration of labelling principles than an applicable method for investigation at microscopic resolution and may only be used in connection with MR scanning of larger objects as organs with high levels of cells loaded with magnetic iron oxide particles.
  • the internalised iron oxide particles can be visualised by fluorescence microscopy. However, this is only possible as long as the tat molecules attached to the dextran encased iron oxide particle exist.
  • the tat peptide is rather fast metabolised leaving the iron oxide particle without possibility of being traced by immunohisto- chemical techniques. Therefore, the magnetic iron oxide technique is not suited for histological investigation. Also, it is known that iron oxide particles can be toxic.
  • organometallic probe in order to ameliorate existing histochemical cell labelling techniques.
  • an organometallic probe comprising a colloidal core with a membrane translocating signal (MTS) molecule attached thereto, wherein said core contains at least one from the group consisting of a metal, a metal selenide or a metal sulphide, for example gold, bismuth sulphide, bismuth selenide, mercury sulphide, mercury selenide, silver sulphide, silver selenide, copper sulphide, or copper selenide, or a combination thereof.
  • MTS membrane translocating signal
  • Metal containment in this context has to be understood as a containment of the metallic form of the substance in the core in contrast to containment of a metal oxide, which is not meant by the term "metal containment".
  • a colloidal gold particle is the most preferred due to its non-toxicity in contrast to iron oxide, which may have toxic side effects.
  • MTS molecules have been described, for example MTS peptide derived from anti-DNA monoclonal antibody as described by A. Avrameas et al: "Polyreactive anti- DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules.” as published in Proc. Natl.Acad.Sci. U.S.A. 95, 5601-6 (1998).
  • Another MTS, namely VP22 herpes virus protein is described by Phelan et al. in the article: “Intercellular delivery of functional p53 by the herpes virus protein VP22.”, which is published in Nat. Biotechnol.16, 440-31998.
  • a HIV-tat molecule is used.
  • Related information for a HIV-1 tat peptide can be found in an article by Fawell et al.: "Tat-mediated delivery of heterologous proteins into cells.” published in Proc.Natl.Acad.Sci. U.S.A. 91, 664-8, (1994).
  • the HIV-1 tat protein has been shown to freely travel through cellular and nuclear membranes as presented by A.D.Frankel and C.O.Pabo in their publication: "Cellular uptake of the tat protein from human immunodeficiency virus.” printed in Cell 55, 1189-93 (1988).
  • Another example of a MTS molecule is the third helix of the homeodomain of Antennapedia.
  • An organometallic probe according to the invention may be used for staining a biological entity, for example a cell, a cell organelle, for example a nucleus, a mitochondrion, a lysosome, a vesicle, for example a secretory vesicle or a synaptic vesicle, a protozoon, a bacterium, or a fungi, into which the probe is internalised after which the probe is subjected to silver enhancement for visual inspection.
  • tracing of the biological entity or its progeny may be performed in, for example, a solution, a cell culture, or an organism or part thereof.
  • the technique also opens up the possibility of tracing whether and if so, where in the organism inoculated cells/bacteria/ fungi settle and how well they proliferate.
  • the biological entities for example lymphocytes
  • the biological entities may be isolated by a biopsy and and probes with the MTS tags internalised before the entities, for example lymphocytes, are replace in the same person or a recipient person. Probes may also be bound to other entities as drugs or virus.
  • the tagged drugs or virus can be traced to the internalised cells in tissues from the exposed, and eventually sacrificed, animal or in biopsies. In the microscope the tagged cells can be identified and the localization of them and their progeny be identified.
  • AMG tags in the following is to be understood as comprising a colloidal gold core according to the invention, though also cores made of bismuth sulphide, bismuth selenide, mercury sulphide, mercury selenide, silver sulphide, silver selenide, copper sulphide, or copper selenide may have comparable properties.
  • a first advantage is the fact that these AMG tags can be silver enhanced to visible dimensions even if present only in a small number, usually less than 10 AMG tags per location are sufficient to identify a tagged cell. In theory, just a single AMG tag should suffice.
  • Iron oxide is not able to catalyse autometallographic silver enhancement. As, nor- mally, cells are loaded with hundreds of thousands of AMG-tags per cell, the obtained signal is impressively strong.
  • the second advantage is that the cells/bacteria and their progeny in many generations can be traced efficiently in tissue sections from biopsies/autopsies, in cell cultures, and in smears from excretes/ejaculates/blood samples and the like because of the above mentioned exceptional sensitivity.
  • This is not possible with iron oxide particles that cannot be traced histologically after removal of the MTS molecule, which typically occurs rather quickly, typically within a few hours, in biological compartments due to degradation, as the MTS is a foreign body in the compartment and subject for chemi- cal attack.
  • iron oxide may be decomposed and even removed as a result of chemical break down.
  • gold particles can be traced by AMG also after the attached MTS molecules has be removed, i.e.
  • Gold cannot be oxidised in a cell and does not react chemically with the organelles or molecules in the cell, which explains why the particles remain unchanged and can be followed in generations of dividing cells.
  • a further advantage is the fact that AMG tags after silver enhancement can be traced at both light microscopic and ultrastructural levels, for example with electron micro- sopy. Furthermore, the preparations can be stored for tens of years and reused for further or repeated investigations, a quality that the described technique using iron oxide particle do not posses nor the techniques with polystyrene spheres.
  • the internalising tags may be delivered as solutions ready for exposure of the cells in question or as a solution for injection.
  • examples are given for the preparation and for the staining technique.
  • Colloidal gold particles can, for exam- pie, be prepared according to the method of G. Frens: "Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions", published in Nat. Phys. Sci. 1973; 241: 20. This procedure results in a colloidal solution composed of gold particles having an average size of 14.5 nm. Colloidal gold particles can be produced in sizes from 0.1 - 100 nm by many different techniques, which is well known and described in prior art.
  • a peptide containing the translocating sequence of the tat peptide was synthesized on an automatic synthesizer (PS3, Rainin, Woburn, MA) by Fmoc chemistry using 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluoro- phosphate (HBTU)/N-hydroxybenzotriazole (HOBt) as activating agent.
  • the peptide is referred to as tat (FITC).
  • Fmoc-Lys(Dde) was anchored to 0.1 mmol of Rink amide MBHA resin® (NovaBiochem®, San Diego, CA) and followed with other amino acids, for example Fmoc-Arg(Pbf), Fmoc-Gln(Trt), Fmoc-Lys(Boc), Fmoc-Gly, and Fmoc-Cys(Trt).
  • the N-terminal was finally capped with t-Boc-Gly.
  • the Dde group on the C-terminal lysine residue was selectively removed with 10 mL of 2% hydrazine in DMF (2> ⁇ 3 min) and the deprotected amino group was reacted with 0.4 mmol of fluorescein isothiocyanate (FITC) (Aldrich, Milwaukee, WI) in 5 mL of DMSO/diisopropylethylamine (20% v/v) overnight.
  • FITC fluorescein isothiocyanate
  • the peptide was cleaved by 5 mL of TFA/thioanisole/ethandithiol/anisole (90/5/3/2) and purified by C18 reversed-phase
  • Rnase A (Sigma®) dissolved in 0.1 ml H2 O was added. At this pH, close to the isoelectric point of the protein, a stabilized gold sol was formed. After centrifugation at 25,000 rpm for 30 min the pellet was suspended in phosphate buffered saline (PBS) at pH 7.5.
  • PBS phosphate buffered saline
  • Exposure of isolated cells/bacteria to AMG-tag-MTS complex The isolated cells/bacteria obtained by the most appropriate technique for the cells/bacteria in question are placed in a suitable medium at 37°C and the AMG-tag-MTS complex dissolved in a phosphate buffer is added. After an hour, the cells/bacteria are washed sev- eral times and are now ready for injection into embryos, organisms, organs or inoculated into cell or bacteria cultures.
  • the AMG-tags present in the target cells/bacteria and their progeny do not demand special precautions of any kind as long as they are not treated with substances that dissolve the AMG-tag used.
  • Lymphocytes were prepared from mouse spleen as described by U. Schoepf, E. Mare- cos, R. Melder, R. Jain, and R. Weissleder: "Intracellular magnetic labelling of lymphocytes for in vivo trafficking studies" published in Bio Techniques 1998; 24: 642-651.
  • the mice were sacrificed under anaesthesia and their spleens were removed.
  • Lymphocytes were obtained by being placed in a rotating tissue chopper and diluted with a medium supplemented with 8% fetal bovine serum.
  • erythrocytes were lysed by resuspending the pellet in 0.83% ammonium chloride in distilled water. One hour later, the solution was transferred to another container leaving the adherent cells in the ammonium chloride jar. This approach results in about 95%> lymphocytes as determined by morphology. After centrifugation the pellet was added to 3 ml medium and 1 ml of the AMG-tag-MTS complex, and the vial incubated at 37°C for 1 h. The suspension was then washed 4 times through a step gradient of 40%) Histopaque-1077 (Sigma®) in a Hank's balanced salt solution.
  • the now gold-tagged lymphocytes were injected into the mice, and 10, 15 and 20 days later, the animals were anaesthetised and perfused transcardially with a 3% glutaralde- hyde solution in a 0.1 M phosphate buffer for 10 minutes.
  • the spleen, mesenteric lymph glands, bone marrow and thymus were removed and placed in the fixation solution for at least one hour. Control tissue from mice not injected with AMG-tagged lymphocytes was included.
  • Tissues that were to be cut in the cryostat were placed in a 30%) solution of sucrose until they sank to the bottom of the vial (1-2 days) and were then frozen in carbon dioxide. Sections from the three sources were placed on glass slides, dipped in a 0.5% gelatine solution and dried.
  • the slides were placed in jars and covered with the autometallographic developer. These jars were kept in a water bath at 26°C in complete darkness. Because the developing time is dependent on the thickness of the sections, it is recommended that dif- ferent developing times are used on parallel series of sections to avoid overlooking delicate traces of gold. After development, the slides are washed in gently running tap- water at 40°C for at least 40 min to remove the protecting gelatine and rinsed in distilled water.
  • One kg crystalline gum arabic is dissolved in 2 1 deiomsed water by intermittent stirring. Five days later the solution is filtered through layers of gauze.
  • Suitable portions of colloid can be frozen in plastic jars and stored for at least 6 months.
  • Silver ion supply Silver lactate, 0.11 g; deionised water, 15 ml. Silver lactate is highly sensitive to light, and the solution should be carefully protected from light.
  • the Epon sections can be counterstained with toluidin blue (1%) After light microscopic analysis a section to be processed for electron microscopy is covered with a drop of Epon solution and a blank Epon block is placed over it. The resin is allowed to polymerise and the section is removed from the glass slide after being quickly heated on a hot plate.
  • the ultrathin sections made in conventional ways, can be stained with uranyl and lead.
  • Epon-embedded sections or metachrylate embedded sections are superior to both paraffin-embedded and frozen sections for the light microscopical localisation of gold.
  • cell organelles for example nuclei loaded by MTS tagged colloidal gold can be removed from its original cell and implanted into another cell including early stages of blastula or striated muscle cells. If studying the progeny of injected cells in an organ- ism, the functional potentials and number of divisions can then be monitored based on the amount of AMG- tags in the cells. Comparably, it will be possible to follow mitochondria and other cell organelles inoculated into a receptor cell.
  • the present invention allows tracing of cells/bacteria/mitochondria and their progeny in organisms and cultures. For instance, it is feasible to AMG-tag sperm cells from two different males with differently sized colloidal gold particles with MTS. The two ejaculates then be mixed and used for insemination for later analysis of possible differences in the capability of sperms from the two males that reach the ampulla of tuba uterine, and the fertilising sperm cell identified as the male pronucleus will be AMG - tagged with either of the two seizes of gold particles. Also, a fraction of sperm cells from a male can be isolated and treated in different ways, AMG-tagged, and later traced, in order to evaluate their capacity.
  • isolated stem cells origin from, for example, embryos or umbilical cords can likewise be tagged and traced.
  • Cancer cells can be AMG- tagged and after injection into the experimental animals, it is possible to observe which organs/tissues are invaded by the cells and where they result in tumours.
  • biopsies from a bone marrow donor can be tagged and injected into the recipient - later biopsies will show the location and dynamics of the injected cells.
  • the invention makes it possible to follow the fate of transplanted cells and their progenitor cells for 15- 20 generations. It allows studies of the behaviour of isolated cells or tissues, for example exposed to a certain drug or toxic molecule.
  • the set-up enables studies after homotransplantation or xenotransplantation and is valuable for embryo- logical studies and when establishing the origin and turnover of cells like macrophages and osteoclasts.
  • the MTS-gold-AMG tags can be used to trace axonal connections in the central nervous system. After local injection of the probes with AMG-tags into the brain, all the cells and axons that are exposed to the tags will be loaded, and the gold particles spread into every part of the cell. In the case of neurons, dendrites and axons will be filled with tags. This line of events leads to a detailed picture of the neurons and their ramifications that are connected to that particular location.
  • AMG-tags in entities reveals the number of generations that has led to the actual entity after multiple division of the cell, bacteria or fungi.
  • 10 7 magnetic iron oxide particles could be loaded per cell.
  • a likewise number is easily pos- sible, which means that at least 15 - 20 generations can be traced.
  • the technique can also be used to irradiate certain tissues, where a particular kind of cells is known to accumulate, by loading the isolated AMG-tagged cells with a radioactive isotope.
  • MTS-gold colloid conjugates can further be attached molecules that selectively bind to receptors, particular for a certain group of cells in an organism or in a culture, or the like, causing uptake of gold in these cells, but not in other cells.
  • the technique according to the invention is also suited for loading liposomes with colloidal gold particles for easy monitoring in tissues.
  • test cells from the donor are marked, for example bone marrow cells, they may be injected into the recipient and traced in biopsies as an extra evaluation of the acceptance of the donor cells by the recipient prior to transplantation.
  • an organometallic probe according to the invention has a broad range of application. Technological progress in the AMG-tag field can be expected on two fronts for further development of the invention, namely development of new efficient AMG-tags, and development of new and perhaps even more efficient MTS moieties.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nanotechnology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Inorganic Chemistry (AREA)
  • Ceramic Engineering (AREA)
  • Dispersion Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une sonde organométallique comprenant un noyau colloïdal, par exemple un colloïde d'or, auquel est directement attachée une molécule de signal de translocation membranaire (MTS), par exemple, un fragment VIH-tat. La sonde organométallique est adaptée aux techniques d'amélioration de la liaison à l'argent.
EP02750835A 2001-07-12 2002-07-11 Sonde organometallique Withdrawn EP1414989A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DKPA200101092 2001-07-12
DK200101092 2001-07-12
PCT/DK2002/000486 WO2003006681A1 (fr) 2001-07-12 2002-07-11 Sonde organometallique

Publications (1)

Publication Number Publication Date
EP1414989A1 true EP1414989A1 (fr) 2004-05-06

Family

ID=8160621

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02750835A Withdrawn EP1414989A1 (fr) 2001-07-12 2002-07-11 Sonde organometallique

Country Status (2)

Country Link
EP (1) EP1414989A1 (fr)
WO (1) WO2003006681A1 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2328457A1 (fr) * 1998-06-20 1999-12-29 Washington University Complexes peptidiques de permeation membranaire destines a l'imagerie medicale, au diagnostic et a la therapie pharmaceutique
AU2002226876A1 (en) * 2000-10-06 2002-04-15 Quantum Dot Corporation Cells having a spectral signature, and methods of preparation and use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03006681A1 *

Also Published As

Publication number Publication date
WO2003006681A1 (fr) 2003-01-23

Similar Documents

Publication Publication Date Title
De Mey Colloidal gold probes in immunocytochemistry
Rash et al. Cell-specific expression of connexins and evidence of restricted gap junctional coupling between glial cells and between neurons
Smith et al. Magnetic resonance microscopy of mouse embryos.
Bendayan et al. Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.
JP5769717B2 (ja) 細胞を磁性化するための材料及び磁気操作
JP3731891B2 (ja) 分泌生成物による細胞の直接選択
JPH06510363A (ja) 磁気性粒子を使用する生物学的材料の単離
Modla et al. Correlative microscopy: a powerful tool for exploring neurological cells and tissues
JPS63502484A (ja) 細胞の由来組識および／または細胞の異常度を特徴付ける方法
Sonomura et al. Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling
Parham et al. Differential staining of tannin in sections of epoxy-embedded plant cells
Kan et al. Preferential association of glycoproteins to the euchromatin regions of cross-fractured nuclei is revealed by fracture-label.
JPH04504049A (ja) インビトロにおける組織の生存能力及び増殖能力を測定する、自生状態法及びシステム
Dvorak et al. RNA is closely associated with human mast cell lipid bodies
CN110361442B (zh) 一种用于质谱流式细胞仪检测的外泌体及其制备方法与应用
Bendayan Electron microscopical localization of nucleic acids by means of nuclease-gold complexes
Winey et al. Cytological analysis of Tetrahymena thermophila
US20210190652A1 (en) Multiplexed Expansion (MultiExM) Pathology
US20030013084A1 (en) Organometallic probe
Morphew 3D immunolocalization with plastic sections
Mason et al. Labeling cell-surface proteins via antibody quantum dot streptavidin conjugates
WO2003006681A1 (fr) Sonde organometallique
Biggiogera et al. Ethidium bromide-and propidium iodide-PTA staining of nucleic acids at the electron microscopic level.
Seeley et al. Use of colloidal gold complexes of wheat germ agglutinin as a label for neural cells
Simpson Morphological studies of possible neuroendocrine structures in Helisoma tenue (Gastropoda: Pulmonata)

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20040209

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

17Q First examination report despatched

Effective date: 20050502

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20050913