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WO2008069992A2 - Procédé et trousse pour administration intracellulaire - Google Patents

Procédé et trousse pour administration intracellulaire Download PDF

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Publication number
WO2008069992A2
WO2008069992A2 PCT/US2007/024681 US2007024681W WO2008069992A2 WO 2008069992 A2 WO2008069992 A2 WO 2008069992A2 US 2007024681 W US2007024681 W US 2007024681W WO 2008069992 A2 WO2008069992 A2 WO 2008069992A2
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WIPO (PCT)
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cells
dmso
qds
cell
transfection
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PCT/US2007/024681
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English (en)
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WO2008069992A3 (fr
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Anja Nohe
Jay Nadeau
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University Of Maine System Board Of Trustees
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Publication of WO2008069992A2 publication Critical patent/WO2008069992A2/fr
Publication of WO2008069992A3 publication Critical patent/WO2008069992A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to a method and a kit for delivering one or more micro-sized and/or nano-sized agents, such as polypeptides and quantum dots, for example, into a cell, such as a mammalian cell, for example.
  • the present invention further relates to a method and a kit for delivering one or more micro-sized and/or nano- sized agents, such as polypeptides and quantum dots, for example, into the cell nucleus and cytoplasm.
  • a cell's plasma membrane functions as a selectively permeable "gatekeeper.” That is, it allows entry of some compounds, such as sugars, lipids and amino acids, into the cell, while excluding others, which may or may not be lethal or otherwise harmful, from the cell.
  • Recent advances in cell biology have allowed for the artificial delivery of compounds across the plasma membrane and into a cell's nucleus or cytoplasm. This artificial intracellular delivery process generally may be referred to as "transfection.”
  • Transfection Our ever-advancing transfection capabilities have helped us improve both our knowledge of cell biology and our ability to develop therapeutic treatments. For example, these capabilities have helped us to elucidate the natural functions of proteins and to deliver drugs into cells.
  • QDs quantum dots
  • nanoparticles typically range from about 1 to 20 nanometers (nm) in size
  • QDs are used to label a protein for the purpose of studying that protein's natural function within the cell.
  • QDs and other micro-sized and nano-sized agents such as DNA, RNA, proteins, for example, into a cell.
  • chemical transfection methods include those that are liposome-mediated and those that involve the use of non-liposomal lipids or dendrimers.
  • Physical methods include those involving electroporation, microinjection, or heat shocking of targeted cells.
  • Viral-based delivery may be by, for example, retrovirus, adeno-associated virus, or lentivirus.
  • nanoparticles Some chemical methods for intracellular delivery of nanoparticles require specific conjugation of these nanoparticles to ligands, which are recognizable via receptor-mediated endocytosis, or to cell-permeating peptides, such as Tat, for example.
  • ligands which are recognizable via receptor-mediated endocytosis, or to cell-permeating peptides, such as Tat, for example.
  • the primary drawback of these specific approaches for nanoparticle delivery is that such manipulation of the particle can impede proper targeting of the particle within the cell.
  • endocytosis generally leads to sequestration in lysosomes, with extra-lysosomal targeting requiring further manipulation, such as the addition of chloroquine, for example.
  • much time is required to generate the constructs that feature a full- length protein fused to Tat or another peptide.
  • What is needed therefore is a method and kit for efficiently delivering micro-sized and/or nano-sized agents, such as polypeptides and QDs, for example, into a cell, including into the cell nucleus.
  • the needed method and kit should cause a large number of cells to be transfected in a single transfection application, while keeping the incidence of cell death low, so that an increased number of healthy and successfully transfected cells may be recovered.
  • the needed method also should require minimal time to complete.
  • the present invention is a method and an optional kit for delivering micro- sized and/or nano-sized agents into a cell, including into the nucleus or cytoplasm.
  • the method and kit may be used, for example, to transfect mammalian cells, including human cells, with at least one transfection agent.
  • the transfection agent may be, but is not limited to being, any one or more of QDs, polypeptides, DNA, RNA, lipids, lipid mixtures, and polymers, for example.
  • the method includes exposing cells to the at least one transfection agent and to dimethylsulfoxide (DMSO), and irradiating the cells by using electromagnetic radiation, which may be delivered by a laser or by a mercury or xenon lamp, for example.
  • DMSO dimethylsulfoxide
  • the method may be carried out for the purpose of transfecting cells only with QDs.
  • the method may be carried out for the purpose of transfecting cells with QDs and with one or more other transfecting agents, such as one or more polypeptides, for example, that are either conjugated to or are not conjugated to the QDs.
  • the concentration of the DMSO to which the cells are exposed in carrying out the transfection method is variable, and therefore is not limited to being any particular concentration. Further, the purity and grade of the DMSO used is also variable, and therefore the DMSO is not limited to being of any particular purity level or grade.
  • the method of the present invention may be used for a variety of clinical and research purposes.
  • the transfection method therefore may be carried out to generate transfected cells that may be used as part of high throughput screening assays, such as ones made for the purpose of rapidly identifying proteins that are involved in a particular biochemical pathway, for example.
  • the method therefore also may be carried out for the purpose of generating transfected cells that are useful for imaging assays, such as ones designed to gather temporal and spatial evidence of a protein's role within a cell, for example.
  • the method also may be carried out for the purpose of generating transfected cells that are useful in diagnostic assays, such as ones made for the purpose of determining whether a particular protein is present or absent in a population of cells, for example.
  • the present invention also is an optional kit that may be used to help carry out the method of the present invention.
  • the kit includes one or more reagents and instructions that are useful for carrying out the present method.
  • FIG. 1 is a flow diagram of a preferred embodiment of a method of the present invention.
  • FIG. 2 shows mammalian cells that were transfected with QDs by carrying out a first example of the method of the present invention.
  • FIGS. 3A-3C shows various populations of mammalian cells after the cells were exposed to anti-Histone 1 antibody and either QDs and no DMSO, DMSO and no QDs, or QDS and DMSO by carrying out a second example of the method of the present invention.
  • FIG. 4 shows a time course analysis of the transfection of mammalian cells with QDs and streptavidin-TRITC antibody by carrying out a third example of the method of the present invention.
  • FIGS. 5A-5D shows the specific localization of anti-Hi stone 1 antibody to the nuclei of mammalian cells that were transfected with QDs and anti-Histone 1 antibody by carrying out a fourth example of the method of the present invention.
  • FIG. 6 is a graph showing the viability of mammalian cells that were transfected with QDs and anti-Histone 1 antibody by carrying out a fifth example of the method of the present invention at one, two and three days following the transfection.
  • FIG. 7 is a graph showing the proliferation capability of mammalian cells that were transfected with QDs and anti-Histone 1 antibody by carrying out the fifth example of the method of the present invention at one and two days following the transfection.
  • a "transfection agent” is any substance that wholly or partially enters, or is capable of wholly or partially entering, a cell by using the present invention.
  • a “quantum dot” or “QD” is any semiconductor nanocrystal. Exemplary QDs are provided herein.
  • a “laser” is any device that utilizes the natural oscillations of atoms or molecules between energy levels for generating a beam of coherent electromagnetic radiation.
  • the present invention is a method and an optional kit for delivering micro-sized and/or nano-sized transfection agents into cells.
  • the method and kit are particularly useful for delivery of one or more of these transfection agents, which may be, for example, polypeptides and QDs, into the cytoplasm and into the nucleus.
  • these transfection agents are transfected into cells by exposing the cells to both DMSO and the transfection agent or agents, and subjecting the cells to electromagnetic radiation, which may be delivered by a laser or a mercury or xenon lamp, for example.
  • the transfection method 10 of the present invention generally includes the steps of: exposing cells to a solution, step 15; adding one or more transfection agents to the solution, step 20; adding DMSO to the solution; step 25; exposing the cells to electromagnetic radiation, step 30; and, optionally, removing the solution from the cells and adding growth medium to the cells, step 35.
  • the cells may be exposed to the DMSO before or after being exposed to the transfection agent.
  • the exposure of the cells to the DMSO and transfection agent may be concomitant, such as would occur when the DMSO and transfection agent co-exist in the same solution and that solution is introduced to the cells, for example.
  • the cells be exposed to cell growth medium, or to any other particular substance, following their exposure to the electromagnetic radiation.
  • the optional step of removing the solution from the cells and adding growth medium to the cells would be useful, for example, where the cells are to be recovered for use in applications that do not immediately follow the transfection of the cells, such as in certain therapeutic applications, for example, but may or may not necessarily be useful where the cells are to be used in applications that do immediately follow the transfection of the cells, such as in certain research applications, for example.
  • the method may be carried out for the purpose of transfecting cells only with QDs.
  • the method described with reference to FIG. 1 is carried out, at least through step 30, using QDs as the transfection agent.
  • Exemplary QDs include semiconductor nanocrystals that have fluorophoric properties. That is, these exemplary QDs absorb light and then re-emit that light at a particular wavelength or within a particular wavelength range.
  • Exemplary QDs contain a plurality of atoms, such as, for example, a few hundred to a few thousand atoms, of a semiconductor material. This semiconductor material may be, for example, cadmium mixed with selenium or tellurium.
  • This semiconductor material also may be coated with a semiconductor shell, such as a shell of cadmium sulfide, lead sulfide or zinc sulfide, for example.
  • a semiconductor shell such as a shell of cadmium sulfide, lead sulfide or zinc sulfide, for example.
  • Exemplary QDs include those of about 1 nm to about 20 nm in diameter. As another example, suitable QDs include those of about 1 nm to about 10 nm in diameter. As another example, suitable QDs include those of about 2 nm to about 5 nm in diameter.
  • Exemplary QDs may emit light at any one or more of a variety of wavelengths. For example, some of these QDs emit light that is within the range of about 300 nm to about 350 nm. Further, some of these QDs emit light that is within the range of about 350 nm to about 400 nm, about 400 nm to about 450 nm, about 450 nm to about 500 nm, about 500 nm to about 550 nm, about 550 nm to about 600 nm, about 600 nm to about 650 nm, or about 650 nm to about 700 nm.
  • Suitable and exemplary QDs may be made, such as being made by the user of the invention, for example, or they may be obtained by the user from any number of vendors.
  • Exemplary methods for making QDs include those described in the following printed references: S. J. Clarke et al., Nature Materials 5:347-8 (2006); J. A. Kloepfer et al., Journal of Physical Chemistry 109:9996-10003 (2005); J. A. Kloepfer et al., Applied and Environmental Microbiology 69:4205-4213 (2003); Y. Chen et al., Nano Lett. 3:581-584 (2003); Semiconductor Quantum Dots, Y.
  • QDs suitable for biological applications are commercially available from, for example, Northern Nanotechnologies, Inc. of Toronto, Ontario, Canada (http://www.nntech.com).
  • Exemplary QDs made available by Northern Nanotechnologies include, but are not limited to being, one color QDs, two color QDs and three color QDs, which emit yellow, green and/or orange light, for example.
  • QDs also are commercially available from other vendors, such as, Evident Technologies, Inc. of Troy, New York (http://www.evidenttech.com), and Invitrogen Corporation of Carlsbad, California (http://www.invitrogen.com), for example.
  • Exemplary QDs made available by Evident Technologies include, but are not limited to being, for example, Evident's core and core-shell EviDots® , EviCompositesTM, EviTagsTM, and EviFluors® brands.
  • Exemplary QDs made available by Invitrogen include that company's Qdot ® 800 ITKTM organic quantum dots (cat. no. Q2177 IMP), Qdot ® 545 ITKTM organic quantum dots (cat. no. Q2179 IMP), Qdot ® 565 ITK TM organic quantum dots (cat. no. Q2173 IMP), Qdot ® 605 ITK TM organic quantum dots (cat. no.
  • Q21701 MP Qdot ® 545 ITK TM carboxyl quantum dots (cat. no. Q2139 IMP), Qdot ® 705 ITKTM carboxyl quantum dots (cat. no. Q21361MP), Qtracker ® 800 non-targeted quantum dots (cat. no. Q21071MP), Qtracker ® 565 non-targeted quantum dots (cat. no. Q2103 IMP), Qdot ® 800 ITKTM amino (PEG) quantum dots (cat. no. Q2157 IMP), and Qdot ® 525 ITK TM amino (PEG) quantum dots (cat. no. Q2154 IMP).
  • the method may be carried out for the purpose of co- transfecting cells with QDs and one or more other types of transfection agent, which may be, but are not limited to being, one or more types of polypeptide, for example.
  • the method described with reference to FIG. 1 is carried out, at least through step 30, using one or more polypeptides and the QDs as the transfection agent.
  • the polypeptides may be naturally occurring polypeptides or they may be artificially synthesized polypeptides. Methods for isolating naturally occurring polypeptides and artificially synthesizing polypeptides are well known to the skilled artisan. See, e.g., Isolation and Purification of Proteins (Biotechnology and Bioprocessing Series), R.
  • the QDs may be separate from the other agent or agents, or they may be bound to the other agent or agents, at the time of their exposure to the cell.
  • the Example section provided herein describes one application of the present method in which QDs and streptavidin-TRITC antibody are added to cells as separate agents (that is, the streptavidin-TRITC antibody is not bound to the QDs) for transfection of those cells with both the QDs and the streptavidin-TRITC antibody.
  • QD bioconjugates such as, for example, QDs bioconjugated to streptavidin-TRITC antibody or some other protein or proteins.
  • QD bioconjugates such as, for example, QDs bioconjugated to streptavidin-TRITC antibody or some other protein or proteins.
  • the skilled artisan would recognize that a large variety of such QD bioconjugates may be made. Any one or more of these QD bioconjugates would be useful for serving as the transfection agent of the present invention.
  • transfection agents include, but are not limited to being, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), lipids and mixtures of lipids, and polymers.
  • DNA includes DNA molecules from all organisms, including viral DNA, from all methods of artificial creation and natural isolation, and of all confirmations, such as linear, circular, relaxed loop, and supercoiled DNA conformations, for example.
  • the DNA when used as a transfection agent, the DNA may be, but is not limited to being, in the form of a plasmid, cosmid, bacterial artificial chromosome (BAC), polymerase chain reaction (PCR) amplicon, or artificially synthesized DNA oligonucleotide, such as, for example, a 20-mer DNA oligonucleotide.
  • BAC bacterial artificial chromosome
  • PCR polymerase chain reaction
  • DNA oligonucleotide such as, for example, a 20-mer DNA oligonucleotide.
  • RNA includes both single-stranded and double-stranded RNA from all organisms, from all methods of artificial creation and natural isolation, and of all types. RNA therefore may be, for example, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short-interfering RNA (siRNA), and microRNA (miRNA).
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • siRNA short-interfering RNA
  • miRNA microRNA
  • Lipids include those transfection agents that are capable of binding to a material, such as DNA, RNA, or protein, for example, and creating liposomes or lipid-based nanoparticles, which can permeate cellular membrane and deliver the material inside the cell.
  • a material such as DNA, RNA, or protein
  • liposomes or lipid-based nanoparticles which can permeate cellular membrane and deliver the material inside the cell.
  • the concentration of the DMSO to which the cells are exposed in carrying out the method of the present invention is selectable and may be varied.
  • the DMSO may be at least at a concentration that is in the range of about 0.1% (v:v) to about 10% (v:v).
  • the DMSO may be at a concentration of about 0.1% (v:v).
  • the DMSO may be at a concentration that is about 10% (v:v).
  • the only limitation regarding the concentration of the DMSO to which the cells are exposed is that the concentration of the DMSO should not be of a value that is lethal to the cells.
  • stock DMSO i. e. , the supply of DMSO from which the DMSO that is to be exposed to cells is diluted
  • stock DMSO may be obtained from the Sigma- Aldrich Corporation of St. Louis, Missouri.
  • Suitable Sigma-Aldrich stock DMSO products include, but are not limited to being, for example, Biotechnology Performance Certified DMSO (cat. no. D2438), BioUltra DMSO (cat. no. 41639), > 99.9% DMSO for molecular biology (cat. no. D8418), and >99.5% DMSO (cat. no. D5879).
  • the only limitation regarding the stock DMSO used in the present invention is that the stock DMSO should not contain any impurities that are lethal to the cells when the method is carried out.
  • composition of the solution that is used to dilute the stock DMSO is selectable and may be varied.
  • Suitable solutions for diluting the DMSO include, but are not limited to being, various cell growth media, such as Dulbecco's Modified Eagle Medium (DMEM) having 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • Other exemplary media include, but are not limited to, Iscove's Modified Dulbecco's Media (IMDM), SFM, EBM, MCD131, M 199, and Ml 99/Fl 2, for example, each one of which may be supplemented with a serum (such as FBS or fetal calf serum) and/or a serum- replacement.
  • IMDM Iscove's Modified Dulbecco's Media
  • the DMSO-dilution solution may include any one or more of a variety of compounds.
  • the DMSO-dilution solution may include, in varying amounts, any one or more of the inorganic salts CaCl 2 , KCl, KNO 3 , MgSO 4 , NaCl, Na 2 SE 3 and NaH 2 PO 4 H 2 O; the amino acids L-alanine, L-arginine HCl, L-asparagine H 2 O, L-aspartic acid, L-cysteine 2HCl, L- glutamic acid, L-glutamine, glycine, L-histidine HCl H 2 O, L-isoleucine, L-leucine, L- lysine HCl, L-methionine, L-phenylalanine, L-proline, L- se
  • a variety of other compounds also optionally may be included in the solution that is used to dilute the DMSO.
  • These compounds include, but are not limited to being, human insulin, human transferrin, ascorbic acid, heparin, hydrocortisone, sodium selenite, monothioglycerol, sodium pyruvate, non-essential amino acids and antibiotics for preventing microbial contamination of the DMSO-dilution solution, such as penicillin and streptomycin, for example.
  • the solution that is used to dilute the stock DMSO also may be, for example, a salt-containing solution that is not a cell growth medium. Therefore, the solution that is used to dilute the stock DMSO may be, for example, a phosphate buffered saline (PBS) solution. Protocols for preparing a large variety of salt solutions are well known to the skilled artisan, and suitable salt solutions are also commercially available. A variety of suitable salt solutions are available from, for example, Invitrogen (for example, Invitrogen products corresponding to cat. nos. 10010-031 and 20012-027).
  • Invitrogen for example, Invitrogen products corresponding to cat. nos. 10010-031 and 20012-027.
  • the diluent solution should not be lethal to the cells that are exposed to the diluted DMSO.
  • the skilled artisan would recognize that there exist a large plurality of salt solutions to which cells may be exposed without causing cell death.
  • the pH of the solution that is used to dilute the stock DMSO, and the pH of any solution to which the cells are exposed before, during or after the transfection method, including the cell growth medium, also is selectable and may be varied.
  • a suitable pH range is about 6.5-8.5, but preferably is about 7.0-7.7, and even more preferably, is about 7.2-7.5.
  • the only limitation of the pH is that the pH should not be of a value that is lethal to the cells.
  • the cells to be transfected is exposed to electromagnetic radiation that may be derived from any source that is capable of emitting electromagnetic radiation.
  • the electromagnetic radiation may be emitted by a laser.
  • An exemplary laser is the confocal diode laser that is supplied with the Olympus Corporation's (Center Valley, Pennsylvania) FluoViewTM brand of microscopes, including Olympus's FluoViewTM 1000-MPE, FluoViewTM 1000, and FluoViewTM 300, models.
  • Other suitable lasers are described in, for example, V. M. Ustinov et al., Quantum Dot Lasers (Series on Semiconductor Science and Technology), Oxford University Press USA (2003), which is incorporated herein by reference in its entirety.
  • the electromagnetic radiation may be emitted by a xenon or mercury lamp.
  • exemplary lamps include, for example, 100 watt and 200 watt mercury short arc lamps and 20 watt xenon short arc lamps that are available from the Advanced Radiation Company (Santa Clara, California).
  • Other exemplary lamps include the large number of lamps that are available from the Hamamatsu Corporation of Japan, including, for example, those mercury-xenon lamps that include a fused silica or ozone- free window and consume 50, 75, 100, 150, or 200 watts or more that are available from Hamamatsu.
  • the present method is particularly useful for, but is not limited to being useful for, transfecting eukaryotic cells. Therefore, the method is useful for transfecting mammalian cells, including, but not limited to, cells of any type derived from human, mouse, rat, feline, canine, bovine, equine, porcine, monkey, or chimpanzee, for example. It is to be understood, however, that the method is not limited to being used for the purpose of transfecting mammalian cells. The method therefore may be carried out for the purpose of transfecting cells derived from non-mammalian organisms, including, but not limited to, fish, nematodes, fly, for example, and also may be used to transfect yeast or plant cells. Further, the method may be carried out for the purpose of transfecting prokaryotic cells, such as bacterial cells, for example.
  • cells that are transfected by using the present invention may be grown or otherwise maintained, including prior to, during and after transfection, according to any one or more protocols that are useful for growing and otherwise maintaining cells. These include the large variety of protocols that are known to the skilled artisan.
  • mammalian cells preferably are grown or otherwise maintained at a temperature of about 37°C while being exposed to a cell growth medium, including, but limited to, any of the cell growth media described above.
  • a cell growth medium including, but limited to, any of the cell growth media described above.
  • those of ordinary skill in the art would recognize that these cells may be grown or otherwise maintained at temperatures that are warmer or cooler than about 37 0 C.
  • mammalian cells may be grown or otherwise maintained at any temperature that is in the range of about 28 0 C to about 41 0 C.
  • Exemplary incubators for maintaining and growing mammalian cells at these temperatures include, but are not limited to, CO 2 incubators that are commercially available from New Brunswick Scientific, Inc. (Edison, NJ; http://www.nbsc. com/co2_incubators.aspx), which include that manufacturer's Innova CO- 14, Innova CO-48, Innova CO- 170, and Excella CO- 170 models.
  • the cells are preferably incubated at about 37°C.
  • these cells may be incubated during the transfection method at any one of a wide range of temperatures, such as those at or between about room temperature (about 20 0 C) to about 41 0 C, for example.
  • An exemplary device for incubating the cells during the transfection method is, but is not limited to being, an open perfusion micro-incubator (such as, for example, the open perfusion micro-incubator corresponding to model PDMI-2, which is available from Warner Instrument Corporation of Hamden, Connecticut).
  • cells may be grown, maintained and/or irradiated while attached to a substrate, such as to a cell culture dish or flask, for example, or to microbeads, for example, and would also recognize that cells may be grown, maintained and/or irradiated while in suspension, such as while in a bioreactor, for example. Cells also may be grown, maintained and/or irradiated while on feeder cells that are attached to a substrate, such as to a cell culture dish or flask or to microbeads, for example.
  • cell lines including mammalian cell lines, whether transfected or not, may be cryopreserved in a dormant state for later use without adverse affect to the cell line, such as the introduction of DNA mutation thereto or reduction of the cell line's ability to proliferate, for example, by using any one or more of a variety of cryopreservation methods known to the skilled artisan.
  • mammalian cell cryopreservation may be achieved generally by: (1) growing the cells to confluency or near confluency on a solid support in a culture medium; (2) washing the cells one or more times with a wash buffer, such as a PBS solution, for example; (3) incubating the cells in trypsin to detach the cells from the solid support; (4) resuspending the cells by mechanical flushing with a pipette while in a freeze medium (such as, for example, a freeze medium including DMSO, such as 10% DMSO, for example, as the cryoprotectant); (5) slowly freezing the cells in a cryoprotectant vial to -80° C at a rate of about -1° C/minute; and (6) immersing the vial in liquid nitrogen for long term storage.
  • a wash buffer such as a PBS solution
  • Suitable vials for storing cells in liquid nitrogen include, for example, polypropylene cryogenic vials (catalog number 5012) that are commercially available from Nalge Nunc International (Rochester, NY; http://www.nalgenunc.com). This cryopreservation protocol may be used to preserve cells transfected through the method of the present invention.
  • Cells including mammalian cells, also may be cryopreserved for long term storage in a culture plate, such as, for example, the 96-well BD FalconTM cell culture plate (catalog number 353936) that is commercially available from BD Biosciences (San Jose, CA; http://www.bdbiosciences.com/index.shtml).
  • a culture plate such as, for example, the 96-well BD FalconTM cell culture plate (catalog number 353936) that is commercially available from BD Biosciences (San Jose, CA; http://www.bdbiosciences.com/index.shtml).
  • cells When cells are to be cryopreserved in a cell culture plate, they may be cryopreserved according to the following protocol, for example: (1) grow the cells to confluency or near confluency in a culture medium in the culture plate; (2) wash the cells one or more times with a wash buffer, such as a PBS solution, for example; (3) incubate the cells in trypsin to detach the cells from the culture plate; (4) resuspend the cells in each well of the culture plate by mechanical flushing with a pipette while in a freeze medium (such as, for example, a freeze medium including DMSO, such as 10% DMSO, for example, as the cryoprotectant); (5) cover the cells resuspended in the freeze medium with paraffin oil; and (6) slowly freeze the cells in the plate to -80° C at a rate of about -1° C/minute.
  • This cryopreservation protocol may be used to preserve cells transfected through the method of the present invention.
  • Cryopreserved cells may be reestablished in culture by using any one or more of a variety of methods that are well known in the art. For example, when mammalian cells are cryopreserved in a culture plate as described above, they may be reestablished in culture by first completely thawing the frozen culture plate by incubating the culture plate at a temperature that is suitable for culturing mammalian cells, such as about 37° C, for example. Following such incubation, the completely thawed mammalian cells may then be mechanically dislodged from the culture plate and from each other, such as by repeatedly pipetting the contents of each well, for example, before being added to a maintenance media. The completely thawed mammalian cells may be grown on feeder cells attached to a solid support. This cell reestablishment protocol may be used to preserve cells transfected through the method of the present invention.
  • transfected cells that are transfected by using the present invention may be used for a variety of clinical and research purposes.
  • the transfection method therefore may be carried out to generate transfected cells that may be used as part of high throughput screening assays, such as ones made for the purpose of rapidly identifying proteins that are involved in a particular biochemical pathway, for example.
  • the method also may be carried out for the purpose of generating transfected cells that are useful for imaging assays, such as ones designed to gather temporal and spatial evidence of a protein's role within a cell, for example.
  • the method may be carried out for the purpose of generating transfected cells that are useful for being used as part of diagnostic assays, such as ones made for the purpose of determining whether a particular protein is present or absent in a population of cells, for example.
  • the transfected cells generated by using the present invention may be used to treat a patient, such as a human patient, for example. This treatment may be made by introducing the cells into the patient as part of a gene therapy application, for example. It is to be understood that these are only meant to serve as examples of the applications that the method of the present invention may be used to carry out, and therefore that the method is not limited to being carried out for these purposes. *
  • the optional kit of the present invention includes one or more reagents and instructions for carrying out the present method.
  • the kit may include one or more of the solution in which the cells are exposed, irradiated, and or otherwise maintained and grown, the DMSO, the QDs, one or more polypeptides, and/or any other transfection agent.
  • the kit further includes instructions that describe how to carry out the present method.
  • the instructions may be directly associated with the kit, such as being supplied with the one or more reagents in paper copy, for example, or they may be indirectly associated with the kit, such as being made available in electronic copy on the internet or published in a journal or trade magazine, for example.
  • the kit may be used as part of any one or more of the variety of clinical and research applications for which the transfected cells may be used.
  • the optional kit may include instructions and/or one or more reagents for carrying out the high throughput screening assays, imaging assays, diagnostic assays described above. It is to be understood that these are only meant to serve as examples of the purposes that the optional kit of the present invention may be used to help achieve, and therefore that the optional kit is not limited to being designed to help achieve only these purposes.
  • the QDs had an emission peak of about 514 nm and the QDs and solution were prepared as described in S. J. Clarke et al., Nature Materials 5:347-8 (2006), which references both J. A. Kloepfer et al., Journal of Physical Chemistry 109:9996-10003 (2005); J. A. Kloepfer et al., Applied and Environmental Microbiology 69:4205-4213 (2003); and Y. Chen et al., Nano Lett. 3:581-584 (2003)). Prior to their addition to the culture dish, the optical density of the QD solution was measured by spectrophotometry to be 0.036.
  • DMSO was then mixed into the DMEM in the dish by swirling (the final concentration of the DMSO was 10%).
  • the dish was then irradiated at a wavelength of 488 nm by using the laser supplied with the Fluoview300 confocal microscope by Olympus for about 1-2 minutes. Following this period, the supernatant (including the DMEM, QDs and DMSO) was aspirated from the cells and replaced with about 2 ml of fresh media (10% FBS in DMEM).
  • EXAMPLE TWO It was a goal of the experiment described in Example Two to determine whether cells may be co-transfected with a protein and QDs according to the process described in Example Two.
  • a plurality of separate populations of C2C12 cells were adhered to the glass bottoms of 35 mm culture dishes, and each population was exposed to about 2 ml of DMEM having 10% FBS.
  • One of these populations of cells (hereinafter, "the QD only population") was then added to the open perfusion micro-incubator equipped to the confocal microscope (Fluoview 300) described in Example One, and was kept heated at 37 0 C.
  • the QD only population was then added to the open perfusion micro-incubator equipped to the confocal microscope (Fluoview 300) described in Example One, and was kept heated at 37 0 C.
  • Approximately 20 ⁇ l of QDs in solution (the QDs and the solution were as prepared as described in Example One herein), and approximately 10 ⁇ l of a 1 mg/ml solution of anti-Histone 1 antibody (obtained from Santa Cruz Biotechnology, Inc.
  • the QD only population was then irradiated at a wavelength of 488 nm by using the laser supplied with the Fluoview300 confocal microscope by Olympus for about 1-2 minutes. Following this period, the supernatant (including DMEM and the QDs) was aspirated from these cells and replaced with about 2 ml of fresh media ( 10% FBS in DMEM).
  • the DMSO only population was added to the same open perfusion micro-incubator equipped to the confocal microscope, and was kept heated at 37 0 C.
  • DMSO was mixed into the DMEM by swirling (the final concentration of the DMSO was 10%).
  • Approximately 10 ⁇ l of a 1 mg/ml solution of anti-Histone 1 antibody was then mixed into the DMEM by swirling.
  • the DMSO only population was then irradiated at a wavelength of 488 nm by using the laser supplied with the Fluoview 300 confocal microscope by Olympus for about 1-2 minutes. Following this period, the supernatant (including DMEM and DMSO) was aspirated from the DMSO only population and replaced with about 2 ml of fresh media (10% FBS in DMEM).
  • the QD and DMSO population A third of these populations of C2C12 cells (hereinafter, "the QD and DMSO population”) was then added to the same open perfusion micro-incubator equipped to the confocal microscope, and was kept heated at 37 0 C.
  • DMSO was mixed into the DMEM by swirling (the final concentration of the DMSO was 10%).
  • the QDs and the solution were as prepared as described in Example One herein), and approximately 10 ⁇ l of a 1 mg/ml solution of anti-Histone 1 antibody was then mixed into the QD and DMSO population.
  • the QD and DMSO population of cells was then irradiated at a wavelength of 488 nm by using the laser supplied with the Fluoview300 confocal microscope by Olympus for about 1-2 minutes. Following this period, the supernatant (including DMEM, QDs and DMSO) was aspirated from the QD and DMSO population and replaced with about 2 ml of fresh media (10% FBS in DMEM).
  • FIGS. 3A and 3B at one day after transfection, there was no evidence that either the QD only population (FIG. 3A) or the DMSO only population (FIG. 3B) of cells had been transfected by the anti-Histone 1 antibody.
  • FIG. 3C the anti-Histone 1 antibody had localized within the nucleus of cells belonging to the QD and DMSO population one day after transfection. (The localized antibody appears as dark spots, as indicated by the arrows.)
  • C2C12 mouse myoblasts were grown to about 70% confluency on the glass bottom of a culture dish in DMEM having 10% FBS.
  • the dish was added to the open perfusion micro-incubator equipped to the confocal microscope (Fluoview 300) described in Example One, and was kept heated at 37°C.
  • the cells were exposed to 100% laser power at 488 nm and at 525 nm.
  • FIG. 4 Panel A shows differential interference contrast (Nomarski) images of the cells at various time points prior to and after the addition of the DMSO.
  • FIG. 4 Panel B shows uptake of the streptavidin-TRITC antibody (bright spots), and
  • FIG. 4 Panel C shows uptake of the QDs (bright spots), by the same cells shown in Panel A.
  • streptavidin-TRITC antibody was present in the nuclei of some cells 60 seconds after the addition of the DMSO.
  • QDs were present in the nuclei of some cells 60 seconds after the addition of the DMSO.
  • C2C12 mouse myoblasts were grown to about 70% confluency on the glass bottom of a culture dish in DMEM having 10% FBS.
  • the dish was added to the open perfusion micro-incubator equipped to the confocal microscope (Fluoview 300) described in Example One, and was kept heated at 37°C.
  • Approximately 10 ⁇ l of a 1 mg/ml solution of anti-Histone 1 antibody (obtained from Santa Cruz Biotechnology, Inc.) and approximately 10 ⁇ l of a 0.1 ⁇ M solution of QDs (the QDs and the solution were as prepared as described in Example One herein) were then mixed into the dish.
  • the cells were exposed to 100% laser power at 488 nm and at 525 nm. While the cells were being exposed to the laser treatment, uptake of the anti-Histone 1 antibody and the QDs was initiated by mixing DMSO to a concentration of 0.1% into the dish containing the laser-treated cells.
  • FIG. 5A shows an overlay of the images shown in FIG. 5A and FIG. 5B.
  • the side and bottom panels (indicated by asterisk) in FIGS. 5A-5C show a z-line slice through a transfected cell.
  • FIG. 5D shows differential interference contrast (Nomarski) images of the localization of the anti- Histone 1 antibody within the transfected cells. (The localized anti-Histone 1 antibody appears as dark spots. One nuclear region exhibiting particularly concentrated anti- Histone 1 antibody is depicted by the arrow.)
  • total protein was obtained from each one of the transfected cell populations that had grown for one or two days (and from the control cells at each one of these time points) by using the BCA Protein Assay Kit obtained from Pierce Biotechnology, Inc. (Rockford, Illinois; http://www.piercenet.com) according to that manufacturer's protocol.

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Abstract

La présente invention concerne un procédé et une trousse permettant l'administration d'un ou de plusieurs agents de transfection dans une cellule, y compris le noyau de cette cellule. Le procédé consiste à exposer la cellule à un ou plusieurs agents de transfection, qui peuvent être des points quantiques ou des polypeptides, par exemple, du diméthylsulfoxyde et un rayonnement électromagnétique. La trousse contient un ou plusieurs réactifs et des instructions permettant la mise en œuvre du procédé.
PCT/US2007/024681 2006-12-01 2007-11-30 Procédé et trousse pour administration intracellulaire WO2008069992A2 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020086842A1 (en) * 2000-06-26 2002-07-04 Christian Plank Method for transfecting cells using a magnetic field
US20020138864A1 (en) * 1998-05-22 2002-09-26 Jae Yong Han Method for transfection of avian primordial germ cells
US20050095578A1 (en) * 2003-10-31 2005-05-05 Koller Manfred R. Method and apparatus for cell permeabilization

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020138864A1 (en) * 1998-05-22 2002-09-26 Jae Yong Han Method for transfection of avian primordial germ cells
US20020086842A1 (en) * 2000-06-26 2002-07-04 Christian Plank Method for transfecting cells using a magnetic field
US20050095578A1 (en) * 2003-10-31 2005-05-05 Koller Manfred R. Method and apparatus for cell permeabilization

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