WO2002100269A1

WO2002100269A1 - Compositions and methods for magnetically labeling cells

Info

Publication number: WO2002100269A1
Application number: PCT/US2002/015597
Authority: WO
Inventors: Joseph A. Frank; Jeff W. M. Bulte
Original assignee: The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health & Human Services
Priority date: 2001-06-13
Filing date: 2002-05-17
Publication date: 2002-12-19

Abstract

A composition that includes magnetic-responsive coated metal oxide particles mixed with at least one transfection agent. According to one particular embodiment, the mixing is done in the presence of a cell culture medium. There is also provided a method of labeling living cells to render the cells magnetic resonance sensitive that involves contacting the cells to be labeled with a mixture of (i) magnetic-responsive coated metal oxide particles and (ii) at least one transfection agent, and allowing the mixture to be internalized by the cells.

Description

COMPOSITIONS AND METHODS FOR MAGNETICALLY LABELING CELLS

Co-pending, commonly-assigned PCT Application No. US00/13826 "Magnetic

Resonance Tracking of Magnetically Labeled Cells" filed May 19, 2000 (published November 30, 2000) discloses related subject matter.

Field of the Disclosure

The present disclosure relates to compositions useful as magnetic probes, particularly for magnetic resonance imaging (MRI) of cells.

Background

MRI is a technique that allows whole body in vivo imaging in three dimensions at near-cellular (microscopic) resolution. In MRI a static magnetic field is applied to the object of interest while simultaneously applying pulses of radio frequency-induced magnetization to change the distributions of the magnetic moments of protons in the object. The change in distribution of the magnetic moments of protons in the object from random to directed and their return to normal (random) constitute the MRI signal. Magnetic resonance (MR) contrast agents assist this return to normal by shortening Ti and/or T₂ relaxation times.

In particular, MR image contrast is largely determined by the nuclear magnetic relaxation times of tissues. The longitudinal relaxation time Tl is defined as the time constant of the exponential recovery of proton spins to their equilibrium distribution along an applied field after a disturbance. The transverse relaxation time T2 is the time constant that describes the exponential loss of magnetization in a plane transverse to the direction of the applied field, following a RF pulse that rotates the aligned magnetization into the transverse plane. MR contrast agents shorten the Tl and T2 relaxation times and their net effectiveness is expressed as relaxivity (R), which represents the reciprocal of the relaxation time per unit concentration of metal, with units of mM^'V¹.

Dextran -coated iron oxide particles are known MR contrast agents. However, such particles by themselves cannot be used to label transplanted living cells since they have no affinity for cellular membranes.

Dextran-coated iron oxide particles are also known to be useful for synthesizing transfection agents. For example, U.S. Patent No. 5,753,477 (Chan) discloses, in general, microparticles coated with biodegradable biopolymers or lipids. Dextran- coated superparamagnetic iron oxide particles are mentioned as possible microparticles (column 5, lines 21-23). According to Chan at column 6, lines 13-15, the dextran coating can be chemically modified or conjugated with other proteins or polyamines such as poly-L-lysine, polyarginine or other biopolymers with positive charges. The coated microparticles are themselves used as transfection agents for intracellular delivery of another bound substance (e.g., nucleic acids) via application of a pulsed magnetic field.

Many therapeutic strategies, such as stem cell transplantation, are based upon introducing exogenous living cells or tissues into a patient or host. A problem common to all therapeutic strategies involving administration of exogenous cells is identifying and monitoring the cells in the host. It is currently difficult or impossible to monitor the location of such cells or tissues in the host after administration. It may also be difficult to establish the survival of these cells in the host. Currently available procedures to locate transplanted cells are invasive and destructive. This problem must be overcome before such cell therapies can achieve their full potential. For example, one class of cells that has been receiving substantial attention are human embryonic stem (ES) cells since they can now be isolated and propagated indefinitely in culture. They can be differentiated into virtually any cell type and have great therapeutic potential to replace or substitute defunct endogenous cell populations. In order to determine the history and fate of transplanted cells, including their migration in vivo, cells are currently labeled ex vivo using a vital dye (e.g. a fluorochrome), a thymidine analogue (e.g. BRDU), or a transfected gene (e.g. LacZ or GFP), which can be visualized using (immuno)histochemical procedures following tissue removal at a particular given time point.

Cellular therapy and diagnostics in humans would be advanced by a technique that can monitor cell fate non-invasively and repeatedly, in order to take a momentary "snapshot" assessment of the cellular biodistribution at a particular given time point. The introduction of such a technique would be facilitated if any novel agents used in the technique could be easily prepared.

Summary of the Disclosure

This disclosure provides a composition that includes magnetic -responsive coated metal oxide particles mixed with at least one transfection agent. Illustrative magnetic- responsive coated metal oxide particles include those that may be utilized by themselves as MR contrast agents such as superparamagnetic dextran-coated iron oxide particles. Although not bound by any theory, it is believed that the transfection agent is electrostatically attracted to the magnetic-responsive coated metal oxide particles.

Both the magnetic-responsive metal oxide particles and the transfection agents of certain embodiments are individually commercially available. A mixture of commercially available magnetic-responsive coated metal oxide particles and transfection agents eliminates the need for any synthesis of specialized coated metal oxide particles.

There is also disclosed a method for making such compositions that involves simply mixing the magnetic-responsive coated metal oxide particles with the transfection agent. According to one particular embodiment, the mixing is done in the presence of a cell culture medium.

The disclosed compositions are useful as a new class of magnetic probes. For example, there is provided a method of labeling cells to render the cells magnetic resonance sensitive that involves contacting the cells to be labeled with a mixture of (i) magnetic-responsive coated metal oxide particles and (ii) at least one transfection agent; and allowing the mixture to be internalized by the cells. Although not bound by any theory, it is believed that the transfection agent located at or near the surface of the magnetic-responsive coated metal oxide particles facilitates the rapid incorporation of the magnetic-responsive coated metal oxide particles into the cell via endocytosis and/or diffusion through the cell membrane. This magnetic labeling renders the cells identifiable and distinguishable by MRI. In particular, the composition can serve as an MR contrast agent useful in the imaging of tissues, organs, cells, antigens, tumors and the blood pool by labeling and detecting living cells in the object of interest. A wide variety of cells, including human neural cells, can be very efficiently magnetically labeled while retaining their normal capability for differentiation.

A further aspect of labeling with the coated metal oxide particle/transfection agent composition involves detecting living cells in a host by contacting the coated metal oxide particle/transfection agent composition with the cells, allowing the coated metal oxide particle/transfection agent composition to be internalized by the cells, and imaging the host with magnetic resonance imaging so as to detect the cells. The disclosed methods provide a universal (e.g., mouse, rat, human), non-specific method of magnetically labeling cells.

Also disclosed is a diagnostic kit for labeling cells to render the cells magnetic resonance sensitive that includes the magnetic-responsive coated metal oxide particles and the transfection agent, and instructions for labeling cells using a mixture of the magnetic-responsive coated metal oxide particles and the transfection agent.

The foregoing features and advantages will become more apparent from the following detailed description of several embodiments that proceeds with reference to the accompanying figures.

Brief Description of the Drawings

Certain embodiments are described below with reference to the following figures: FIGS. 1A-E are digital micrograph images of Prussian Blue staining of human cervix carcinoma (HeLa) cells, wherein (A) are cells mixed only with Feridex® iron oxide particles, (B) are cells labeled with a Feridex® iron oxide particles/cationic liposome transfection agent mixture in accordance with a disclosed embodiment, (C) are cells labeled with a Feridex® iron oxide particles/poly-L-lysine mixture in accordance with a disclosed embodiment, (D) are cells mixed only with MION-46 L iron oxide particles, and (E) are cells labeled with a MION-46 L iron oxide particles/poly-L-lysine mixture in accordance with a disclosed embodiment;

FIGS. 2A-2C are graphs depicting the relaxometry (1/T2) results of unlabeled cells (identified as "control") compared to cells labeled with a Feridex® iron oxide particles/cationic liposome transfection agent mixture, Feridex® iron oxide particles/poly-L-lysine transfection agent mixture or Feridex® iron oxide particles/Superfect® dendrimer transfection agent mixture in accordance with disclosed embodiments, wherein (A) are HeLa cells, (B) are rat oligodendrocyte progenitor (CG- 4) cells and (C) are proteolipid protein stimulated mouse T lymphocytes (ML); and

FIG. 3 are digitized MR images of cells obtained at 1.5 Tesla, using a gradient echo sequence with TR=300, TE=20, and flip=20°, wherein (1) are unlabeled ML cells (2e7 cells/ml), (2) are ML cells labeled with a Feridex® iron oxide particles/Superfect® dendrimer transfection agent mixture in accordance with a disclosed embodiment (2e7 cells/ml), (3) are same as (2) except le7 cells/ml, (4) are same as (2) except 5e6 cells/ml, (5) are the same as (2) except 2.5e6 cells/ml, (6) are unlabeled CG-4 cells (2e6 cells/ml), (7) are CG-4 cells labeled with a Feridex® iron oxide particles/poly-L-lysine transfection agent mixture in accordance with a disclosed embodiment (2e6 cells/ml),

(8) are CG-4 cells labeled with a Feridex® iron oxide particles/cationic liposome transfection agent mixture in accordance with a disclosed embodiment (2e6 cells/ml),

(9) are unlabeled HeLa cells (2e7 cells/ml), (10) are HeLa cells labeled with a Feridex® iron oxide particles/poly-L-lysine transfection agent mixture in accordance with a disclosed embodiment (2e7 cells/ml), (11) are HeLa cells labeled with a Feridex® iron oxide particles/cationic liposome transfection agent mixture in accordance with a disclosed embodiment (2e7 cells/ml), (12) are unlabeled HeLa cells (2e6 cells/ml), (13) are HeLa cells labeled with a Feridex® iron oxide particles/poly-L-lysine transfection agent mixture in accordance with a disclosed embodiment (2e6 cells/ml), and (14) are HeLa cells labeled with a Feridex® iron oxide particles/cationic liposome transfection agent mixture in accordance with a disclosed embodiment (2e6 cells/ml).

Detailed Description of Several Embodiments

The following definitions are provided for ease of understanding and to guide those of ordinary skill in the art in the practice of the embodiments. "Endocytic transfection agent" encompasses any transfection agent that delivers a foreign molecule (such as DNA) or other material into a cell via endocytosis and/or diffusion through the cell membrane. Endocytic transfection agents are distinguished from biolistic transfection agents since biolistic transfection agents are projected into a cell via a ballistic device as opposed to endocytosis and/or diffusion. The magnetic-responsive coated metal oxide particles include a metal oxide particle and a coating material that is in contact with the surface of the metal oxide particle. The metal oxide particles typically are coated with the coating material prior to mixing with the transfection agent (i.e., pre-coated metal oxide particles are mixed with the transfection agent). The metal of the metal oxide particle may include transition or lanthanide metals. Illustrative transition or lanthanide metals include iron, cobalt, gadolinium, europium and manganese. The magnetic-responsive metal oxide particles may be paramagnetic, ferrimagnetic, superparamagnetic or anti-ferromagnetic.

The coating material may be in contact with the metal oxide particle surface via any type of chemical bonding and/or physical attractive force such as, for example, covalent bonding, ionic bonding, hydrogen bonding, colloidal mixtures or complexing. Illustrative coating materials include polysaccharides, polyvinyl alcohols, polyacrylates, polystyrenes, and mixtures and copolymers thereof. According to a particular embodiment the coating material is a polysaccharide such as, for example, starch, cellulose, glycogen, dextran, and derivatives thereof. According to a particular embodiment the metal oxide is an iron oxide, especially a superparamagnetic iron oxide. Superparamagnetic iron oxides are (on a millimolar metal basis) the most MR-sensitive tracers currently available. Superparamagnetic particles possess a large ferrimagnetic moment that, because of the small crystal size, is free to align with an applied magnetic field (i.e., there is no hysteresis). The aligned magnetization then creates microscopic field gradients that dephase nearby protons and shorten the T2 NMR relaxation time, over and beyond the usual dipole-dipole relaxation mechanism that affects both Tl and T2 relaxation times. Examples of superparamagnetic iron oxides include MION-46L (available from Harvard Medical School), Feridex® (commercially available from Berlex Laboratories, Inc. under license from Advanced Magnetic, Inc), Endorem® ferumoxides (commercially available from Guerbet Group), Clariscan® (commercially available from Nycomed Amersham), Resovist® (commercially available from Schering AG), Combidex® (commercially available from Advanced Magnetics), and Sinerem® (commercially available from Guerbet Group under license from Advanced Magnetics). MION-46L is a dextran-coated nanoparticle with a superparamagnetic maghemite- or magnetite-like inverse spinel core structure. The core structure has a diameter of about 4.6±1.2 nm in diameter and the overall particle size (including the dextran coating) is about 8 to about 20 nm. Feridex® is a FDA-approved aqueous colloid of superparamagnetic iron oxide associated with dextran for intravenous administration. Resovist® consists of superparamagnetic iron oxide particles coated with carboxydextran.

Transfection agents are known and typically are used as carriers for introducing DNA into a cell. The transfection agent may have sufficient molecular size so that it includes a plurality of binding sites for the cell membrane. Although the molecular size for specific transfection agents will vary, most transfection agents can have a molecular weight of at least about 1 kDa, particularly at least about 5 kDa, and more particularly at least about 10 kDa. Illustrative transfection agents include cationic polyaminoacids (e.g., polyallylalanines, poly-L-alanines, poly-L-arginines, poly-L-lysines, and copolymers thereof), spermidines, salmon sperm DNA, poly-L-ornithines, diethylaminoethyl-dextrans, cationic liposomes or lipids, non-liposomal lipids, dendrimers, polynucleotides, and mixtures thereof. Examples of dendrimer transfection agents include those dendrimers having a relatively high electrostatic charge due to (activated) amino and/or carboxyl terminal groups on the outside perimeter of the dendrimer molecule. Such dendrimers can be activated, for instance, by heating up to about 60°C to selectively remove a portion of the peripheral tertiary amine terminal groups. PolyFect® transfection reagent and SuperFect® transfection reagent are examples of commercially available activated dendrimers (available from Qiagen GmbH, Hilden, Germany). A commercially available example of a cationic liposome formulation is LipofectAMTNE PLUS reagent from Life Technologies, Inc. A commercially available example of a non-liposomal lipid is Effectene™ transfection reagent from Qiagen GmbH, Hilden, Germany. According to particular embodiments, the transfection agent is a non-viral transfection agent. When the magnetic-responsive coated metal oxide particles are mixed with the transfection agent, it is believed that the transfection agent is associated with the surface of the magnetic-responsive coated metal oxide particles via electrostatic attraction. Thus, according to one embodiment, the transfection agent does not chemically bond to, or modify, the coating material on the surface of the magnetic-responsive metal oxide particles. The transfection agent has a net negative or positive electrostatic charge and the magnetic-responsive coated metal oxide particles has an oppositely corresponding net negative or positive electrostatic charge. The differential between the net electrical charges should cause a sufficient physical attraction so that the magnetic-responsive metal oxide particles and the transfection agent molecules are in close proximity to each other to form "clusters" that are distributed substantially uniformly throughout the mixture.

According to a particular embodiment, the disclosed compositions do not include any therapeutic, diagnostic or bioactive agents other than the magnetic- responsive coated metal oxide particles or transfection agents. Such compositions are particularly effective as magnetic probes as detailed below. Alternatively, bioactive agents such as nucleic acids (e.g., DNA), receptors or antigens can be bound to the coated metal oxide particles/transfection agent mixture. The inclusion of such bioactive agents could provide monitoring of DNA delivery into specific cells and tissues. As described above, the coated metal oxide particle/transfection agent compositions can be used as magnetic probes. The magnetic probes can achieve a high degree of intracellular magnetic labeling that is non-specific (i.e., not dependent on targeted membrane receptor binding) and that can be used on virtually any mammalian cell. The magnetic probe could be used in cell tagging, as an MR contrast agent, cancer therapy hyperthermia, cellular therapy, magnetic guidance of cells, ultrasound imaging, microwave radiation and nuclear isotope imaging using ⁵⁹Fe preparations. A particularly useful application is as an MR contrast agent that enables non-invasive and repeated identification and monitoring of cells with a high degree of precision.

When the coated metal oxide particle/transfection agent mixture is utilized for intracellular labeling, the coated metal oxide particles and transfection agent may be mixed together in the presence of a cell culture medium. The specific cell culture medium is selected based on the appropriate medium for the desired cell line for labeling. Selection of appropriate cell culture media is well within the skill of the art. The pH of the coated metal oxide particle/transfection agent/cell culture buffered media may be about 6.5 to about 7.5. The mixing typically may be done at ambient room temperature and atmosphere.

One benefit of the disclosed composition is that commercially available magnetic- responsive coated metal oxide particles and transfection agents may be mixed together as received without any pre-treatment or additional ingredients (other than the cell culture medium). For example, further chemical modification of the coating on the coated metal oxide particles is not required. Furthermore, the mixing may be accomplished without the presence of an organic solvent. The amount of coated metal oxide particles mixed with the transfection agents should be sufficient to provide uptake of the coated metal oxide particles by the cell and may vary widely depending upon the particular transfection agent. One particular embodiment includes labeling living cells with the coated metal oxide particle/transfection agent mixture to render the cells MR sensitive. Such magnetically labeled cells may be prepared by simple incubation of cells with the coated metal oxide particle/transfection agent mixture in cell cultures. Suitable cell incubation techniques are well known. For example, a cell of interest can be cultured in a standard media that includes the iron oxide/transfection agent mixture at a dose ranging from about 5 to about 100 μg Fe/ml, more particularly about 5 to about 25 μg Fe/ml. Alternatively, the coated metal oxide particle/transfection agent mixture can be injected into tumors and other areas to label cells in situ or by injecting into blood vessels, ventricles or other brain or body cavities. The coated metal oxide particle/transfection agent mixture may be internalized by a cell via endocytosis and/or diffusion with subsequent entrance into the cytoplasm and localization in endosomes.

The magnetically labeled cells may be exogenously applied to a host and monitored within the host using MRI. For example, such cells may be injected or otherwise applied to the host.

Another option is to label cells in the host in situ so as to allow labeling of structures within the host. This would allow monitoring of labeled structures and cells. For example, tumors could be labeled to monitor effectiveness of treatment and follow metastasis. The cells can be labeled in situ for therapeutic, diagnostic, or experimental purposes. Another embodiment encompasses infusing the magnetic probes into such areas as tumors, so that the growth, metastasis, or regression of the tumor can be monitored. Such a procedure could be part of a treatment protocol to monitor disease progress.

The living cells for labeling and detection in accordance with the disclosure are those that are of therapeutic, diagnostic, or experimental value when introduced into a patient or host. The term "cell" is understood to mean embryonic, fetal, pediatric, or adult cells or tissues, including but not limited to, stem cells, precursors cells, and progenitor cells. It is also understood that the term "cells" encompasses virus particles and bacteria. The term "host' can mean any mammalian patient or experimental subject, including human patients or subjects. The living cells of the current invention can be bone marrow cells, hematopoietic cells, tumor cells, lymphocytes, leukocytes, granulocytes, hepatocytes, monocytes, macrophages, fibroblasts, neural cells, mesenchymal stem cells, neural stem cells, and combinations thereof. It is understood that the term "neural cells" includes neurons and neuroglia. The term "neuron" encompasses central and peripheral neurons, central nervous system neurons, and neuropithelial cells. The term "neuroglia" encompasses oligodendrocytes, astrocytes, ependymal cells, microglia, stellate cells, Schwann cells, and neurilemma. Preferably the cells are neural stem cells or oligodendrocyte progenitors. Oligodendrocytes are glial cells of the central nervous system, which form sheaths around nerve fibers and capsules around nerve cell bodies.

The cells can be applied to the host to cure or diagnose a disease or to supply cell type that is lacking or deficient in the host. The cells can also provide a drug or substance that is needed in the host for diagnostic, therapeutic, or experimental purposes. Cells can be immune cells to specific proteins in the host's body, such as proteins found in malignant tissue or molecules associated with a disease state such as bacterial or veal proteins or glycoproteins.

In one embodiment the cells can be stem cells. Stem cells are cells that retain their ability to divide and to differentiate into specialized mature cells. Preferably the cells are multipotent cells from the nervous which retain their ability to differentiate into oligodendrocytes. Oligodendrocytes are cells that myelinate or provide protective sheaths to neurons and axons in the central nervous system.

In another aspect the cells are carcinoma cells. Such cells are neoplastic and divide indefinitely. Preferred tumor cells is small cell carcinoma cells. Labeling such cells in vitro is an experimental tool to study how such cells behave in experimental conditions. Cancer cells can also be labeled in vivo to allow clinical investigators to track possible metastases.

In a further aspect, the method includes applying such cells, which are labeled with the MR contrast agent, for any therapeutic, diagnostic, or experimental purposes. The cells can be dispersed, or can be part of a tissue or organ or labeled cells can be applied to any tissue or organ after the cells are labeled. The cells can be directly applied to the area to be treated or studied by means of surgery or injection into the circulation or injection into a structure, organ, or body cavity in situ. When cells are integrated ex vivo into a tissue or organ, such tissue or organ can then be surgically applied or transplanted into a host. Preferably, the cells are applied directly to a body structure. Most preferably the cells are applied to the central nervous system. A further embodiment involves using MRI to monitor the movement, disposition and survival of the cells in the host. When cells are used that are immune cells, which react with a component of a disease process in the host, MRI monitoring can be used diagnostically to locate the cells attached to the disease process in the host. Immune cells are understood to encompass lymphoid or myeloid hematopoietic cells. Examples of such disease processes are malignant and metastatic diseases, degenerative diseases and infectious diseases. The cells can be used experimentally, in animal hosts, to study the development of disease as a basis for designing therapeutic strategies. Tumor or neoplastic cells can be applied to animals as an experimental technique to study the behavior of neoplastic growth and metastasis in the organism.

Cells can be used to replace injured or diseased cells in the host, examples are diseases of the nervous system, injuries to the nervous system, injuries or diseases of bone, muscle, heart, circulation, internal organs, skin, interstitial tissue, mucosa, lungs, and gastrointestinal tract. When cells are used in this way, the host can be scanned with MRI to establish the location of the cells, the movement or migration if any of the cells, and the survival of the cells. The host can be scanned with MRI as frequently and during as long a period of time as required or desirable to monitor the cells. Cells that are loaded with therapeutic vectors can be monitored as necessary to establish their migration if any in the host and to establish their continued survival and ability to produce therapeutic proteins or drugs in the host.

A diagnostic kit can be provided for enabling a user to render cells magnetic resonance sensitive. One kit variant could include a first container for a magnetic- responsive metal oxide particles reagent, a second container for a transfection agent reagent, and instructions for labeling the cells. The reagents typically would be provided in the form of commercially available standard stock solutions. Another kit variant could include a first container that holds a mixture of the metal oxide particles and the transfection agent, provided the mixture is sufficiently stable. The kit instructions would include mixing directions, the dilutions required for various cell types, and incubation times. Optional kit components could include tube holders, magnet(s) (such as a neodymium iron boron magnetic disk (1 inch diameter by 0.25 inches thick, lifts 15 lbs.) available from Edmund Scientific as catalogue number #c30351-07), graduated sterile tubes, and syringe(s). Each component of the kit could be packaged together or separately. The amount of the various kit components would vary depending upon how many tests could be practically performed with the kit.

An illustrative example of the kit could include 1 cc of Feridex® iron oxide particles at 11.2mg/ml and lg of poly-L-lysine powder. The user could mix the poly - L-lysine powder with serum free culture media to a concentration of 1.5 mg/ml to provide a stock solution. An alternative illustrative kit could include a container holding 1 cc of Feridex® iron oxide particles pre-mixed with 1 gram of poly-L-lysine in 4 cc of water. The user could dilute this stock mixture to lOOcc in serum free culture media prior to cell labeling. The kits can be used as described below.

First, obtain an equal volume of serum free media and an equal volume of media with cells. According to particular embodiments, the initial cell density could be greater than 106/ml in media incubated in wells or flasks. The Feridex® iron oxide particles at a 50 μg/ml dose are mixed with the serum free media. The poly-L-lysine (from stock solution of 1.5 mg/ml) then is added to the Feridex®-containing media at 1:500 ratio of poly-L-lysine to media. The media + Feridex® + poly-L-lysine is mixed by a rotator for 30 minutes. Any large particles of Feridex®/poly-L-lysine complex remaining after 30 minutes may be fractionated by repeated pipetting. An equivolume of the Feridex®/poly-L-lysine mixture is added to the cells to achieve a final concentration of Feridex® at 25 μg/ml and poly-L-lysine at 1:1000. The resulting mixture is incubated for 4 hours to 12 hours. The cells then are collected and washed 2- 4 times with sterile phosphate-buffered saline. The cells are re-suspended in complete media in a 15 ml tube and the tube is placed in a magnetic rack for 2-3 minutes (not more than 3 minutes). The total cells are collected by a pipette without agitating the attracted debris along the wall of the tube near magnet. The cells then are centrifuged. At this point, a volume of the cells may be made available for Prussian blue staining to determine the extent of magnetic labeling. A volume of the cells also may be administered to a test subject or patient for MRI monitoring within the subject or patient.

The specific examples described below are for illustrative purposes and should not be considered as limiting the scope of this invention.

Examples

Dendrimer transfection agents (PolyFect® and SuperFect®), a cationic liposome transfection agent (LipofectAMINE PLUS), a non-liposomal lipid transfection agent (Effectene™), and a poly-L-lysine transfection agent were each mixed with dextran- coated superparamagnetic iron oxide particles (MION-46L or Feridex®) and a cell culture media suitable for the cells identified below. Sufficient iron oxide particles were added to provide a concentration of 25 μg Fe/ml cell culture medium and sufficient transfection agent was added to provide a concentration of 1 ml transfection agent/250 ml cell culture medium (diluted from stock transfection agent). The resulting mixture was allowed to incubate for 60 minutes to allow for full development of the iron oxide particles/transfection agent "clusters". Proteolipid protein stimulated mouse T lymphocytes, human lymphocytes, rat oligodendrocyte progenitor (CG-4) cells, and human cervix carcinoma (HeLa) cells were incubated in the resulting mixtures for 48 hours. As a comparison, cell culture media were prepared with MION-46L and Feridex® iron oxide particles at 25 μg Fe/ml, but with no transfection agent. The same cell lines identified above were incubated for 48 hours in the non-transfection agent media. Magnetic cellular labeling was evaluated using Prussian Blue staining for iron, T2 relaxometry (1.0 Tesla, CPMG, TE=2 and 10 milliseconds, 42 MHz resonance frequency, V2 echo times of tau=l and tau=5), and MR imaging (1.5 Tesla) of the resulting cell suspensions.

The cells that were incubated in the media that only contained the iron oxide particles (no transfection agent) displayed low cellular labeling for Feridex® iron oxide particles and undetectable cellular labeling for MION-46L iron oxide particles when observed with Prussian Blue staining (see FIGS. 1A and ID). Cells incubated in the media that contained both the iron oxide particles and the transfection agent, however, displayed numerous iron-containing intracytoplasmic vesicles (for both MION-46L and Feridex®) when observed with Prussian Blue staining (see the dark particles in FIGS. IB, IC and IE) and MRI (see the significantly darker images in FIGS. (3)(2) - (3)(5), (3)(7), (3)(8), (3)(10), (3)(11), (3)(13) and (3)(14)). Following labeling with the iron oxide/transfection agent mixtures, the cells were unaffected in their viability and proliferation rate. Compared to unlabeled control cell suspensions, there was a dramatic increase in the 1/T2 values of labeled cells as shown in FIGS. 2A-2C indicating significant uptake of the iron oxide particles.

In another example, human mesenchymal (MSC) stem cells (obtained from BioWhittaker, Walkersville, MD) were co-cultured with Feridex® particles/transfection agent mixtures or with Feridex® particles alone (no transfection agent). The transfection agents were SuperFect®, LipofectAMINE PLUS, and poly-L-lysine. Each of the transfection agents was individually mixed with Feridex® particles for 60 minutes in a cell culture medium at room temperature. For LipofectAMINE PLUS, the lipofectamine was added to the media only for the final 15 minutes. Dilutions of 1:1250 of the transfection agents from stock solutions as supplied by the manufacturer were mixed with the Feridex® particles. Sufficient Feridex® particles were added to provide a concentration of 25 μg Fe/ml cell culture medium. The MSC cells were incubated in the resulting mixtures for 2 hours. As a comparison, cell culture media were prepared with Feridex® particles at 25 μg Fe/ml, but with no transfection agent. Magnetic cellular labeling was evaluated using Prussian Blue staining for iron, T2 relaxometry (1.0 Tesla, CPMG pulse sequence, 42 MHz resonance frequency, Vi echo times of tau=l and tau=5 microseconds), and MR imaging (1.5 Tesla; fast spin echo TR=3000 milliseconds, TE=45 milliseconds, echo rain length = 8, and a multi-slice gradient echo(GRE) TR=300 milliseconds, TE=20 milliseconds and flip angle of 20 degrees) of the resulting cell suspensions.

The MSC cells that were incubated in the media that only contained the Feridex® iron oxide particles (no transfection agent) displayed low or virtually undetectable cellular labeling when observed with Prussian Blue staining. Cells incubated in the media that contained both the Feridex® iron oxide particles and the transfection agent, however, displayed numerous iron-containing intracytoplasmatic vesicles when observed with Prussian Blue staining and MRI. Following labeling with the Feridex® iron oxide/transfection agent mixtures, the MSC cells were unaffected in their viability and proliferation rate as demonstrated by a 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide (MTT) toxicity and proliferation assay. Compared to unlabeled control cell suspensions, there was a dramatic increase in the 1/T2 values of labeled cells indicating significant uptake of the iron oxide particles. Having illustrated and described the principles of our invention with reference to several embodiments, it should be apparent to those of ordinary skill in the art that the invention may be modified in arrangement and detail without departing from such principles.

Claims

What is claimed is:

1. A composition comprising:

(a) magnetic-responsive coated metal oxide particles; and (b) at least one transfection agent.

2. A composition according to claim 1, wherein the magnetic-responsive coated metal oxide particles comprise metal oxide particles coated with a material selected from polysaccharide, polyvinyl alcohol, polyacrylate, polystyrene, and mixtures thereof.

3. A composition according to claim 2, wherein the coating material consists essentially of a polysaccharide.

4. A composition according to claim 1, wherein the magnetic-responsive coated metal oxide particles comprise paramagnetic, ferrimagnetic, superparamagnetic or anti-ferromagnetic iron oxide particles.

5. A composition according to claim 1, wherein the transfection agent is selected from polyaminoacid, spermidine, salmon sperm DNA, poly-L-ornithine, diethylaminoethyl-dextran, cationic liposome or lipid, non-liposomal lipid, dendrimer, polynucleotide, and mixtures thereof.

6. A composition according to claim 1, wherein the transfection agent has a molecular weight of at least about 1 kDa.

7. A composition according to claim 1, wherein the transfection agent comprises an endocytic transfection agent.

8. A composition according to claim 1, wherein the transfection agent is not chemically bonded to the surface of the magnetic-responsive metal oxide particles.

9. A composition comprising: (a) superparamagnetic dextran-coated iron oxide particles; and

(b) at least one transfection agent selected from polyaminoacid, spermidine, salmon sperm DNA, poly-L-ornithine, diethylaminoethyl-dextran, cationic liposome or lipid, non-liposomal lipid, dendrimer, polynucleotide, and mixtures thereof.

10. A mixture prepared by mixing together:

(a) magnetic-responsive coated metal oxide particles; and

(b) at least one transfection agent, wherein the transfection agent is electrostatically attracted to the magnetic-responsive coated metal oxide particles.

11. A mixture according to claim 10, wherein the magnetic-responsive coated metal oxide particles comprise superparamagnetic dextran-coated iron oxide particles and the transfection agent comprises an endocytic transfection agent selected from polyaminoacid, spermidine, salmon sperm DNA, poly-L-ornithine, diethylaminoethyl-dextran, cationic liposome or lipid, non-liposomal lipid, dendrimer, polynucleotide, and mixtures thereof.

12. A mixture according to claim 10, wherein the magnetic-responsive coated metal oxide particles and the transfection agent are mixed together in the absence of an organic solvent.

13. A mixture according to claim 10, wherein the magnetic-responsive coated metal oxide particles comprise metal oxide particles coated with a material selected from polysaccharide, polyvinyl alcohol, polyacrylate, polystyrene, and mixtures thereof.

14. A mixture according to claim 10, wherein the coating material consists essentially of a polysaccharide.

15. A mixture prepared by mixing together:

(a) magnetic-responsive coated metal oxide particles; and

(b) at least one transfection agent, wherein the magnetic-responsive coated metal oxide particles and the transfection agent are mixed together in the presence of a cell culture medium.

16. A composition comprising:

(a) magnetic-responsive coated metal oxide particles; and

(b) at least one transfection agent, wherein the composition does not include an additional therapeutic, diagnostic or bioactive agent.

17. A magnetic probe comprising a mixture of the following ingredients:

(a) at least one magnetic resonance contrast agent; and

(b) at least one transfection agent.

18. A magnetic probe according to claim 17, wherein the transfection agent is selected from selected from polyaminoacid, spermidine, salmon sperm DNA, poly-L- ornithine, diethylaminoethyl-dextran, cationic liposome or lipid, non-liposomal lipid, dendrimer, polynucleotide, and mixtures thereof.

19. A method for making a composition comprising mixing together:

(a) magnetic-responsive coated metal oxide particles; and

(b) at least one transfection agent.

20. A method according to claim 19, wherein the magnetic-responsive metal oxide particles comprise metal oxide particles coated with a material selected from polysaccharide, polyvinyl alcohol, polyacrylate, polystyrene, and mixtures thereof.

21. A method according to claim 20, wherein the coating material consists essentially of polysaccharide.

22. A method according to claim 19, wherein the magnetic-responsive coated metal oxide particles comprise paramagnetic, ferrimagnetic, superparamagnetic or anti-ferromagnetic iron oxide particles.

23. A method according to claim 19, wherein the magnetic-responsive coated metal oxide particles and the transfection agent are mixed together in the presence of a cell culture medium.

24. A method according to claim 19, wherein the magnetic-responsive coated metal oxide particles and the transfection agent are mixed together in the absence of an organic solvent.

25. A method of labeling cells to render the cells magnetic resonance sensitive comprising: contacting the cells to be labeled with a mixture of (i) magnetic-responsive coated metal oxide particles and (ii) at least one transfection agent; and allowing the mixture to be internalized by the cells.

26. A method according to claim 25, wherein the cells are selected from stem cells and neural cells.

27. A method according to claim 25, wherein the magnetic-responsive coated metal oxide particles enter the cellular cytoplasm.

28. A method according to claim 25, wherein the contacting step comprises culturing the cell to be labeled in a medium that includes the mixture of (i) magnetic- responsive coated metal oxide particles and (ii) at least one transfection agent.

29. A method according to claim 25, wherein the transfection agent is selected from selected from polyaminoacid, spermidine, salmon sperm DNA, poly-L- ornithine, diethylaminoethyl-dextran, cationic liposome or lipid, non-liposomal lipid, dendrimer, polynucleotide, and mixtures thereof.

30. A method according to claim 25, wherein the magnetic-responsive coated metal oxide particles comprise metal oxide particles coated with a material consisting essentially of polysaccharide.

31. A method according to claim 25, wherein the transfection agent is electrostatically attracted to the magnetic-responsive coated metal oxide particles.

32. A method of labeling cells to render the cells magnetic resonance sensitive comprising: contacting the cells to be labeled with a mixture of (i) magnetic-responsive coated metal oxide particles and (ii) at least one endocytic transfection agent; and allowing the mixture to be internalized by the cells in the absence of an applied magnetic field.

33. A method according to claim 32, wherein the cells are selected from stem cells and neural cells.

34. A method according to claim 32, wherein the magnetic-responsive coated metal oxide particles enter the cellular cytoplasm.

35. A method according to claim 32, wherein the contacting step comprises culturing the cell to be labeled in a medium that includes the mixture of (i) magnetic- responsive coated metal oxide particles and (ii) at least one endocytic transfection agent.

36. A method of detecting living cells in a host comprising: contacting the cells to be labeled with a mixture of (i) magnetic-responsive coated metal oxide particles and (ii) at least one transfection agent; allowing the mixture to be internalized by the cells; and imaging the host with magnetic resonance imaging so as to detect the cells.

37. A method according to claim 36, wherein the cells are selected from stem cells and neural cells.

38. A method according to claim 36, wherein the magnetic-responsive coated metal oxide particles enter the cellular cytoplasm.

39. A method according to claim 36, wherein the contacting step comprises culturing the cell to be labeled in a medium that includes the mixture of (i) magnetic- responsive coated metal oxide particles and (ii) at least one transfection agent.

40. A method according to claim 36, wherein the transfection agent is selected from polyaminoacid, spermidine, salmon sperm DNA, poly-L-ornithine, diethylaminoethyl-dextran, cationic liposome or lipid, non-liposomal lipid, dendrimer, polynucleotide, and mixtures thereof.

41. A method according to claim 36, wherein the magnetic-responsive coated metal oxide particles comprise metal oxide particles coated with a material consisting essentially of polysaccharide.

42. A method according to claim 36, wherein the transfection agent is electrostatically attracted to the magnetic-responsive coated metal oxide particles.

43. A method of detecting living cells in a host comprising: contacting the cells to be labeled with a mixture of (i) magnetic-responsive coated metal oxide particles and (ii) at least one endocytic transfection agent; allowing the mixture to be internalized by the cells in the absence of an applied magnetic field; and imaging the host with magnetic resonance imaging so as to detect the cells.

44. A method according to claim 43, wherein the cells are selected from stem cells and neural cells.

45. A method according to claim 43, wherein the magnetic-responsive coated metal oxide particles enter the cellular cytoplasm.

46. A method according to claim 43, wherein the contacting step comprises culturing the cell to be labeled in a medium that includes the mixture of (i) magnetic- responsive coated metal oxide particles and (ii) at least one endocytic transfection agent.

47. A method of detecting living cells in a host comprising: contacting the cells to be detected with a mixture of (i) magnetic-responsive coated metal oxide particles and (ii) at least one transfection agent; allowing the mixture to be internalized by the cells; introducing such cells into the host; and imaging the host with magnetic resonance imaging so as to detect the cells.

48. A diagnostic kit for labeling cells to render the cells magnetic resonance sensitive comprising: (a) magnetic-responsive coated metal oxide particles;

(b) a least one transfection agent; and

(c) instructions for labeling cells using a mixture of the magnetic-responsive coated metal oxide particles and the transfection agent.

49. The diagnostic kit of claim 48, wherein the magnetic-responsive metal oxide particles comprise iron oxide particles and the transfection agent is selected from polyaminoacid, spermidine, salmon sperm DNA, poly-L-ornithine, diethylaminoethyl- dextran, cationic liposome or lipid, non-liposomal lipid, dendrimer, polynucleotide, and mixtures thereof.

50. The diagnostic kit of claim 48, wherein the magnetic-responsive coated metal oxide particles are provided as a first reagent and the one transfection agent is provided as a separately contained second reagent.

51. The diagnostic kit of claim 48, wherein the magnetic-responsive coated metal oxide particles and the transfection agent is provided as a mixture in a single container.

52. The diagnostic kit of claim 48, wherein the instructions include directions for: mixing the magnetic-responsive coated metal oxide particles, the transfection agent, and the cells; the dilutions required for various cell types; and incubation times.