MAGNETIC CELLS FOR LOCALIZING DELIVERY AND TISSUE REPAIR

Abstract

Normal or genetically modified cell(s) having magnetic nanoparticle(s) bound (affixed) to their surfaces and methods of delivery to target tissues, e.g. for treatment of disease and/or injury.

Claims

1.-27. (canceled)

28. A method of delivering a cell to a target tissue in an animal having a condition selected from selected from cirrhosis, heart failure, adenocarcinoma, carcinoma, lymphoma, skin wounds, stroke and spinal cord injury, said method comprising: administering a magnetic particle-comprising cell having at least one magnetic nanoparticle affixed to the surface of said cell to the animal; and applying a magnetic field to said target tissue under conditions such that said magnetic particle-comprising cell is delivered to specific regions of said target tissue.

29. The method of claim 28, wherein the magnetic particle is affixed to the cell surface by means of a cell-surface specific binding agent.

30. The method of claim 29, wherein the cell-surface specific binding agent is an antibody.

31. The method of claim 28 wherein the magnetic particles have a mean diameter of no more than 500 nm.

32. The method of claim 30 wherein the magnetic particles have a mean diameter of no more than 200 nm.

33. The method of claim 28 wherein the magnetic particles comprise iron in any ferromagnetic form.

34. The method of claim 28 wherein the magnetic nanoparticles have a surface coating.

35. The method of claim 34 wherein the surface coating is chemically modified to allow the binding of an antibody, or antibody fragment, or protein or sugar fragment that binds to cells.

36. The method of claim 28 wherein the magnetic particle comprising cells are administered by injection or infusion or surface application.

37. The method of claim 28 wherein the animal has cirrhosis and hepatocytes are delivered to a liver, wherein the animal has heart failure and cardiac myocytes are delivered to a heart of the animal, wherein the animal has adenocarcinoma, carcinoma, or lymphoma and toxin-containing cells are delivered to a tumor of the animal, wherein the animal has a skin wound and fibroblasts and epithelial cells are delivered to the skin of the animal; wherein the animal has a stroke and stem cells are delivered to a brain of the animal, or wherein the animal has a spinal cord injury and stem cells, neurons, or immune cells are delivered to a spinal cord of the animal.

38. The method of claim 28 wherein the animal is a mammal.

39. The method of claim 38 wherein the mammal is a human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGS. 1A-1B: RGCs express trkB FIG. 1A RGCs immunolabled with anti-trkB (red; DAPI nuclear counterstaining is blue). FIG. 1B RGCs were cultured in growth media including BDN F. CNTF, insulin and forskolin for 24 hours. 1 m magnetic nanoparticles coated with anti-trkB were subsequently added to the medium for 2 hours, after which time the cultures were washed with fresh medium and the RGCs examined with DIC microscopy.

[0024] FIG. 2A force-distance relationship was constructed based on Stokes' law F=6ltT1Rv, where F is the force due to friction acting on a particle of radius R at a distance d from the tip of the pole piece, traveling at velocity v through a fluid viscosity of .

DETAILED DESCRIPTION

General Methods

Cells

[0025] Cells for magnetization and use in the method can be obtained according to known protocols. For examples below, RGCs were purified to homogeneity (>995%), separating them from all other retinal neurons as well as all other CNS glial cells (Meyer-Franke et al., 1995; Goldberg et al., 2002b; Goldberg et al., 2002a). Purification of RGCs will allow more rapid identification of nanoparticle binding and endocytosis, and will allow us to better characterize the force versus axon growth rate. In other examples, CNS glia, both astrocytes and oligodendrocytes (Goldberg et al., 2002b: Goldberg et al., 2002a) can be purified for testing (Goldberg et al., 2002b; Goldberg et al., 2002a).

Magnetic Nanoparticles

[0026] Magnetic nanoparticles in various forms are already in use clinically and in research applications without any demonstrated toxicity. For example, superparamagnetic particles containing monocrystalline iron oxide nanoparticles (MION) of diameters <50 nm have been used as MRI contrast agents. These particles have demonstrated neurologic non-toxicity and axonal transport of ferrous-based agents (Neuwelt et al. 1994) Published studies supporting the use of the MRI contrast agent Ferridex (Advanced Magnetics and Berlex Laboratories) have found no deleterious effects. Furthermore, magnetically directed drug delivery, using tagged pharmaceuticals in the form of magnetic microspheres and magnetic polymer carriers, has shown success in delivering anti-neoplastic drugs and radio-isotopes to magnetically targeted areas in vivo (Schutt et al., 1997; Lubbe et al., 2001).

Coatings

[0027] Means for applying the contemplated coatings to magnetic nanoparticles are well known to those of skill in the art. Commercial kits are available having the necessary agents and instructions, for example, as detailed in the description above and examples below (see, e.g., Example 1).

Magnets

[0028] Magnets for use in the medical arts and in particular for localizing magnetic particles in tissue are familiar to those of skill in the art. Suitable magnets are described, for example in Consigny (U.S. Pat. No. 6,203,487). The clinical device will include either a superconducting magnet or fixed/rare earth magnet with sufficient field density uniformity and magnetic field gradient to direct the cells and hold them in place. Specifics of magnetic field strength will vary by need, such that stronger fields/gradients will be used when the magnet is required to act at greater distances, and weaker fields/gradients may be used when the magnet can be localized closer to the implanted particles and/or target tissues. We anticipate directing the cells to the target tissue and then modulating the underlying held to further refine their movement and shape the tissue.

EXAMPLES

Example 1

Coating Magnetic Nanoparticles for Surface Attachment

[0029] For coating various magnetic nanoparticles for surface attachment to neurons, a procedure analogous to that effective for coating 1 m particles activated with carboxylic acid (Dynal Biotech, Oslo, NORWAY) or for coating 50 nm particles (e.g. Miltenyi Biotech) with anti-TrkB (BD Bioscience, San Jose, Calif., USA) can be used. The coating procedure is performed according to the manufacturer's standard protocols. Briefly, particles are washed twice with 25 mM MES at approximate pH6 buffer for approximately 10 min each time. Approximately 150 g of anti trkB in MES buffer is used for functionalizing particles, and slow tilt rotated for approximately 30 min. Then, 0.3 mg of EDC in MES buffer is added, and incubated overnight at 4 C. with tilt rotation. Finally, particles are washed in PBS for four times and PBS is added to a final 1 mg/ml. We found that 1 m magnetic particles coated in this manner can strongly bind to RGCs (FIG. 1). It is expected that this and similar protocols can be used to coat magnetic nanoparticles down to 25 nm diameter and smaller.

Example 2

Optimal Functionalization (Surface Coating) of Commercially Available Superparamagnetic Nanoparticles to Maximize Binding to Retinal Ganglion Cells

[0030] Commercially available surface activated superparamagnetic nanoparticles as small as 25 mn (MicroMod Partikeltechnologie GmbH, GERMANY) can be coated according to manufacturers' protocols with functional molecules selected for their ability to strongly and specifically bind neurons. Briefly, tosyl-activated or carboxyl-activated magnetic nanoparticles can be used for attaching antibodies, proteins and other biomolecules that contain primary amino or sulphydryl groups. We will use manufacturers' suggested protocols for nanoparticle and protein/antibody concentrations as a starting point to covalently attach the following proteins: antibodies to the trkB receptor, antibodies to the surface adhesion molecule L1, antibodies to surface integrin receptors, and cholera toxin subunit B, which binds to the GMI ganglioside on the surfaces of RGCs and other neurons. We have already successfully shown that we can functionalize magnetic nanoparticles using these techniques (see above). The coupling of the functional group will be verified by staining the nanoparticles with fluorescently tagged secondary antibodies directed against the primary antibody/protein. Non-functionalized magnetic nanoparticles will be used as controls. We will confirm that the coating process did not disrupt the ability of these antibodies/molecules to bind their targets.

Example 3

Measurement of Binding Specificity of Magnetic Nanoparticles in Purified and Mixed Cultures

[0031] To assay for nanoparticle binding by neurons, retinal ganglion cells (RGCs) can be cultured according to standard protocols (Meyer-Franke et al., 1995; Goldberg et al., 2002b). We will add functionalized nanoparticles generated as described above to the RGCs 2 hours after plating, leave them for an additional 1 hour at 37 C., and then exchange the media to remove excess unbound nanoparticles. We will leave the neurons in culture for 1 hour to 3 days, to examine whether the nanoparticles remain attached with time. At the end of the culture period we will use three techniques to confirm nanoparticle binding: (1) direct visualization using high-magnification microscopy available in the lab, (2) commercially available iron staining kits (Sigma) in the case of nanoparticles with exposed iron surfaces; and (3) standard immunohistochemistry with fluorescent secondary antibodies directed against the antibodies/proteins coating the nanoparticles. Using these 3 techniques we will estimate at a gross level the amount of nanoparticle binding by counting nanoparticles or comparing stained cells.

[0032] To assay for neuron-specific binding, we will use mixed retinal and cortical cell cultures, both of which we are currently using in the lab. Although most of the studies for initial simplicity will focus on the use of RGCs, we wish to generate at a minimum some indication that the data generated for RGCs will be testable more broadly on other CNS neurons. Approximately 2 hours after plating we will add functionalized magnetic nanoparticles, as above, and exchange the media after 1 hour at 37 C. to remove excess unbound nanoparticles. After 1 hour to 3 days, we will do double immunohistochemistry to determining binding specificity, using antibodies against the neuron-specific surface molecule thy-1 to identify RGCs or cortical neurons.

Example 4

Measurement of Binding Strength in Varying Magnetic Fields

[0033] The magnet will first be calibrated to the magnetic nanoparticles to be used, as nanoparticles in different regions in the dish will experience different forces. We will initially calibrate two different magnets: (1) a calibrated permanent magnet, and (2) a sharpened magnetized tip. We will use uncoated magnetic nanoparticles for calibration by suspending them in a high viscosity polydimethylsiloxane solution (PDMS, Sigma). We will use 12,000 centistoke PDMS for 1 m nanoparticles, and 1,000 centistoke PDMS solution for nanoparticles smaller than 1 m. We will place the permanent magnet in the middle of a 35 mm Petri dish with glass bottom containing magnetic nanoparticles and PDMS solution. The movement of the nanoparticles towards the magnet will be digitally recorded using videomicroscopy, from which we will calculate position versus time (velocity) of the nanoparticles. We will then plot velocity versus position from magnet to fit a curve, which can then be used to estimate the force versus position (distance) curves based on Stokes' law:

F=6Rv

wherein F is the force due to friction, is the fluid viscosity, R is the nanoparticle radius, and v is the nanoparticle velocity. This will give us the force-distance relationship for the specific magnet/nanoparticle in use (see, e.g. FIG. 2).

[0034] To measure binding strength of magnetic nanoparticle to neurons, we will add functionalized nanoparticles to the RGC cultures as described above. We will use a calibrated permanent magnet to apply a known force to RGC-nanoparticle pairs. We will note whether the nanoparticle was attached to an axon or the cell body, as binding strength may vary according to the cellular site of attachment. By varying the distance between the nanoparticle and the magnet, we can vary the applied force, for example to increase the force until the nanoparticles detach from the neurons. We will record the time, t, since application of force and the force at which the nanoparticle detaches from the cell/axon. We will use this data for statistical analysis of the binding force of the nanoparticle to a cell for a variety of nanoparticle sizes coated with one of the above mentioned molecules.

[0035] Using this technique, we have demonstrated that surface activated nanoparticles can be strongly and specifically attached to neurons and other cells. Optimal nanoparticle size should be able to be determined through routine experimentation. Likewise, antibody and protein coatings can be optimized for individual applications.

Example 5

Delivery of Magnetic Particle-Comprising Cells to a Target Tissue

[0036] Magnetic particle-comprising cells as described above can be administered to the subject by any suitable means known in the art, for example, by injection (local or systemic), topical application, infusion, etc. It is expected that for applications involving the eye, topical application or local injection will be preferred. Following administration of the cells, one or more magnets will be positioned so as to cause the cells to migrate to or remain in or at the desired target tissue. The required strength of the magnet and time period necessary for the magnetic force to be applied in order to effect the desired outcome (in most instances, cells being fixed in or attached to the target tissue) can be determined by routine experimentation.

[0037] Three such examples ate offered in detail.

[0038] (a) Delivery of donor or autologous corneal endothelial cells to the corneal endothelial surface of the patient with inadequately functioning endothelium, as in Fuch's Endothelial Dystrophy or Pseudophakic Bullous Keratopathy. Corneal endothelial cells would be isolated from human donor corneas (Joyce et al., 1990; Joyce et al., 1996; Chen et al., 2001; Joyce, 2003; Joyce and Zhu, 2004; Zhu and Joyce, 2004) or derived from human stem cells in cultures by other technologies (Yokoo et al., 2005; Yamagami et al., 2006). Such corneal endothelial cells would be bound with magnetic nanoparticles, for example 50 nm or 360 nm magnetic nanoparticles bought commercially or constructed using published methods (Schroder et al., 1986; Douglas et al., 1987; Sestier et al, 1998; Perrin et al., 1999, McCloskey et al., 2000; Tibbe et al., 2001). Binding of cells to coated nanoparticles would be based on specific antibody-antigen nanoparticle coatings, for example using antibodies against cadherin-11, integrin-beta-1, platelet-derived growth factor 1-alpha receptor, or neuropilin-1, all of which are expressed by corneal endothelial cells [our unpublished data]. Such magnetic nanoparticle-coated endothelial cells would be injected into the anterior chamber of the eye in a manner that can be done in a clinic, for example with a 30 gauge needle, without a requirement for incisional surgery. 10.sup.3-10.sup.6 cells will be delivered by injection in a volume of 3-300 L but more typically around 10.sup.4-10.sup.5 cells in a volume of 50-100 L. A suitable magnet, for example a rare earth magnet of suitable strength, would be affixed in a patch to the surface of the eye external to the eyelid centered over the cornea. Over the course of a 1 hour to 7 days but more typically 16 hours to 3 days, the magnetic field would help affix the donor, nanoparticle-bound endothelial cells to the surface of the host/patient endothelial surface, after which time natural endothelial cell adhesion would take place, removing the need for additional magnetic field application. The external magnet would be removed. With time, the nanoparticles on the surface of the donor cells would degrade from the surfaces by natural proteolytic mechanisms, and be washed away in the fluid of the anterior chamber. Their small size would allow outflow through the trabecular meshwork and other natural outflow pathways without clogging these pathways or elevating intraocular pressure. The delivery of the magnetic endothelial cells to the internal corneal surface would allow improved pump function of the corneal endothelium and removal of fluid (edema) from the cornea. The cornea would subsequently become more clear, improving vision, and less edematous, decreasing the pain typically associated with this condition.

[0039] (b) Delivery of donor or autologous stem cells, photoreceptors, or retinal pigment epithelial (RPE) cells to the subretinal space in patients with photoreceptor/RPE dysfunction, as in age-related macular degeneration or retinitis pigmentosa. As in (a), such cells would be bound with magnetic nanoparticles, and injected subretinally, or perhaps through the bloodstream intravenously. Surgical implantation of a magnet or magnetic coil (electromagnet) would precede such injection, for example by affixing a rare-earth magnet by means of a sutured plate to the sclera behind the macula using a surgical technique in current use for the attachment of radioactive plaques in the treatment of ocular melanoma (Giblin et al., 1989; Shields et al., 1993; Shields et al., 1996; Shields et al., 1997). The magnetic field will cause localization and retention of the implanted cells at the site of degeneration, typically the macula. After healing and integration processes took hold, the magnet might be surgically removed. Alternatively the magnet could be left in place for future, additional cell treatments. The small, nano-scale, surface bound particles would as above degrade from the surfaces by natural proteolytic mechanisms allowing excretion from the eye. In this treatment paradigm, the delivery of magnetic cells to the posterior aspect of the retina would allow the improved function of the photoreceptors, enhancing visual acuity and visual field in these patients.

[0040] (c) Delivery of donor or autologous stem cells or retinal ganglion cells to the retinal surface, for such diseases as glaucoma or ischemic optic neuropathy, or other optic neuropathies. As in (b), such cells would be bound with magnetic nanoparticles, and injected intravitreally. Rather than simply floating around in the vitreous or sinking to the base of the eye, a posteriorly place magnet would pull the cells to the surface of the retina, perhaps over the macula, or in the retinal region of an acquired visual field deficit. Magnet and placement can be, for example as described in (b), above. Sequential localization of the magnetic field towards the head of the optic nerve could pull axons along their normal wiring pathways to the brain. As above, the small, nano-scale, surface bound particles would as above degrade from the surfaces by natural proteolytic mechanisms allowing excretion from the eye. In the treatment of glaucoma or other optic neuropathies with this version of the invention, the improved number of retinal ganglion cells, and the improved integration of these magnetic cells into the proper location of the eye, will allow for improved vision, and will contribute to the neuroprotection of the remaining retinal neurons preventing their cell death.

[0041] References, patents and other publications cited herein are hereby incorporated by reference.

REFERENCES

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