Patent Application: US-86600409-A

Abstract:
normal or genetically modified cell having magnetic nanoparticle bound to their surfaces and methods of delivery to target tissues , e . g . for treatment of disease and or injury .

Description:
cells for magnetization and use in the method can be obtained according to known protocols . for examples below , rgcs were purified to homogeneity (& gt ; 99 . 5 %), 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 in various forms are already in use clinically and in research applications without any demonstrated toxicity . for example , superparamagnetic particles containing monociystalline iron oxide nanoparticles ( mion ) of diameters & lt ; 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 ). 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 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 field to further refine their movement and shape the tissue . for coating various magnetic nanoparticles for surface attachment to neurons , a procedure analogous to that effective for coating 1 μm 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 &# 39 ; s standard protocols . briefly , particles are washed twice with 25 mm mes at approximate ph6 buffer for approximately 10 min each time . approximately 150 μg 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 μm magnetic particles coated in this manner can strongly bind to rgcs ( fig1 ). it is expected that this and similar protocols can be used to coat magnetic nanoparticles down to 25 nm diameter and smaller . optimal functionalization ( surface coating ) of commercially available superparamagnetic nanoparticles to maximize binding to retinal ganglion cells commercially available surface activated superparamagnetic nanoparticles as small as 25 nm ( micromod partikeltechnologie gmbh , germany ) can be coated according to manufacturers &# 39 ; 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 &# 39 ; 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 gm1 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 . measurement of binding specificity of magnetic nanoparticles in purified and mixed cultures 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 . 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 . 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 μm nanoparticles , and 1 , 000 centistoke pdms solution for nanoparticles smaller than 1 μm . 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 &# 39 ; law : wherein f is the force due to friction , η is the fluid viscosity , r is the nanoparticle radius , and ν is the nanoparticle velocity . this will give us the force - distance relationship for the specific magnet / nanoparticle in use ( see , e . g . fig2 ). to measure binding strength of magnetic nanoparticles 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 . 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 . 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 . ( a ) delivery of donor or autologous corneal endothelial cells to the corneal endothelial surface of the patient with inadequately functioning endothelium , as in fuch &# 39 ; 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 3 - 10 6 cells will be delivered by injection in a volume of 3 - 300 μl , but more typically around 10 4 - 10 5 cells in a volume of 50 - 100 μl . 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 . ( 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 . ( 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 . references , patents and other publications cited herein are hereby incorporated by reference . chen k h , azar d , joyce n c ( 2001 ) transplantation of adult human corneal endothelium ex vivo : a morphologic study . cornea 20 : 731 - 737 . douglas s j , davis s s , illium l ( 1987 ) nanoparticles in drug delivery . crit rev ther drug carrier syst 3 : 233 - 261 . giblin m e , shields j a , augsburger j j , brady l w ( 1989 ) episcleral plaque radiotherapy for uveal melanoma . aust n z j opthalmol 17 : 153 - 156 . goldberg j l , klassen m p , hua y , barres b a ( 2002a ) amacrine - signaled loss of intrinsic axon growth ability by retinal ganglion cells . science 296 : 1860 - 1864 . goldberg j l , espinosa j s , xu y , davidson n , kovacs g t , barres b a ( 2002b ) retinal ganglion cells do not extend axons by default : promotion by neurotrophic signaling and electrical activity . neuron 33 : 689 - 702 . joyce n c ( 2003 ) proliferative capacity of the corneal endothelium . prog retin eye res 22 : 359 - 389 . joyce n c , zhu cc ( 2004 ) human corneal endothelial cell proliferation : potential for use in regenerative medicine . cornea 23 : s8 - s19 . joyce n c , meklir b , neufeld a h ( 1990 ) in vitro pharmacologic separation of corneal endothelial migration and spreading responses . invest opthalmol vis sci 31 : 1816 - 1826 . joyce n c , meklir b , joyce s j , zieske j d ( 1996 ) cell cycle protein expression and proliferative status in human corneal cells . invest opthalmol vis sci 37 : 645 - 655 . lubbe a s , alexiou c , bergemann c ( 2001 ) clinical applications of magnetic drug targeting . j surg res 95 : 200 - 206 . mccloskey k e , chalmers j j , zborowski m ( 2000 ) magnetophoretic mobilities correlate to antibody binding capacities . cytometry 40 : 307 - 315 . meyer - franke a , kaplan m r , pfrieger f w , barres b a ( 1995 ) characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture . neuron 15 : 805 - 819 . neuwelt e a , weissleder r , nilaver g , kroll r a , roman - goldstein s , szumowski j , pagel m a , jones r s , remsen l g , mccormick c i , et al . 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