U.S. patent application number 11/210173 was filed with the patent office on 2006-02-23 for magnetically-controllable delivery system for therapeutic agents.
Invention is credited to Kenneth A. Barbee, Zachary G. Forbes, Gennady Friedman, Benjamin B. Yellen.
Application Number | 20060041182 11/210173 |
Document ID | / |
Family ID | 35910545 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060041182 |
Kind Code |
A1 |
Forbes; Zachary G. ; et
al. |
February 23, 2006 |
Magnetically-controllable delivery system for therapeutic
agents
Abstract
A magnetic delivery system for delivering a magnetizable
particle to a location in a body, the device includes a
magnetizable object implanted in the body, wherein the magnetizable
object includes a plurality of segments distributed throughout the
magnetizable object and wherein the segments are configured to
provide a magnetic gradient for attracting the magnetizable
particle and an external source of a magnetic field capable of (i)
magnetizing the magnetizable particle and (ii) increasing a degree
of magnetization of the magnetizable object and thereby creating
the magnetic gradient. A drug delivery system including the
magnetic delivery system and a magnetizable particle associated
with a therapeutic agent and/or a cell. A cell delivery system
based on the magnetic delivery system and a magnetizable particle
associated with a cell. A method of using the magnetic delivery
system for delivery of a therapeutic agent and/or a cell to a
targeted location in a body.
Inventors: |
Forbes; Zachary G.;
(Philadelphia, PA) ; Yellen; Benjamin B.;
(Philadelphia, PA) ; Barbee; Kenneth A.;
(Philadelphia, PA) ; Friedman; Gennady; (Richboro,
PA) |
Correspondence
Address: |
CAESAR, RIVISE, BERNSTEIN,;COHEN & POKOTILOW, LTD.
11TH FLOOR, SEVEN PENN CENTER
1635 MARKET STREET
PHILADELPHIA
PA
19103-2212
US
|
Family ID: |
35910545 |
Appl. No.: |
11/210173 |
Filed: |
August 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US04/11861 |
Apr 16, 2004 |
|
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11210173 |
Aug 22, 2005 |
|
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60463505 |
Apr 16, 2003 |
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60680833 |
May 13, 2005 |
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Current U.S.
Class: |
600/12 ;
604/891.1; 623/1.46; 623/23.53 |
Current CPC
Class: |
A61F 2/82 20130101; A61N
1/30 20130101; A61L 31/022 20130101; A61L 27/50 20130101; A61L
31/14 20130101; A61M 37/00 20130101; A61N 2/00 20130101; A61M
2037/0007 20130101; A61F 2210/009 20130101 |
Class at
Publication: |
600/012 ;
623/023.53; 623/001.46; 604/891.1 |
International
Class: |
A61N 2/10 20060101
A61N002/10; A61F 2/06 20060101 A61F002/06; A61F 2/28 20060101
A61F002/28 |
Claims
1. A magnetic delivery system for delivering a magnetizable
particle to a location in a body, the device comprising: a
magnetizable object implanted in the body, wherein the magnetizable
object includes a plurality of segments distributed throughout the
magnetizable object and wherein the segments are configured to
provide a magnetic gradient for attracting the magnetizable
particle; and an external source of a magnetic field capable of (i)
magnetizing the magnetizable particle and (ii) increasing a degree
of magnetization of the magnetizable object and thereby creating
the magnetic gradient.
2. The magnetic delivery system of claim 1, wherein a size of each
segment is selected to commensurate with a size of the magnetizable
particle such that at least one spatial dimension of each segment
does not exceed by more than about 10,000 times at least one
spatial dimension of the magnetizable particle.
3. The magnetic delivery system of claim 2, wherein the size of
each segment is selected to commensurate with the size of the
magnetizable particle such that at least one spatial dimension of
each segment does not exceed by more than about 100 times at least
one spatial dimension of the magnetizable particle.
4. The magnetic delivery system of claim 1, wherein the
magnetizable object is in the shape of a cylinder, a cylindrical
rod, a cube, a cubical rod, a spring, a circular disc, a mesh, a
ring, a wire, a sphere or a combination thereof.
5. The magnetic delivery system of claim 1, wherein the
magnetizable object is a member selected from the group consisting
of a stent, a pacemaker, a catheter, a tube, a vascular graft, an
artificial joint, an artificial bone, a prostate seed, an aneurysm
coil, a surgical staple, and a suture.
6. The magnetic delivery system of claim 1, wherein the
magnetizable object comprises a cluster of gradient forming
particles, wherein a surface of each gradient forming particle
represents a segment of the magnetizable object.
7. The magnetic delivery system of claim 1, wherein the
magnetizable object is made from at least one of materials selected
from the group consisting of cobalt, nickel, iron, manganese,
samarium and neodymium.
8. The magnetic delivery system of claim 1, wherein the
magnetizable object is in a shape of a support made from a metal, a
rare earth element, a ceramic, a polymer or a combination
thereof.
9. The magnetic delivery system of claim 1, wherein the
magnetizable object is in a shape of a coating on the support,
wherein the coating is made from a metal, a rare earth element, a
ceramic, a polymer or a combination thereof.
10. The magnetic delivery system of claim 1, wherein the segments
comprise patterns of indentations and/or ridges of various length,
width, depth and shape.
11. The magnetic delivery system of claim 1, wherein the segments
comprise patterns of materials with different degrees of
magnetization.
12. The magnetic delivery system of claim 1, further comprising the
magnetizable particle located in the body.
13. The magnetic delivery system of claim 12, wherein at least one
of the magnetizable object and the magnetizable particle is
magnetized only in the presence of the external magnetic field.
14. The magnetic delivery system of claim 12, wherein at least one
of the magnetizable object and the magnetizable particle is
permanently magnetized.
15. The magnetic delivery system of claim 12, wherein the
magnetization of at least one of the magnetizable object and the
magnetizable particle is increased in the presence of the external
magnetic field.
16. The magnetic delivery system of claim 12, wherein the
magnetizable particle is in a form of a superparamagnetic colloidal
fluid.
17. The magnetic delivery system of claim 12, wherein the
magnetizable particle has a diameter of less than 10
micrometers.
18. The magnetic delivery system of claim 12, wherein the
magnetizable particle has a diameter from about 10 nm to about 1000
nm.
19. The magnetic delivery system of claim 12, wherein the
magnetizable particle has a diameter from 10 nm to 500 nm.
20. The magnetic delivery system of claim 12, wherein the
magnetizable particle comprises at least one of materials selected
from the group consisting of Fe.sub.2O.sub.3, Fe.sub.3O.sub.4,
FeNi, FePt, Fe, CoNi alloy and optionally a biodegradable
polymer.
21. The magnetic delivery system of claim 12, wherein the
magnetizable particle comprises a therapeutic agent.
22. The magnetic delivery system of claim 21, wherein the
magnetizable particle is in a form of a superparamagnetic colloidal
fluid.
23. The magnetic delivery system of claim 12, wherein the
magnetizable particle is loaded within a cell, thereby forming a
magnetic cell.
24. The magnetic delivery system of claim 22, wherein the magnetic
cell comprises a therapeutic agent, such that the therapeutic agent
is associated with the cell, the magnetizable particle or both.
25. A method of using the magnetic delivery system of claim 1 for
delivery of a therapeutic agent, the method comprising: providing
the external source of the magnetic field; implanting the
magnetizable object in the body, wherein the magnetizable object
includes a plurality of segments distributed throughout the
magnetizable object and wherein the segments are configured to
provide a magnetic gradient for attracting a magnetizable particle
comprising a therapeutic agent; and providing an external magnetic
field by initializing the external source and thereby (i)
magnetizing the magnetizable particle and (ii) increasing the
degree of magnetization the magnetizable object and thereby
creating the magnetic gradient for attracting and advancing the
magnetizable particle toward the magnetizable object.
26. The method of claim 25, further comprising: administering the
magnetizable particle comprising the therapeutic agent to the body;
and attracting and advancing the magnetizable particle toward the
magnetizable object using the magnetic gradient and thereby
delivering the therapeutic agent to the location in the body.
27. The method of claim 26, wherein a size of each segment is
selected to commensurate with a size of the magnetizable particle
such that at least one spatial dimension of each segment does not
exceed by more than about 10,000 times at least one spatial
dimension of the magnetizable particle.
28. The method of claim 26, wherein the magnetizable particle is in
a form of a superparamagnetic colloidal fluid.
29. The method of claim 26, wherein the magnetizable particle has a
diameter from about 10 nm to about 1000 nm.
30. The method of claim 26, wherein the magnetizable particle has a
diameter from 10 nm to 500 nm.
31. The method of claim 26, wherein the magnetizable object is a
member selected from the group consisting of a stent, a pacemaker,
a catheter, a tube, a vascular graft, an artificial joint, an
artificial bone, a prostate seed, an aneurysm coil, a surgical
staple, and a suture.
32. The method of claim 26, wherein at least one of the
magnetizable object and the magnetizable particle is magnetized
only in the presence of the external magnetic field.
33. The method of claim 26, wherein at least one of the
magnetizable object and the magnetizable particle is permanently
magnetized.
34. The method of claim 26, wherein the magnetizable particle
comprises a cell such that the magnetizable particle is loaded
within a cell and the therapeutic agent is associated with the
cell, the magnetizable particle or both.
35. The method of claim 25, wherein implanting the magnetizable
object is accomplished by administering a cluster of gradient
forming particles wherein a surface of each gradient forming
particle represents a segment of the magnetizable object.
36. A method of using the magnetic delivery system of claim 1 for
delivery of a cell to a body, the method comprising: providing the
external source of the magnetic field; implanting the magnetizable
object in the body, wherein the magnetizable object includes a
plurality of segments distributed throughout the magnetizable
object and wherein the segments are configured to provide a
magnetic gradient for attracting a magnetizable particle comprising
a therapeutic agent; administering the magnetizable particle loaded
within the cell to the body; providing an external magnetic field
by initializing the external source and thereby (i) magnetizing the
magnetizable particle and (ii) increasing the degree of
magnetization the magnetizable object and thereby creating the
magnetic gradient; and attracting and advancing the magnetizable
particle toward the magnetizable object using the magnetic gradient
and thereby delivering the cell to the location in the body.
37. The method of claim 36, wherein a size of each segment is
selected to commensurate with a size of the magnetizable particle
such that at least one spatial dimension of each segment does not
exceed by more than about 10,000 times at least one spatial
dimension of the magnetizable particle.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of the PCT
application titled "MAGNETICALLY CONTROLLABLE DRUG AND GENE
DELIVERY STENTS" having the application Ser. No. PCT/US2004/011861
and filed on Apr. 16, 2004, which claims the benefit of provisional
Application No. 60/463,505, filed on Apr. 16, 2003, titled
MAGNETIZABLE IMPLANTS AND METHODS FOR TARGETED DELIVERY OF
MAGNETICALLY SUSCEPTIBLE THERAPEUTIC AGENTS which are incorporated
herein in their entireties.
[0002] This application claims the benefit of provisional
Application No. 60/680,833, filed on May 13, 2005, titled METHOD OF
MAGNETIC DELIVERY OF CELLS AND BIOLOGICS, which is incorporated
herein in its entirety.
SPECIFICATION
BACKGROUND OF THE INVENTION
[0003] 1. Field of Invention
[0004] The present invention relates to magnetically controllable
delivery systems and methods of using thereof to attract and
deliver therapeutic agents attached to, or encapsulated within,
magnetic particles (i.e., carriers) at selected sites in a body or
a subject. More specifically, this invention relates to the use of
two sources of magnetic force to deliver therapeutic agents
including cells by utilizing magnetizable particles associated with
the therapeutic agents.
[0005] 2. Description of Related Art
[0006] The best approach for treating tumors and other localized
ailments is to administer drugs only at the site of complication.
By delivering the drug locally, the toxicity of the drug to the
rest of the body can be reduced while maintaining the desired
therapeutic benefit at the site of the ailment. Many drugs
developed by the pharmaceutical industry have shown remarkable
success during in vitro testing and animal trials, but have yielded
undesirable results in clinical trials due to systemic toxicity of
the drug to the body. Thus, the ability to deliver large
concentrations of drugs locally (i.e., only at the site of the
ailment) is of major importance for both the pharmaceutical
industry and for clinicians.
[0007] However, known drug delivery vehicles are not capable of
local delivery of high concentrations of drugs by minimally
invasive techniques. This is especially true when repeat dosing is
required. The magnetic delivery system described herein overcomes
many of these difficulties, and provides a method for concentrating
drugs at selected sites in the body with minimal stress on the
patient.
[0008] Stents are commonly used in a variety of biomedical
applications. For example, stents are routinely implanted in
patients to keep blood vessels open in the coronary arteries, to
keep the esophagus from closing due to strictures of cancer, to
keep the ureters open for maintenance of kidney drainage, and to
keep the bile duct open in patients with pancreatic cancer. Such
stents are usually inserted percutaneously under radiological
guidance.
[0009] Stents comprise a tube shaped object made of metal (e.g.,
316 L Stainless Steel), an alloy (e.g., Nickel-Titanium) or polymer
(e.g., polyurethane), in a wide range of physiologically
appropriate diameters and lengths, which are inserted into a vessel
or passage to keep the lumen open and prevent closure due to a
stricture or external compression. General stent design varies in
the number of intersections and the interstrut area, the in-strut
configuration, and the metal-to-artery ratio. The two different
expansion principles for stents are balloon-expansion and
self-expansion, and the design types can be categorized into five
types: ring, tubular, multi-design, coil, and mesh (Regar et al.
Br. Med. Bull. 2001 59:227-48; Hehrlein et al. Basic Res. Cardiol.
2002 97:417-23; Gershlick et al. Atherosclerosis 2002 160:259-71;
Garas et al. Pharmacology and Therapeutics 2001 92:165-78).
[0010] Stents have been routinely used over the last ten years in
percutaneous transluminal coronary angioplasty (PTCA), a procedure
for the treatment of severe, symptomatic coronary stenosis (Garas,
S. M. et al. Pharmacology and Therapeutics 2001 92:165-178). The
PTCA procedure was first introduced in the 1970s as an alternative
to coronary-artery bypass surgery for the clearing of coronary
vessels blocked by plaque. PTCA has proven to be a much less
invasive procedure, with patients able to return to work the week
following the procedure, as opposed to the lengthy hospital stay
required with bypass surgery (Fricker, J. Drug Discovery Today 2001
6:1135-7). Stents are used extensively in PTCA procedures due to
their unique ability to master a major complication of balloon
angioplasty ((sub) acute vessel closure), and a superior long-term
outcome in comparison to balloon angioplasty (Regar et al. Br. Med.
Bull. 2001 59:227-48).
[0011] However, in-stent restenosis (the re-closing of the vessel)
remains a major limitation, particularly in coronary stenting.
Restenosis is generally considered a local vascular manifestation
of the biological response to injury. The injury as a result of
catheter insertion consists of denudation of the intima
(endothelium) and stretching of the media (smooth muscle). The
wound-healing reaction consists of an inflammatory phase, a
granulation phase, and a remodeling phase. The inflammation is
characterized by growth factor and platelet activation, the
granulation by smooth muscle cell and fibroblast migration and
proliferation into the injured area, and the remodeling phase by
proteoglycan and collagen synthesis, replacing early fibronectin as
the major component of extracellular matrix. Coronary stents
comprise mechanical scaffolding that almost completely eliminates
recoil and remodeling. However, neo-intimal growth or proliferation
is still a problem. Neo-intimal proliferation occurs principally at
the site of the primary lesion within the first 6 months after
implantation, a major checkpoint for patient health post-surgery
(Regar et al. Br. Med. Bull. 2001 59:227-48). Neo-intima forms
during the first week after PTCA and the progress is well under way
after 4 weeks, with continued progression over the following months
(Hehrlein et al. Basic Re. Cardiol. 2002 97:417-23). This
neo-intima is an accumulation of smooth muscle cells within a
proteoglycan matrix that narrows the previously enlarged lumen. Its
formation is triggered by a series of molecular events including
leukocyte infiltration, platelet activation, smooth muscle cell
expansion, extracellular matrix elaboration, and
re-endothelialization (Regar et al. Br. Med. Bull. 2001
59:227-48).
[0012] Three major drug delivery techniques under consideration for
the prevention of restenosis are (i) prevention of thrombus
formation; (ii) prevention of vascular recoil and remodeling; and
(iii) prevention of inflammation and cell proliferation (Garas et
al. Pharmacology and Therapeutics 2001 92:165-78). In vitro and in
vivo animal model experimentation has shown promise in all three
categories, mainly in antiproliferation treatments. However,
clinical success has been limited (Garas et al. Pharmacology and
Therapeutics 2001 92:165-78), primarily due to systemic
toxicity.
[0013] Local drug delivery provides limited systemic release,
thereby reducing the risk of systemic toxicity. Known techniques
for local drug delivery include direct coating of the stent with
drug, coating of the stent with a drug-containing biodegradable
polymer, and hydrogel/drug coating. Biodegradable stents have also
been described (Regar et al. Br. Med. Bull. 2001 59:227-48;
Hehrlein et al. Basic Res. Cardiol. 2002 97:417-23; Gershlick et
al. Atherosclerosis 2002 160:259-71; Garas et al. Pharmacology and
Therapeutics 2001 92:165-78; Schwartz et al. Circulation 2002
106:1867-73; Fricker, J. Drug Discovery Today 2001 6:1135-7).
Problems with these technologies, however, include the inflammatory
response generated due to large polymer concentrations, the
inability to deliver effective concentrations, one-time dosage
limitations, and, in the case of the biodegradable stent,
mechanical compromise. An additional concern with the
polymer-coated drug-eluting stents is limitation of the growth of
the cell layer necessary to cover the stent and prevent the bare
metal from coming in long contact with the blood, thereby leading
to clot formation (Schwartz et al. Circulation 2002 106:1867-73;
Fricker, J. Drug Discovery Today 2001 6:1135-7).
[0014] The ability to apply forces on magnetic particles with
external magnetic fields has been harnessed in various biomedical
applications including prosthetics (Herr, H. J. of Rehab. Res. and
Devel. 2002 39(3):11-12), targeted drug delivery (Goodwin, S. J. of
Magnetism and Magnetic Materials 1999 194:209-217) and
antiangiogenesis strategies (Liu et al. J. of Magnetism and
Magnetic Materials 2001 225:209-217; Sheng et al. J. of Magnetism
and Magnetic Materials 1999 194:167-175). U.S. Pat. No. 4,247,406
describes an intravascularly-administrable,
magnetically-localizable biodegradable carrier comprising
microspheres formed from an amino acid polymer matrix containing
magnetic particles embedded within the matrix for targeted delivery
of chemotherapeutic agents to cancer patients. Microspheres with
magnetic particles, which are suggested to enhance binding of a
carrier to the receptors of capillary endothelial cells when under
the influence of a suitable magnetic field, are also described in
U.S. Pat. No. 5,129,877.
[0015] U.S. Pat. Nos. 6,375,606; 6,315,709; 6,296,604; and
6,364,823 describe methods and compositions for treating vascular
defects, and in particular aneurysms with a mixture of
biocompatible polymer material, a biocompatible solvent, adhesive
and preferably magnetic particles to control delivery of the
mixture. In these methods, a magnetic coil or ferrofluid is
delivered via catheter into the aneurysm. This magnetic device is
shaped, delivered, steered and held in place using external
magnetic fields and/or gradients. This magnetic device attracts the
mixture to the vascular defect wherein it forms an embolus in the
defect thereby occluding the defect.
[0016] A model for inducing highly localized phase transformations
at defined locations in the vascular system by applying 1) external
uniform magnetic fields to an injected superparamagnetic colloidal
fluid for the purpose of magnetization and 2) using embedded
particles to create high magnetic field gradients was described by
inventors (Forbes et al. Abstract and Poster Presentation at the
6th Annual New Jersey Symposium on Biomaterials, Oct. 17-18, 2002,
Somerset, N.J.). This work describes the use of uniform magnetic
fields in combination with large magnetic particles (greater than 2
micron in diameter) to form chains along the direction of applied
field and in turn use this to embolize micro-vessels (50-100
microns in diameter). The use of these magnetizable implants in
drug delivery was also described previously by authors Z. Forbes,
B. B. Yellen, G. Friedman, and K. Barbee (IEEE Trans. Magn. 39(5):
3372-3377 (2003)).
[0017] Known methods and devices for delivery of magnetizable drug
or agent-containing magnetic carrier to specific locations in the
body rely upon a single source of magnetic field to both magnetize
the carriers and to pull them by magnetic force to the specific
location. Previous attempts to use magnetic particles in these
applications have relied on high gradient magnetic fields produced
by magnets external to the body to direct magnetic particles to
specific locations (see Flores, 2002; Gallo, et al., (1997); Lubbe,
et al., (2001); Mossbach, et al., (1979); Rudge, et al., (2001)).
The main disadvantage of this approach is that externally generated
magnetic fields apply relatively small and insufficiently local
forces on micron and nano-scale magnetic particles, and thus these
methods have limited applications.
[0018] Chen (U.S. Pat. No. 5,921,244) discloses inserting a magnet
(an electromagnet or a permanent magnet) or a plurality of magnets
into an opening in a body to attract magnetic fluid/particles. The
plurality of magnets is described to be disposed along the
longitudinal axis of the magnetic probe. The plurality of magnets
actually forms a larger magnet. Chen does not describe using a
plurality of sources of magnetic fields or simultaneously creating
a far penetrating field and a strong magnetic field gradient, which
cannot be accomplished with a single source.
[0019] Gordon (U.S. patent Publication No. US 2002/0133225)
describes a device comprising an implant having a magnetic field
and a medical agent carried by a magnetically sensitive carrier.
The carrier is introduced into the blood flow of the organism
upstream from the target tissue, and the carrier and medical agent
migrate via the blood flow to the target tissue. Gordon discloses
an implant comprising a magnetized material (e.g., a ferromagnetic
or a superparamagnetic material). Examples describe making a stent
from ferromagnetic materials and magnetized by using an external
magnet or made from a magnetized material. Gordon does not disclose
optimizing the surface of the implant for providing a stronger
magnetic field gradient. Gordon does not describe using a plurality
of sources of magnetic fields.
[0020] Single source capture methods, however, are at odds with the
underlying physics of magnetic particle capture, which depends on
the simultaneous imposition of very strong far-reaching magnetic
fields and strong spatial magnetic field gradients. The purpose of
the far-reaching field is to increase the magnetic moment of
individual drug-containing particles in the vicinity of the field
to the point of magnetic saturation. Far-penetrating fields are
most typically generated with large magnetic sources. However, the
force on a magnetized particle also depends on production of strong
magnetic field gradients, which are most easily generated with very
small magnetic sources. Thus, the ability to simultaneously produce
far-penetrating magnetic fields that have strong magnetic filed
gradients is very difficult to accomplish with a single source. For
this reason, Chen teaches use of relatively large electromagnets
implanted in tissue beds to attract magnetic fluid circulating
within the blood vessels that are relatively far away, which is a
less effective method for capturing magnetic carriers.
[0021] The current invention recognizes that the ability to
simultaneously produce far-penetrating magnetic fields that have
strong magnetic filed gradients is very difficult to accomplish
with a single source and offers solutions to this problem. The
present invention further differs from previous techniques in that
the goal is to deliver therapeutic agents to a desired tissue site
without obstructing flow through the blood vessel.
[0022] Despite the foregoing developments, there is still a need in
the art for improved methods of delivery of therapeutic agents
utilizing magnetic forces.
[0023] All references cited herein are incorporated herein by
reference in their entireties.
BRIEF SUMMARY OF THE INVENTION
[0024] Accordingly, the invention provides a magnetic delivery
system for delivering a magnetizable particle to a location in a
body, the device comprising a magnetizable object implanted in the
body, wherein the magnetizable object includes a plurality of
segments distributed throughout the magnetizable object and wherein
the segments are configured to provide a magnetic gradient for
attracting the magnetizable particle and an external source of a
magnetic field capable of (i) magnetizing the magnetizable particle
and (ii) increasing a degree of magnetization of the magnetizable
object and thereby creating the magnetic gradient.
[0025] In certain embodiments, the magnetic delivery system further
comprises the magnetizable particle located in the body.
[0026] In certain embodiments, at least one of the magnetizable
object and the magnetizable particle is magnetized only in the
presence of the external magnetic field.
[0027] In certain embodiments, at least one of the magnetizable
object and the magnetizable particle is permanently magnetized.
[0028] In certain embodiments, the magnetization of at least one of
the magnetizable object and the magnetizable particle is increased
in the presence of the external magnetic field.
[0029] Further provided is a method of using the magnetic delivery
system for delivery of a therapeutic agent, the method comprising
providing the external source of the magnetic field, implanting the
magnetizable object in the body, wherein the magnetizable object
includes a plurality of segments distributed throughout the
magnetizable object and wherein the segments are configured to
provide a magnetic gradient for attracting a magnetizable particle
comprising a therapeutic agent, and providing an external magnetic
field by initializing the external source and thereby (i)
magnetizing the magnetizable particle and (ii) increasing the
degree of magnetization the magnetizable object and thereby
creating the magnetic gradient for attracting and advancing the
magnetizable particle toward the magnetizable object.
[0030] Further provided is a method of using the magnetic delivery
system for delivery of a cell to a body, the method comprising
providing the external source of the magnetic field, implanting the
magnetizable object in the body, wherein the magnetizable object
includes a plurality of segments distributed throughout the
magnetizable object and wherein the segments are configured to
provide a magnetic gradient for attracting a magnetizable particle
comprising a therapeutic agent, and administering the magnetizable
particle loaded within the cell to the body, providing an external
magnetic field by initializing the external source and thereby (i)
magnetizing the magnetizable particle and (ii) increasing the
degree of magnetization the magnetizable object and thereby
creating the magnetic gradient, and attracting and advancing the
magnetizable particle toward the magnetizable object using the
magnetic gradient and thereby delivering the cell to the location
in the body. In certain embodiments of the method, a size of each
segment is selected to commensurate with a size of the magnetizable
particle such that at least one spatial dimension of each segment
does not exceed by more than about 10,000 times at least one
spatial dimension of the magnetizable particle.
[0031] Methods of administration of magnetizable particle or
magnetic cells include but not limited to systemic delivery (e.g.,
by injection, catheterization, etc).
[0032] In a preferred embodiment, the invention provides a
magnetizable implant, preferably a stent, for targeting of magnetic
therapeutic agents to a selected site of implantation of the
magnetizable implant in a subject through creation of a high field
magnetic gradient as well as creation of a relatively uniform
magnetic field for magnetizing the magnetic therapeutic agents. In
the present invention, two magnetic fields are independently
produced in order to improve capture efficiency and uniformity of
captured therapeutic agent as a coating on the implant, as well as
to allow for miniaturization of the implant.
[0033] Further, the object of the present invention is to provide
magnetizable objects for implantation in a body or a subject and
methods for use of these magnetizable objects in delivering a
therapeutic agent encapsulated in or dispersed in a magnetic
carrier/particle to the implanted magnetizable object. The
magnetizable object of the present invention comprises a
biocompatible metal or polymeric structural supporting implant
coated with or comprising segments of a magnetizable compound. The
magnetizable object is specifically designed to produce strong
magnetic field gradients through creation of magnetizable features
distributed throughout the implant. An exemplary embodiment of a
magnetizable object of the present invention is a magnetic stent,
the geometry of which produces a strong magnetic field gradient
when modified to comprise magnetic or magnetizable segments. In
this embodiment, the stent itself preferably comprises a
magnetizable compound. In another exemplary embodiment, a
magnetizable compound is uniformly coated on the implant, and the
magnetized state of the coating is locally segmented through
magnetic recording to provide regions of high field gradients
distributed throughout the implant. In another embodiment, the
magnetizable coating of the implant is etched into a pattern to
produce strong local field gradients at desired locations on the
implant surface. In yet another embodiment, the structure of a
non-magnetic supporting material of the implant creates
well-defined magnetized segments in an otherwise uniform coating of
magnetic material. The magnetizable compound in the implant may be
permanently magnetized or remain magnetized only in the presence of
an externally applied field.
[0034] Optimization of the segments (i.e., features) of the implant
to produce strong magnetic field gradients reduces the penetration
of the magnetic field into the surrounding tissue. Thus, in the
present invention, in order to magnetize the magnetic carrier of a
therapeutic agent to saturation, a second relatively uniform
magnetic source is used which can penetrate deep into the selected
delivery site and/or tissue of interest. In one embodiment, this
second relatively uniform source is applied externally through use
of large electromagnets. In another embodiment, the second
relatively uniform magnetic source is implanted internally. In this
embodiment, the second relatively uniform magnetic source may be
separate from the implant or part of the implant that also creates
strong magnetic field gradients.
[0035] Further, the invention provides a method of delivering an
activated cell to a location in a body, the method comprising
providing the activated cell comprising a magnetizable particle;
providing an external magnetic field; providing an implantable
surface having a plurality of magnetizable features characterized
by regions of high field gradients, said magnetizable features
distributed throughout the implantable surface; advancing the
activated cell by influence of the external magnetic field and the
high field gradients toward the location in the body and thereby
delivering the activated cell.
[0036] Magnetic cell delivery is accomplished by the use of the two
source method for magnetic drug delivery to magnetizable
implantable surfaces. Biological cells, including, but not limited
to endothelial and stem cells, are loaded with magnetizable
particles (e.g., superparamagnetic nanoparticles) and form magnetic
cells. Cells can be isolated from the patients themselves or
obtained from maintained cell lines. Magnetizable particles now
provide cells with a large collective magnetic moment in the
presence of the uniform fields used within this method to deliver
magnetic cells to magnetic implants within the body. The cells are
then injected into the body by arterial puncture, catheter release,
or intravenous injection. The externally applied magnetic field
does not serve to direct the cells to the implant. It saturates
their magnetic moment, along with the magnetic moment of the
implanted magnetizable implant. The high local magnetic field
gradients to the magnetic moment and geometry of the implant
provide the strong magnetic forces necessary to capture the cells
out of blood flow. These cells can serve as a delivery vehicle for
magnetic particles loaded with therapeutics or biologics (e.g.,
drugs, radioisotopes, antibodies, retroviruses, etc) or they can be
used in promoting healthy tissue growth for endothelialization of
an implant, wound healing, or otherwise needed tissue
regeneration.
[0037] Magnetic nanoparticles are delivered by endocytosis into
cells, which can be then delivered magnetically to implantable
surfaces, e.g., stents. These cells can be used as a vehicle for
mass transport of drug loaded particles, or as a means to deliver
various cell types such as adult or embryonic stem cells, as well
as endothelial cells. This invention can be used for wound healing,
in vivo engineering of new healthy tissues, targeted delivery of
cells loaded with magnetic drug for local targeting of pathologies
such as heart disease, cancer, and nervous system disorders,
targeting of autologous and allogous cell types for wound healing
and in vivo tissue engineering.
BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS
[0038] The invention will be described in conjunction with the
following drawings in which like reference numerals designate like
elements and wherein:
[0039] FIGS. 1A and 1B are diagrams of an exemplary embodiment of
the magnetizable object of the present invention depicting a
magnetic stent as the magnetizable object implanted in an artery.
In this embodiment, the magnetizable stent has segments bearing a
magnetizable compound which was electro-deposited as a mesh-like
structure on some of the struts of the stent. Darkened bars depict
the segments (or deposits) of the magnetizable compound which has
been deposited on the stent. As shown in FIG. 1A, blood flow
through the artery brings a magnetic drug (which is a therapeutic
agent associated with a magnetic carrier/particle) in proximity
with the implanted stent. As shown in FIG. 1B, as blood flows
through the stent, in the magnetic carrier bearing the therapeutic
agent is attracted to the segments that have the magnetizable
compound. Thus, the drug can be captured on the surface of the
segments or in a close proximity to the segments.
[0040] FIGS. 2A and 2B are scanning electron microscopy images of a
capture of magnetic carriers/particles (e.g., beads) obtained using
a wire mesh electroplated with a magnetizable compound such as
Cobalt Nickel (Co--Ni) alloy. FIG. 2A shows the mesh before
exposure to the magnetic carriers/particles, and FIG. 2B shows the
mesh after exposure to 2.8-micron magnetic carriers/particles.
[0041] FIG. 3 is a schematic illustration of the preferred
embodiment of the magnetic delivery system of the invention.
[0042] FIG. 4A is a schematic illustration of a top view of
micro-channels having walls seeded with magnetic particles.
[0043] FIG. 4B is a chart depicting a flow of micro-particle
solutions through micro-channels shown in FIG. 4A.
[0044] FIGS. 5A and 5B are fluorescent microscopy images
demonstrating a capture of 2 .mu.m and 350 nm diameter magnetic
particles on the electroplated 316L Stainless Steel mesh (5A) and
unplated mesh (5B) when exposed to magnetic field under 15 cm/s
flow velocity at 1% concentration in DI water. Minimal capture was
seen on unplated mesh (5B) where a 500 Gauss field was still
applied.
[0045] FIG. 6 includes images of endothelial cells with
internalized magnetic nanoparticles: vascular endothelial cells
with internalized fluorescent magnetic nanoparticles (top left);
confocal image of nuclear stained endothelial cells with
internalized magnetizable nanoparticles (top right); magnetic
endothelial cells captured to the wires of a magnetized stent mesh
(bottom left and right).
[0046] FIG. 7A is an image of BAECs with internalized 350 nm
diameter nile red polystyrene magnetizable particles, 1 hour after
being passed and re-plated at a 1:6 splitting ratio, shown herein
at 20.times. magnification.
[0047] FIG. 7B is an image of BAECs with internalized 350 nm
diameter nile red polystyrene magnetizable particles, 1 hour after
being passed and re-plated at a 1:6 splitting ratio, shown herein
at 40.times. magnification.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The invention was driven by a desire to develop a system
capable of targeted delivery of magnetizable particles to a
location within a body. Inventors have discovered that magnetizable
particles are attracted to regions of the strongest magnetic field
gradients and devised a two source system that produces strong and
highly localized field gradients inside the body utilizing (1) a
magnetizable object implanted in a body as an internal source of a
magnetic gradient and (2) an external source of a magnetic field.
Unlike the one source systems known in the prior art, this
invention utilizes two sources of magnetic influence for targeted
delivery of magnetizable particles to areas on the magnetizable
object implanted in a body and/or in the near proximity
thereto.
[0049] Accordingly, the invention provides a magnetic delivery
system for delivering a magnetizable particle to a location in a
body, the device comprising a magnetizable object implanted in the
body, wherein the magnetizable object includes a plurality of
segments distributed throughout the magnetizable object and wherein
the segments are configured to provide a magnetic gradient for
attracting the magnetizable particle and an external source of a
magnetic field capable of (i) magnetizing the magnetizable particle
and (ii) increasing a degree of magnetization of the magnetizable
object and thereby creating the magnetic gradient.
[0050] This invention provides the ability to dose the site
repeatedly over time with various substances (the same or a
different kind) presenting a clear clinical advantage over known
delivery systems. The long shelf-life of controlled release spheres
and non-drug-coated stents, compared to the expensively sterilized
and briefly storable drug-eluting stents is another benefit of this
invention.
[0051] Further, the magnetic delivery system of the invention can
be used together with drug-eluting implants to provide an
enhancement for additional doses along the lifetime of the implant.
The use of drug eluting implants is limited because of
complications related to implantation, cracking of the polymer
layer, limited dose size, shelf life, and the fact that they can
only provide a single dose. The magnetic delivery system of the
invention can be used with endovascular and extravascular implants
for treatment of localized tumors. Implants in these cases would
have the sole or primary function of facilitating local magnetic
drug delivery, and could be implanted by catheter, or in cases
where open chest surgery is already required to excise tumors,
implanted extravascularly over vessels or organs. This could
provide a future option for local chemotherapy at the same site
should carcinomas be found to be re-growing during remission.
[0052] It should be understood that the benefits of magnetic
delivery system of the invention must not come at the cost of
increased risk in other arenas, such as chemical tolerance of a
magnetic coating or final compositions of polymer and magnetite
crystals. It is preferred to utilize FDA approved magnetic or
magnetizable particle composites, as well as soft magnetic coatings
and magnetic alloys in order to explore the range of manufacturing
capabilities that maintain the fundamental essence of the
technology such as controllable local delivery of magnetizable
particles loaded with a drug and/or a cell to the segments of a
magnetizable object. While both soft magnetic coatings and varied
alloy composition appear to possess functionality for adapting
implants to this magnetic drug delivery system, it is possible that
their chemical effects and responses to MRI will differ. As
biocompatibility is important in clinical testing, this system
provides desired flexibility in the design which makes it much more
attractive to the industry.
[0053] Regarding MRI, under development is a technology which uses
magnetic material to enhance MRI safety and quality (Biophan, MA).
This opens the possibility of achieving a balance between such
enhancements and the point of magnetization of an implant that
would create safety issues relative to movement or torquing of the
implant. The current invention provides enough flexibility in the
design that the options of patient receiving an MRI would not be
compromised. One skilled in the art using the guidance provided in
this disclosure would be able to design a magnetic drug delivery
system that would not preclude safe and effective MRI procedures
for patients receiving the implants in accordance with the
invention. Similar concerns can be addressed for other types of
treatment or diagnostic methods wherein magnetic interference may
be a problem.
[0054] Advantages of the invention further include reduced immune
cell and capillary concerns regarding the ability of magnetic drug
particles to remain in tact to reach their target in blood flow,
and highly accurate targeting of rare cell types not available in
mass quantities, improved functionality of stem cell therapy.
Definitions:
[0055] The term "magnetizable object" as used herein refers to an
object capable of creating strong magnetic gradient near its
surface (i.e., a local gradient) for attracting magnetizable
particles which is implanted in a body. The magnetizable object has
a plurality of segments distributed throughout the magnetizable
object. The segments are configured to provide a magnetic gradient
for attracting the magnetizable particle. This term is used
interchangeably with the term "magnetic implant" in the
specification.
[0056] Magnetizable objects should not be equated with magnetizable
particles unless specifically indicated. For example, in certain
embodiments, the magnetizable object is formed by a cluster of
gradient forming particles, wherein a surface of each gradient
forming particle represents a segment of the magnetizable
object.
[0057] The term "gradient forming particles" as used herein refers
to a group or a cluster of particles sized less than 10 micrometers
in a diameter each and capable of creating a magnetic gradient for
attracting the magnetizable particle. Thus, gradient forming
particles play a role of the plurality of segments. Gradient
forming particles are made from the same materials as the
magnetizable objects made from and can have a variety of shapes,
preferably a sphere.
[0058] The term "segment" as used herein with relationship to the
magnetizable object of the invention refers to an area on the
magnetizable object that is characterized by higher magnetization
as compared to other areas of the magnetizable object. In other
words, in the absence of segments, the magnetizable object is not
capable of providing a magnetic gradient (in the presence of
magnetic field) that is sufficiently strong to capture magnetizable
particles. Thus, a combination of segments with higher
magnetization with other areas of lower magnetization on the
magnetizable object creates localized high gradients. It should be
understood that most surfaces are not ideal and have naturally
uneven areas which may magnetize differently. However, for the
purposes of this invention, such surfaces are not contemplated as
they are not capable of providing sufficiently strong gradient to
attract the magnetizable particle.
[0059] Non-limiting examples of segments are patterns of
indentations and/or ridges of various length, width, depth and
shape on the magnetizable object. In preferred embodiments,
segments are made by application (e.g., deposition) of a
magnetizable compound on a surface of the implant. Referring to a
plain view of the magnetizable object such as a stent or a spiral,
each coil can serve as a segment. In certain embodiments, the
segments comprise patterns of materials with different degrees of
magnetization.
[0060] By the term "magnetizable compound", as used herein, it is
meant a material that conducts magnetic flux strongly. Examples of
magnetizable compounds useful in the magnetizable objects (i.e.,
implants) of the present invention include, but are not limited to,
cobalt, iron, iron oxides, nickel, and rare earth magnetic
materials and various soft magnetic alloys (e.g., Ni--Co). In one
embodiment, the magnetizable compound is magnetized only in the
presence of externally applied magnetic fields. Examples of these
types of magnetizable compound include, but are not limited to,
superparamagnets and soft ferromagnets. In other embodiment,
magnetizable compounds known as ferromagnets, which can be
permanently magnetized, are used.
[0061] The term "coating", as used herein, includes coatings that
completely cover a surface, or a portion thereof (e.g., continuous
coatings, including those that form films on the surface), as well
as coatings that may only partially cover a surface, such as those
coatings that after drying leave gaps in coverage on a surface
(e.g., discontinuous coatings). The later category of coatings may
include, but is not limited to a network of covered and uncovered
portions. Coatings can be flat or raised above the surface or
embossed on the surface (e.g., a ridge) or it can be in a shape of
dots or other shapes creating a pattern. A combination of various
coatings can also be used.
[0062] Coating can be made from a magnetizable compound (e.g.,
stainless steel, soft magnetic alloys) and a non-magnetizable
compound (a polymer). Selecting the appropriate combination of
coating and support materials, it is important that the
magnetizable object prepared based on the selection will have a set
of segments on its surface that will enable the creation of a
localized magnetic gradient. For example, if the support or a
surface of the magnetizable object is made from a magnetizable
compound, material(s) of the segment can have a higher or a lower
degree of magnetization or they can be made from non-magnetizable
materials. On the other hand, if the support or a surface of the
magnetizable object is made from a non-magnetizable compound,
material(s) of the segment must be made from a magnetizable
compound.
[0063] The term "magnetizable particle" is used interchangeably
with the term "magnetic carrier" and the term "magnetic particle"
throughout this disclosure.
[0064] Exemplary therapeutic agent loaded magnetizable particles
comprise a biodegradable matrix (e.g., polymer, protein, DNA)
containing, for example, 0-40% by weight therapeutic agent (e.g.,
drug) and 20% magnetite by weight. Exemplary drugs of low water
solubility such as paclitaxel, or commonly used chemotherapeutics
like doxirubicin can be used for applications such as coronary
artery disease and hepatic cancer, respectfully.
[0065] The term "activated cell" as used herein means a cell
containing magnetizable particles. This term is used
interchangeably with the term "magnetic cell."
[0066] The term "endocytosis" means the uptake particles by cells
and includes receptor mediated endocytosis.
[0067] Magnetizable Object
[0068] The magnetizable object of the invention is an implant
capable of creating strong magnetic gradient near its surface
(i.e., a local gradient) when implanted, such that the magnetic
gradient attracts magnetizable particles. The magnetizable object
has a plurality of segments distributed throughout the magnetizable
object. The segments are configured to provide a magnetic gradient
for attracting the magnetizable particle. This term is used
interchangeably with the term "magnetic implant" or ":magnetic
device" in the specification.
[0069] In certain embodiments, the magnetizable object is in the
shape of a cylinder, a cylindrical rod, a cube, a cubical rod, a
spring, a circular disc, a mesh, a ring, a wire, a sphere or a
combination thereof. In certain embodiments, the magnetizable
object is a stent, a pacemaker, a catheter, a tube, a vascular
graft, an artificial joint, an artificial bone, a prostate seed, an
aneurysm coil, a surgical staple, and a suture.
[0070] It is also contemplated in this invention that the
magnetizable object is does not a have a continuous surface and can
be formed from distinct entities such as, for example, a cluster of
gradient forming particles, wherein a surface of each gradient
forming particle represents a segment of the magnetizable
object.
[0071] In certain embodiments, a size of each segment is selected
to commensurate with a size of the magnetizable particle such that
at least one spatial dimension of each segment does not exceed by
more than about 10,000 times at least one spatial dimension of the
magnetizable particle. In certain embodiments, the size of each
segment is selected to commensurate with the size of the
magnetizable particle such that at least one spatial dimension of
each segment does not exceed by more than about 100 times at least
one spatial dimension of the magnetizable particle.
[0072] Those skilled in the art would be able to select material
for making the magnetizable object such that it would be magnetized
in the presence of an external magnetic field as those materials
are know or are being developed(e.g., metals, metal alloys and rear
earth elements). In certain embodiments, the magnetizable object is
made from at least one of materials selected from the group
consisting of cobalt, nickel, iron, manganese, samarium and
neodymium.
[0073] In certain embodiments, the magnetizable object is in a
shape of a support made from a metal, a rare earth element, a
ceramic, a polymer or a combination thereof. In certain
embodiments, the magnetizable object is in a shape of a coating on
the support, wherein the coating is made from a metal, a rare earth
element, a ceramic, a polymer or a combination thereof. A coating
is defined above and is preferably made from a magnetizable
compound.
[0074] The magnetizable object of the present invention comprises a
structural supporting implant made from a biocompatible metal,
ceramic, a rare earth element, a polymer or a combination thereof.
The magnetizable object of the present invention further comprises
a magnetizable compound in a shape of a segment or a coating.
[0075] In one embodiment, the magnetizable compound is segmented on
the structural supporting implant to provide regions of high field
gradients distributed throughout the implant. In another
embodiment, the magnetizable compound is uniformly coated on the
structural supporting implant, and the magnetized state of the
coating is either locally recorded (e.g., embossed and/or indented)
to provide regions of high field gradients distributed throughout
the implant or uniformly magnetized. In yet another embodiment, the
implant itself comprises the magnetizable compound.
[0076] In one embodiment, the structurally supporting implant
itself is made entirely from alloys of magnetizable compounds.
[0077] In another embodiment, a layer of magnetizable compound is
placed on the structural supporting implant as a coating with a
thickness ranging from about 2 nanometers to about 200 microns or
in segments. When placed on as segments of magnetizable compound,
it is preferred that these segments extend through and around the
structural supporting implant from one end of the implant to the
other. Similar arrangements include, but are not limited to,
multiple rings extending over the implant and a mesh-like structure
surrounding the implant such as depicted in FIGS. 1 and 2.
[0078] The magnetizable compound can be applied to the structural
supporting implant by various methods including, but not limited
to, electro-deposition, evaporation and sputtering, and by chemical
reactions.
[0079] Magnetic particles may not always be uniformly attracted to
implants that are simply coated with magnetic material. This is due
to the fact that magnetic domains in such coatings are hard to
control. Those skilled in the art would be able to select various
method of patterning the magnetic coating to control domain
patterns in the devices of the present invention without undue
experimentation. One method involves laser-assisted
electrodeposition of alloys of magnetic metals such as Co, Ni and
Fe. In this method, the implant, preferably a stent is used as a
cathode during the deposition and a voltage slightly below
electroplating threshold is applied. Magnetic material can then be
electroplated only in those spots that are exposed to a focused
laser beam. Another method for patterning of the implant with the
magnetizable compound involves the use of magnetic nanoparticles
and nanorods separately prepared. These may either be purchased or
made by electroplating into nanotemplates. Magnetic nanoparticles
are then deposited onto the implant through a process called
dielectrophoresis. In this process the implant is placed into an
aqueous solution containing magnetic nanoparticles in between two
insulated electrodes. Application of a relatively high frequency
(100 KHz-1 MHz) electric field creates strong, high frequency
electric field gradients on the implant that attracts the
nanoparticles.
[0080] Another method for patterning the implant involves recording
of magnetic domain pattern on the implant using methods closely
related to those that are employed in magnetic information storage
devices. One such approach involves laser assisted thermomagnetic
recording. In this method, the implant is first uniformly
magnetized by a strong external field. Subsequently selected spots
are heated by a laser in the presence of a reversed magnetic field.
The strength of the reversed field is sufficient to reverse
magnetization of heated spots, but insufficient to reverse
magnetization of unheated spots. Spots that have been heated by a
laser are then magnetized in opposition to the rest of the implant
magnetization.
[0081] In a preferred embodiment of the device of the present
invention, the implant is a stent comprising a metallic tube such
as, but not limited to, a corrugated stainless steel tube, coated
with a magnetizable compound such as cobalt, iron, iron oxides,
nickel, or other rare earth magnetic materials or alloys, followed
by a passivating layer of a biocompatible material. In this
embodiment, it is preferred that the stent maintain its capability
for balloon expansion and complete mechanical integrity so that it
is useful not only in selective targeting of therapeutic agents,
but also in keeping the lumen into which it is inserted open.
Accordingly, this stent-based delivery system, when used in
procedures such as PTCA, preserves the beneficial properties of
stents (preventing vascular recoil and remodeling) while delivering
therapeutic agents, preferably drugs or radionuclides, that inhibit
or measure initial thickening, respectively.
[0082] The segments of magnetizable compound coated or otherwise
deposited on the magnetizable object (e.g., a stent) of the
invention provide the stent with the capability to attract
arterially injected magnetic carriers including, but not limited to
magnetic particles, magnetic liposomes and ferrofluids
encapsulating or attached to the therapeutic agent, over the entire
stent, thus distributing the therapeutic agent over the entire
stent so that possible clogging of the artery and/or stent by
deposited therapeutic agent is decreased.
[0083] In an alternative embodiment of the device of the present
invention, the implant comprises a plurality of biocompatible metal
or polymer beads, spikes or pellets wherein at least one segment of
each bead, spike or pellet comprises the magnetizable compound.
Various means for implanting such a device into the selected site
are known and can be selected by one of skill in the art based upon
the site of implantation. For example, for implantation into a
blood vessel wall, the device may be delivered via a catheter-based
system. In this embodiment, it is preferred that the catheter be
equipped with a balloon coated with the plurality of implants so
that upon expansion of the balloon the implants are lodged into the
blood vessel. Alternatively, the balloon can be coated with a
plurality of implants which have been modified to further comprise
a specific receptor for the endothelial lining which bind to the
endothelial lining upon contact. In another embodiment, the device
can be implanted by injection of the plurality of magnetizable
beads administered to the site of interest and lodged into the
tissue by application of external magnetic field gradients.
Alternatively, a device of the present invention comprising a
plurality of implants can be injected directly into the site of
interest by a means similar to a biopsy needle so as to provide a
region of high internal magnetic field that attracts magnetic
therapeutic agent-containing particles. In these embodiments, it is
preferred that the plurality of implants be scattered over the site
of treatment so that the therapeutic agent is dispersed over the
treatment site and possible clogging of the artery by deposited
agent is decreased.
[0084] Another aspect of the present invention relates to the use
of these devices in the targeted delivery of a therapeutic agent to
a selected site in a subject. In this aspect, a device of the
present invention is first implanted into a subject at a selected
site. The site of implantation in the subject is selected based
upon where targeted treatment is desired and the mode of
administration for the therapeutic agent.
[0085] For example, for PTCA procedures, the treatment site is a
coronary artery at a region of stenosis. The therapeutic agent is
preferably a drug that is administered intravascularly to prevent
restenosis at this site. The implant, preferably a stent, is thus
also implanted in the coronary artery at the site of stenosis. In a
preferred embodiment, the device of the present invention is
implanted by catheterization in accordance with well-known
procedures.
[0086] While a primary application of these devices is for
treatment of cardiovascular disease and/or restenosis, alternative
functions for implants of the present invention are envisioned.
These include, but are not limited to, treatment of tumors (benign
and malignant), bacterial or viral infections, cysts, internal
wounds, and anti-rejection treatments for transplant patients.
These treatments can be performed by implantation of the device at
the mouth of the site of blood supply, or placement of a device
with a plurality of implants within or on the site itself, followed
by systemic administration of therapeutic agent attached to or
encapsulated in a magnetic carrier such as magnetic particles,
magnetic liposomes or a ferrofluid.
[0087] There are numerous other potential sites of implantation
envisioned for a magnetizable/magnetic device of the present
invention. It is important to note that in all of these cases, a
magnetic carrier such as magnetic particles, magnetic liposomes or
a ferrofluid is carrying or bound with the therapeutic agent. These
include, but are in no way limited to, the lymphatic system for
treatment of swollen or infected glands, and damaged or occluded
vessels; in the bile ducts for pancreatic cancer patients; in the
ureter or urethra for kidney drainage or kidney/bladder infections;
in or on the surface of the larynx, trachea, or lung surface for
treatment of respiratory disorders or cancers with the use of an
inhalable solution of magnetic particles bound with drug. Further,
a device of the present invention placed within the esophagus could
be used to capture magnetic particles contained within a viscous
creeping solution for treatment of cancers or infection.
[0088] In addition, devices of the present invention surgically
placed within the brain for local delivery of a therapeutic agent
could be used to capture magnetic particles, preferably
nanoparticles, with lipid-soluble or other permeability-enhancing
coatings to allow targeting of intra-arterial chemotherapeutics.
While intra-arterial chemotherapeutics are already practiced in
various manners to treat brain tumors, magnetic targeting of such
treatments may limit healthy neural tissue exposure to chemotherapy
while maximizing dosage levels.
[0089] External Source
[0090] The external source of a magnetic field of the present
invention is capable of (i) magnetizing the magnetizable particle
and (ii) increasing a degree of magnetization of the magnetizable
object and thereby creating the magnetic gradient. Those skilled in
the art using guidance provided in this disclosure will be able to
select the proper source and its capabilities without undue
experimentation. The preferred external source is an
electromagnet.
[0091] Magnetizable Particle
[0092] In certain embodiments, the magnetizable particle has a
diameter of less than 10 micrometers. In certain embodiments, the
magnetizable particle has a diameter from about 10 nm to about 1000
nm.
[0093] It is also preferred that magnetic carriers such as magnetic
particles or magnetic liposomes comprise magnetite. Magnetite is a
member of the spinel group with the standard formula Fe2O3 or
Fe3O4. Magnetite particles to be incorporated in the magnetic
particles or magnetic liposomes encapsulating the therapeutic agent
are preferably around 10 nm in diameter and are dispersed within
the magnetic particle or magnetic liposome to account for 10-50% of
sphere volume.
[0094] In cases where use of a magnetic carrier comprising
microspheres or nanospheres for encapsulation of the therapeutic
agent is required, the microspheres or nanospheres preferably
comprise a biodegradable polymer, such as poly(lactic acid) PLA
and/or poly(lactic-co-glycolic acid) PLGA, which cause minimal
inflammatory response upon degradation. As will be understood by
those of skill in the art upon reading this disclosure, numerous
other biodegradable polymers are known, such as polyhydroxybutyrate
and elastomeric poly(ester-amide), which may also be used in these
microspheres or nanospheres. Ultimate selection of the
biodegradable polymer for encapsulation of the drug is based upon
desired degradation times, side effects, and drug conjugation.
[0095] In certain embodiments, the magnetizable particle has a
diameter from 10 nm to 500 nm. In certain embodiments, the
magnetizable particle comprises at least one of materials selected
from the group consisting of Fe.sub.2O.sub.3, Fe.sub.3O.sub.4,
FeNi, FePt, Fe, CoNi alloy and optionally a biodegradable polymer.
In certain embodiments, the magnetizable particle is in a form of a
superparamagnetic colloidal fluid.
[0096] In certain embodiments, the magnetizable particle comprises
a therapeutic agent and wherein the magnetic delivery system
functions as a targeted drug delivery system.
[0097] In certain embodiments, the magnetizable particle is loaded
within a cell, thereby forms a magnetic cell and wherein the
magnetic delivery system functions as a targeted cell delivery
system. In certain embodiments, the magnetic cell comprises a
therapeutic agent, such that the therapeutic agent is associated
with the cell, the magnetizable particle or both and wherein the
magnetic delivery system functions as a targeted drug and cell
delivery system.
[0098] Those skilled in the art should be able to make magnetizable
particles utilizing known methods and materials and the guidance of
this disclosure.
[0099] Therapeutic Agent
[0100] The therapeutic agent to be delivered by the method if the
invention is encapsulated in, attached to, or dispersed in a
magnetic carrier/particle. For example, the therapeutic agent may
be encapsulated in magnetic particles including, but not limited
to, microspheres and nanospheres or magnetic liposomes.
Alternatively, the therapeutic agent may be dispersed in a
ferrofluid or in a colloidal fluid. In embodiments wherein the
magnetic carrier involves magnetic particles and/or liposomes, it
is preferred that the particles and/or liposomes be less than 10
micrometers in size to prevent clogging of any small
arterioles.
[0101] Selection of a therapeutic agent to be encapsulated within
the magnetic carrier such as magnetic particles or magnetic
liposomes or dispersed in a magnetic carrier such as ferrofluid and
used with the devices of the present invention is dependent upon
the use of the device and/or the condition being treated and the
site of implantation of the magnetizable device. For example,
multiple therapeutic agents have been experimented with and tested
for prevention of restenosis following PTCA and any of these can be
used with the stents of the present invention. Some examples of
such therapeutic agents include, but are not limited to,
antiplatelet agents such as aspirin, glycoprotein receptor
antagonists, and cilostazol for prevention of thrombus formation by
interfering with platelet aggregation; anticoagulants such as
heparin, hirudin, and coumadin for prevention of thrombus formation
by blocking the coagulation pathway; calcium channel antagonists
for reducing vascular recoil and remodeling; growth factor
inhibitors, such as trapidil, an inhibitor of PDGF;
immunosuppressants such as Rapamycin (Sirolimus; Rapamune.RTM.);
anti-inflammatory agents; and anti-proliferation agents such as
Actinomycin D (Cosmegen.RTM.), Estrogen (Estrodiol.RTM.), and
Paclitaxel (Taxol.RTM.). Thus, for PTCA procedures and tumor
treatments using a magnetizable stent of the present invention, a
preferred therapeutic agent for encapsulation may be Actinomycin D,
Rapamycin or Paclitaxel. Other therapeutic agents that can be
administered, include, but are not limited to radioactive
materials, gene vectors, genetically modified viruses such as
retroviruses, and living cells, such as endothelial cells, that are
attached to magnetic particles. The ability to attract endothelial
cells to the implant will greatly decrease the time it takes to
form an endothelial layer on the stent, and may inhibit the growth
and migration of smooth muscle cells that are largely responsible
for the neo-intimal growth.
[0102] Magnetically targeted therapeutic agent delivery achieved
through use of the present invention allows for reduced initial
inflammation response often experienced in clinical tests of
polymer/drug-coated stents, as the amount of polymer necessary for
targeted delivery can be reduced. In addition, since the magnetic
vehicles are deliverable via arterial injection, minimally invasive
means for delivery of the therapeutic agents at selected times can
be used. Thus, in a procedure such as PTCA, a therapeutic agent may
not need to be delivered until sufficient time has elapsed for
endothelium growth over the stent. Further, multiple doses of the
therapeutic agent or combinations of therapeutic agents can be
administered.
[0103] In some embodiments, the device of the present invention
and/or magnetic carrier of the therapeutic agent may require
magnetization just prior to, during or following administration of
the therapeutic agent. In these embodiments, the device and/or
magnetic carrier of the therapeutic agent is magnetized by a
magnetic field applied externally to the subject.
[0104] The utility of the devices of the present invention in
targeting a therapeutic agent to the site of their implantation is
based upon obtaining an attractive magnetic force upon the injected
magnetic carrier of the therapeutic agent that overcomes the drag
resistance in moving towards the wall. The magnetic force can be
optimized by using a device that contains a plurality of magnetic
features, producing strong magnetic field gradients that pull the
therapeutic agent encapsulated or attached to the magnetic carrier
to desired locations on the surface of the implant of the device.
The implant of the device is designed to be in direct or proximal
contact with the transporting medium for the therapeutic agent so
that the therapeutic agent-containing magnetic carrier will be
attracted most strongly. For example, in one embodiment as depicted
in FIGS. 1A and 1B, the transporting medium is blood of a blood
vessel that has been injected with a colloidal solution of
therapeutic agent-containing magnetic particles. In another
embodiment, the transporting medium is that of an aerial passageway
that has been exposed to a gaseous solution of therapeutic
agent-containing magnetic particles in the form of a nasal spray or
inhalation. In yet another embodiment, the transporting medium
comprises lymphatic or cerebrospinal fluid that has been injected
with therapeutic agent-containing magnetic particles that are
attracted to a device of the present invention in the lymph nodes
or brain.
[0105] In a preferred embodiment of the invention, high magnetic
field gradients within the body are applied to the magnetizable
particle which is in a form of an injected superparamagnetic
colloidal fluid carrying a therapeutic agent with the aid of
uniform magnetic field created by the external magnet toward a
target, an endovascular implant (e.g., a stent). The design of the
magnetizable object involves patterning the surface of endovascular
implants with a soft magnetic coating capable of producing high
local magnetic field gradients within the body. A conceptual
diagram of the preferred embodiment can be found in FIG. 3.
[0106] In the preferred embodiment of the invention, the magnetic
delivery system is used for the treatment or prevention of coronary
restenosis following angioplasty. The magnetic delivery system of
the invention is a viable alternative or enhancement to
drug-eluting stents, offering increased control of dose size, the
ability to treat a site repeatedly, and a wide array of
applications for treatment of other pathologies.
[0107] As described in detail below, the magnetic delivery system
utilizing micron and sub-micron scale magnetic particles for
site-specific delivery of pharmaceuticals and magnetic cells is
described based on the theoretical and experimental models (e.g.,
parallel plate and pipe flow analysis, and cell culture
models).
[0108] In the preferred embodiment, the present invention is used
for treatment and prevention of coronary restenosis. The
magnetizable object is in a shape of a stent as described below.
The magnetic delivery system will operate by first implanting the
stent at designated sites in the cardiovascular system, and then
attracting injected or otherwise administered doses of magnetically
susceptible drugs as magnetizable particles to the sites of the
implant the with the aid of the external magnetic field and
magnetic field gradient created by the implant (i.e., stent). In
vitro experiments demonstrated that magnetizable particles were
attracted to regions of the strongest magnetic field gradients.
[0109] Ability of local magnetic field gradients to trap magnetic
particles can be demonstrated based on computational models.
Adaptation of these models to the analysis of a magnetizable steel
mesh (i.e., the magnetizable object) provided insight for implant
magnetization, external magnetic field parameters, particle and
vessel sizes, as well as preliminary dose concentrations for
beginning in vitro flow simulations.
[0110] Determination of the attractive magnetic forces in the
preferred embodiment that is necessary to capture agent-containing
magnetic particles was achieved by modeling the force on a single
magnetic particle in blow flow due to the magnetic field from a bar
of magnetic material as follows.
[0111] A rigid sphere transported along in Poiseuille flow through
a tube has been shown to be subject to radial forces which tend to
carry it to a certain equilibrium position at about 0.6 tube radii
from the axis, irrespective of the radial position at which the
sphere enters the tube (Segre, G. and Silberberg, A. J. Fluid Mech.
1962 14:136). Further, it has been shown that the trajectories of
the particles are portions of one master trajectory, and that the
origin of the forces causing the radial displacements is in the
inertia of the moving fluid (Segre, G. and Silberberg, A. J. Fluid
Mech. 1962 14:136).
[0112] Before the effects of multiple particles inside a lumen
coated three dimensionally with magnetic segments can be modeled,
it must first be ensured that were there only one particle, and one
bar of magnetic material, that the magnetic attraction would be
strong enough that in fluid flow the particle would be able to
overcome the drag resistance and attach to the implant.
Accordingly, the magnetic force has been modeled as Fz, the force
in the z direction on a particle by a bar of magnetic material
directly placed along the lumen of the vessel. From basic
electromagnetic theory it is known that the force on a magnetizable
spherical bead in an external magnetic field is given by Equation
1. F -> = .mu. 0 .function. ( m -> .gradient. ) .times.
.times. H -> . Eq . .times. 1 m -> = 3 .times. .chi. .chi. +
3 .times. V .times. H -> Eq . .times. 2 ##EQU1##
[0113] In Equations 1 and 2, .mu.o is the magnetic permeability of
free space, H is the total external magnetic field, m is the
magnetic moment of the particle attracted to the implant, V is the
volume of the particle, and .chi. is the magnetic susceptibility of
the particle. As can be seen from Equation 1, the force is directly
proportional to both the magnetic moment of the particle and the
magnetic field gradient. The trajectory of the particle is computed
numerically according to Eq. 3 by modeling the movement of the
particle towards the wall in a velocity flow field. A particle is
captured if its trajectory terminates on the site of the implant:
.DELTA. .times. .times. z = F z 6 .times. .pi..eta..alpha. .times.
.times. v x .times. .DELTA. .times. .times. x Eq . .times. 3
##EQU2##
[0114] Using values ranging from extreme to moderate to account for
varying inducible magnetic field strength, particle size, and blood
flow velocity, simulations have shown that for a variety of
different physiologically significant parameters, F.sub.z can
indeed overcome the vertical drag resistance. These equations must
then be modeled based around a circumference equally spaced with
magnetic segments, and a fluid containing multiple magnetic
particles.
[0115] In vitro experiments of various kinds have been conducted
which demonstrate the utility of devices of the present invention
in drug delivery. In one set of experiments, a 316L stainless steel
stent was coated with cobalt by electro-deposition. This mesh was
then placed within a flow chamber, and magnetic particles were
flown through the chamber at varying velocities to assess the
capability of the mesh to attract them. Aggregation of magnetic
particles on the magnetic stent was increased as compared to a
non-magnetic stent.
[0116] In another set of experiments, magnetizable beads were
embedded in microchannels and magnetic microspheres were injected
into the channel under applied magnetic field in order to determine
if the embedded beads could capture the microspheres. Again the
magnetic microspheres aggregated on the magnetized embedded
beads.
[0117] Experiments using microfabricated fluidic channels were
carried out to show a uniqueness of using the magnetizable stent
with the surface-modified nanoperticles. Microfluidic channels of
various sizes (50.times.50, 100.times.100, 500.times.500 .mu.m
cross-section) were fabricated in polydimethyl siloxane (PDMS). The
walls of the micro-channels were seeded with
superparamagnetic/latex composite microparticles (5 .mu.m diameter)
(See Forbes Z G, Yellen B B, Barbee K A, Friedman G, An Approach to
Targeted Drug Delivery Based on Uniform Magnetic Fields, IEEE
Transactions on Magnetics, 39 (5): 3372-3377, (2003)). Top views of
the micro-channels and of the entire experimental set-up are
schematically illustrated in FIG. 2. Various concentration
solutions of commercially available magnetic particles of various
sizes (1 .mu.m, 3 .mu.m, 5 .mu.m, 7 .mu.m) were observed to flow in
the channels while applying external relatively uniform magnetic
field varied up to 300 Oe (0.03 Tesla). When no magnetic field is
applied, magnetic particle solution flows freely. When the applied
magnetic field exceeded 30 Oe aggregation of particles is observed.
Stronger fields lead to faster and more pronounced attraction of
magnetic particles in solution toward the walls. In lower
concentration particle solution, particle chains attracted to the
walls form relatively slowly (10 s of seconds). In higher
concentration particle solution, aggregation of particles on the
micro-channel walls occurs very quickly. Examples of aggregation of
magnetic carriers in the micro-channels are shown under various
flow conditions and fields were observed.
[0118] The possibility of making stents that could attract magnetic
particle carriers under similar conditions was also confirmed using
the technique. Commercially available (e.g., Cordis Corp) stainless
steel stents were first confirmed not to attract commercially
available magnetic particles (100 nm, 1 .mu.m and 5 .mu.m
diameter). A basic set-up for electroplating these stents was
developed and stents were coated with 1 .mu.m thick layer of
cobalt. The cobalt coated stents were placed into the magnetic
particle solution and strongly attracted magnetic particles when a
permanent magnet applying a relatively uniform field was placed
above the stents. The cobalt coated stents remained covered by the
magnetic particles even after washing.
Explanation of Basic Principles of the Proposed Method
[0119] The magnetic force dragging isolated magnetic carriers
toward the stent is: {right arrow over (F)}=({right arrow over
(M)}.gradient.){right arrow over (B)} (1) where the magnetic moment
{right arrow over (M)}.apprxeq..lamda.{right arrow over (B)} of the
drug carrying particle is approximately proportional to the total
field {right arrow over (B)} up until saturation which occurs for
most nanoparticles in the range of about 600 Oe (0.06 Tesla). The
total field {right arrow over (B)}={right arrow over
(B)}.sub.ext+{right arrow over (B)}.sub.stent experienced by the
magnetic carriers consists of contributions due to the stent and
due to the external magnet. However, while the field of the
external magnet is much larger than the field of the stent, it is
largely uniform (.gradient.{right arrow over
(B)}.sub.ext.apprxeq.0). The stent, on the other hand, produces
very large gradients near itself because of the presence of very
small magnetized features on it. Thus, from (1) and from above
arguments, the force on magnetic drug carriers can be approximately
written as {right arrow over (F)}.apprxeq..lamda.({right arrow over
(B)}.sub.ext.gradient.){right arrow over (B)}.sub.stent (2)
[0120] The above formula makes it clear that forces capturing drug
carriers are maximized when a strong external uniform magnetic
field is superimposed on a field produced by an insert with tiny
features maximizing field gradients. Inventors believe that this
can not be achieved by a single external magnet. Numerical
simulations have been carried out to study efficiency of magnetic
carrier capture by magnetized inserts. These simulations have
indicated that capture of magnetic carriers as small as 100 nm is
feasible with stents that are patterned with magnetized features
that are 2-3 .mu.m in diameter and about 200-500 nm in
thickness.
[0121] It was observed that magnetic carriers may not always be
uniformly attracted to stents that are simply coated with magnetic
material. This is due to the fact that magnetic domains in such
coatings are hard to control. This invention provides several means
of patterning the magnetic coating to control domain patterns.
[0122] One embodiment involves laser assisted electrodeposition of
alloys of magnetic metals such as Co, Ni and Fe. Stents will be
used as cathodes during the deposition and voltage slightly below
electroplating threshold will be applied. Magnetic material can
then be electroplated only in those spots that are exposed to a
focused laser beam.
[0123] Another approach to stent patterning involves the use of
magnetic nanoparticles and nanorods separately prepared. These may
either be purchased or made by electroplating into nanotemplates.
Magnetic nanoparticles (e.g., ferromagnetic) will then be deposited
onto the stents through a process called dielectrophoresis. In this
process the stent will be placed into an aqueous solution
containing magnetic nanoparticles in between two insulated
electrodes. Application of a relatively high frequency (100 KHz-1
MHz) electric field will create strong high frequency electric
field gradients on the stent that will attract the
nanoparticles.
[0124] Another approach involves recording of magnetic domain
pattern on stents using methods closely related to those that are
employed in magnetic information storage devices. One such approach
will involve laser assisted thermomagnetic recording. In this
method the stent is first uniformly magnetized by a strong external
field. Subsequently selected spots are heated by a laser in the
presence of a reversed magnetic field. The strength of the reversed
field is sufficient to reverse magnetization of heated spots, but
insufficient to reverse magnetization of unheated spots. In the
end, spots that have been heated by a laser, will be magnetized in
opposition to the rest of the stent magnetization.
[0125] The design of the magnetizable object of the invention
(i.e., magnetic implant) began with the selection of
stent-simulating materials of different geometries, mesh sizes, and
metallic content, suitable for in vitro flow experimentation. In
preferred embodiment, stainless steel materials ranging from 316
(316L SS) to 302 (302 SS) grades were chosen, in grid-like mesh
geometries, as well as in the form of a compression spring. A soft
magnetic alloy of Cobalt-Nickel was selected as a convenient
material for increasing the saturation magnetization of the
materials, while retaining a very low state of magnetization in the
absence of the externally applied magnetic field. An electroplating
setup was developed utilizing a cobalt anode, borate bath
containing scaled concentrations of cobalt and nickel, and
controlled by a potentiostat. By combining the use of very weakly
magnetic materials (316L SS) and highly magnetic materials (302 SS)
with varied plating heights of soft magnetic alloy, flow
experiments were conducted to determine the scalability of magnetic
capture over a range of saturation magnetizations. Two flow systems
were used to test the capture of magnetic particles onto the wires
of model stent materials. The first system employed was a parallel
plate flow chamber (PPFC) adaptable to various channel heights and
capable of sustaining high volumetric flow rates needed to obtain
physiologically significant flow velocities. These experiments
provided qualitative results from fluorescent microscopy that
validated model predictions that magnetic particles could be
captured using the proposed design. The system was validated for
particle sizes ranging from 130 nm to 2 .mu.m in diameter, but
optimized for 350 nm and 2 .mu.m diameter superparamagnetic
particles stained with rhodamin for nile red fluorescence. A
characteristic set of experimental data is shown in FIGS. 5A and
5B. These particles were tested and validated for magnetic capture
to both 316L and 304 steel meshes, magnetic capture through layers
of silicone in order to simulate scar tissue, capture of particles
concentrated in porcine blood, and for a range of dose
concentrations and volumes.
[0126] The second system for flow analysis of the proposed design
utilized three-dimensional implants placed within a pipe flow
system. Large vessel scaled implants molded from 304 grade steel
into 5 mm diameter tubes, as well as 3 mm diameter 302 grade steel
compression springs were chosen for testing. These materials
maintain a much higher inherent saturation magnetization due to
their alloy content, but were also electroplated with various
heights of a soft magnetic alloy (e.g., Co--Ni alloy) to examine
capture over a range of magnetic properties. The pipe flow analysis
also allowed to analyze uncaptured magnetic particles from a single
dosage pass using a MicroMag Alternating Gradient Magnetometer
(AGM) (Princeton Measurements, NJ). AGM analysis provided capture
efficiency percentages, as well as insight into numerical capture
capabilities and its variance with dose concentration and material
magnetization. A skilled in the art would be able to use these data
and guidance provided herein in selection of system parameters
(e.g., materials, segments' design, etc.) for applications in vivo
without undue experimentation.
[0127] Further, the magnetic delivery system was tested for
biocompatibility. While the risks of the use of Cobalt-Nickel
coatings and magnetite-based magnetic particles can be further
studied in vivo, prior art literature gave insight into tissue
tolerance. Experiments were conducted to examine any effects on the
growth, morphology, and behavior of endothelial cells as a
non-limiting example due to the magnetic field gradients of the
wires of an electroplated mesh as the magnetizable object. It was
found that endothelial cells not only survived the delivery of a
high concentration of magnetic particles to cultures, but were
actually found to compartmentalize the particles into the cell. The
endothelial cells with internalized magnetic particles (i.e.,
magnetic cells) maintained normal growth, morphology, and behavior
and were captured magnetically to mesh surfaces. Images of magnetic
cells are shown in FIG. 6.
[0128] It was discovered that endothelial cells were able to
tolerate magnetic gradients when grown in the presence of a
magnetically plated mesh. Further, when cells came in contact with
delivered magnetic particles, cells were able to compartmentalize
and uptake the particles. These cultures were also found to attach,
spread, and divide at rates consistent with control cultures, and
to respond to fluid shear stimulation. Magnetic endothelial cells
were also successfully delivered magnetically to the surface of
magnetic mesh, both in static culture as well as in high flow rate
in the parallel plate flow chamber. Significant numbers of these
magnetic endothelial cells were able to survive and grow, even
after exposure to such potentially traumatic magnetic forces. These
results demonstrate that magnetic cells can be used as delivery
vehicles for a drug associated with a cell or a magnetic particle,
as well as for delivery of cells to a desired location in the
body.
[0129] In a preferred embodiment, magnetizable nanoparticles were
delivered by endocytosis into cells, which can be then delivered
magnetically to implantable surfaces, e.g., stents. These magnetic
cells can be used as a vehicle for mass transport of drug loaded
particles, or as a means to deliver various cell types such as
adult or embryonic stem cells, as well as endothelial cells.
Experiments conducted with magnetizable nanoparticles and BAEC
cells are described in detail below.
[0130] The implant material selected for biocompatibility studies
was the woven, 316L stainless steel wire mesh (140 .mu.m wire
diameter, 400 .mu.m apertures.) This particular material was
selected due to its large strut spacing, and extremely low
magnetization. As a result, a large difference in response to
applied magnetic fields, and subsequently in capture ability, can
be compared between a virtual non-magnetic 316L mesh and a CoNi
electroplated 316L mesh.
[0131] Because of the likely chemical reactivity of the CoNi
coating to cells and culture medium, and in order to provide a
"level playing field" on which cells could grow and easily be
studied, poly (dimethyl siloxane) (PDMS) (Dow Corning, MI) was used
to passivate the mesh. Depending on the desired cover slip size
(0.25 mm thick, ranging from 5 mm to 25 mm in diameter in the below
listed experiments), a volume of PDMS and curing agent (10:1
polymer to curing agent mass ratio) was dropped onto the glass
cover slip. The slip was placed by vacuum upon a wafer spinner at
the micro-fabrication facilities of the Drexel University Clean
Room, and was spun for an appropriate period (5 to 25 seconds
depending on the size of the slip) to obtain an even layer of 150
.mu.m of PDMS.
[0132] A 5 mm diameter circular punch-out of mesh (at 140 .mu.m
thickness) was then dropped on top of the PDMS layer, sinking into
the polymer. The preparation was then cured under vacuum at room
temperature overnight, and measured with digital calipers to verify
height. This creates a roughly 10 .mu.m tall boundary layer between
the mesh and the cell culture surface, and while not identically
physiologically relevant, provides an excellent working model for
studying endothelial cell cultures in close proximity to magnetic
gradients, as well as for endothelial cells with internalized
magnetic particles in close proximity to magnetic forces when an
external field is applied.
[0133] Spherotech (Spherotech, IL) magnetic particles were selected
for all pipe flow experiments. Spherotech magnetic particles were
20% .gamma.-Fe.sub.2O.sub.3 magnetite by weight, labeled with nile
red fluorescent pigment and had a nominal diameter of 350 nm with
approximately 10% variance in size. Particles come in 2 mL water
solutions concentrated at 1% w/v or 4.8.times.10.sup.11
particles/mL. These particles have a carboxylate per nm.sup.2 of
surface area, which can be used as a linker for therapeutics such
as peptides, antibodies or other biomolecumes.
[0134] Bovine aortic endothelial cells (BAECs) were selected as a
culture model. These cells were previously isolated by standard
technique at the University of Pennsylvania. All experiments were
performed at low-passages (<10). Cells were routinely cultured
in low glucose DMEM (Sigma, MO) supplemented with 10% Qualified
Heat inactivated Fetal Bovine Serum (Sigma, MO) and 1% 2.5 mM
L-glutamine (Sigma, MO). After expansion of the culture using 100
.mu.g/mL streptomycin (Sigma, MO), and 100U/mL penicillin (Sigma,
MO) per 500 mL batch of medium, it was determined that BAECs could
be cultured with ease without the use of antibiotics or
antimycotics, and were removed from future batches of culture
medium.
[0135] Cells were routinely cultured in 75 cm.sup.2 flasks in a
Fisherbrand cell culture incubator at 37.degree. C. and 5.0%
CO.sub.2 (Fisher, IL). In preliminary experiments determining the
preparation of mesh for culturing, the BAECs were shown to grow
poorly on silicone and glass surfaces, and when these surfaces were
treated with coated with a 1% Rat Tail Type I Collagen (Sigma, MO),
cells grew robustly, maintaining a growth rate and visible
morphology alike to BAECs cultured in T75 flasks.
[0136] 5 mm punch-outs of magnetic and non-magnetic mesh were
prepared, and treated with PDMS as described above. Prior to use,
each of the 6 cover slips (25 mm diameter.times.0.25 mm thick, 3
with magnetic mesh, 3 with non-magnetic mesh) were washed with soap
and water, and rinsed thoroughly. Following cleaning, each mesh was
placed in a glass Petri dish within a laminar flow hood, and soaked
in 70% ethanol for 30 minutes. After 30 minutes, all ethanol was
aspirated, and the cover slips were allowed to dry for 20
minutes.
[0137] Two separate 6-well plates were obtained (one for magnetic
mesh, the other for non-magnetic mesh), and 3 slips of each
experimental group were placed in each. A preparation of 1% by
volume Rat Tail Type I Collagen (Sigma, MO) in Phosphate Buffered
Saline (Sigma, MO) was used to add 150 .mu.L to each well. After 30
minutes, the collagen solution was aspirated, the slips rinsed
twice with an equal volume of PBS, and allowed to dry for 10
minutes within the laminar flow hood.
[0138] BAECs, routinely cultured as described above, were then
seeded to each cover slip at 1:2 split ratio (adjusted to cm.sup.2
growth area). 3 cm long by 0.5 cm tall by 0.5 cm wide pieces of
neodymium permanent magnets were placed under each well, for both
non-magnetic and magnetic mesh, and separated by plastic spacers.
These pieces applied an approximately 500 Gauss magnetic field at
the center of the mesh as measured by a handheld Gaussmeter
(Lakeshore Cryotronics, OH). These magnets were kept in place,
under the wells, and placed in the cell culture incubator for 30
minutes while cells were allowed to attach to the surface of the
collagen-coated PDMS. 30 minutes was selected as a modest period
for which a magnetic drug delivery injection may be performed,
saturating the magnetic moment of the material for that period. At
the conclusion of 30 minutes, the magnets were removed and the
samples were left in culture, and imaged by phase contrast at 24
hours.
[0139] Magnetizable particles uptake into or chemical attachment on
to cell cultures has been used as a means of mechanically stressing
cells (Wang, et al., 1993), and also considered as a means for
cellular localization (Consigny, 1999; Frank, 2004; Mertl, 1999),
but has not yet been successfully utilized in a magnetic delivery
method.
[0140] A method for weak surface attachment of Bovine Serum Albumin
was adapted to 350 nm diameter magnetic particles from the 3-5
.mu.m diameter particles used by the authors (Consigny, et al.,
1999). As mentioned above, the particles maintained a carboxyl
group per nm.sup.2 of surface area. A 1% by volume solution of
Bovine Serum Albumin (BSA) (Sigma, MO) in Phosphate Buffered Saline
(PBS) (Sigma, MO) was prepared according to the literature.
[0141] Prior to labeling the magnetic particles with albumin, each
sample of particles was rinsed 3 times with 70% ethanol, using
neodymium permanent magnets for separation between rinses. For
every 10.sup.11 particles, 1 mL of BSA/PBS dilution was added to
the 15 mL centrifuge tube in which the particles were cleaned.
Following the addition of the solution, the sample was gently
shaken on a gyrating shaker (Fisher, IL) for 1 hour. After shaking,
the particles were separated from the solution using a neodymium
permanent magnet, and re-suspended in appropriate volume of culture
medium, and stored in a 37.degree. C. water bath (Fisher, IL).
[0142] The day before particle preparation, BAECs were routinely
cultured as described above, and seeded on 6 tissue culture treated
cover slips (25 mm diameter.times.0.25 mm thick) at a 1:2 split
ratio (adjusted to cm.sup.2 growth area), and cultured for 1 day to
reach confluence. At confluence, all cover slips were seeded with
magnetic particles at a 4.times.10.sup.3 particle to cell ratio,
using particle preparations as described above. No magnets were
placed beneath the cover slips.
[0143] 24 hours after particle seeding, each cover slip was rinsed
three times with warmed culture medium, and submerged in Earle's
Balanced Saline Solution (Sigma, MO) supplemented with 10% Fetal
Bovine Serum (Sigma, MO). Each cover slip was imaged fluorescently
to assess internalization and cell survival. After imaging, fresh,
warm culture medium was added back to each cover slip.
[0144] Following imaging, each cover slip was split at a 1:6 split
ratio and re-seeded to fresh cover slips. Cells were imaged at 1
hour, monitored for growth over 3 days by a standard phase contrast
Nikon microscope (Nikon, Japan), and imaged fluorescently as
described above, at 72 hours.
[0145] Alternate BAEC samples were prepared as described above, but
prepared on glass slides containing 8 wells (0.69 cm.sup.2 per
well), and labeled with albumin-treated magnetic particles as
described above, but at a 2.times.10.sup.3 particle per cell ratio,
in order to obtain a clear view of particle internalization and
orientation, by confocal microscopy at 60 and 120.times.. After 24
hours, and particles had been uptaken by the cells, the well covers
were removed from the slide, and a drop of Vectorshield Mounting
Medium with DAPI (Vector Labs, CA) was applied to each of the 8
cultures, and a cover slip was fixed over the sample with nail
polish and allowed to set for 24 hours at 4.degree. C. Confocal
imaging was performed on a Leica TCS SP2 Confocal Microscope with
Louise Bertrand of the Department of Neurobiology and Anatomy at
Drexel University College of Medicine.
[0146] An average loading of particles per cell was estimated using
the MicroMag Alternating Gradient Magnetometer (AGM) (Princeton
Measurements, NJ). BAECs were routinely cultured as described
above, and seeded onto nine 5 mm diameter by 0.25 mm thick glass
cover slips resting in the bottom of wells in a 48 well plate. Each
slip was treated with Rat Tail Type I Collagen as described above.
The slips remained in culture for 24 hours until the cells had
reached confluence. Cells were loaded with particles by methods as
described above, at a loading density of 4.times.10.sup.3 particles
per cell. Approximately 8.times.10.sup.3 BAECs can grow upon a 5 mm
diameter cover slip. After 24 hours, resting medium was aspirated,
the slips were gently rinsed with culture medium three times, top
and bottom, following by a gentle alcohol swabbing of the bottom of
slip, to remove any particles that may have been attached. Each of
slips was then measured for saturation magnetization by AGM. The
results were then averaged, and based on the known quantity of
cells per slip, used to give an estimate of the average particle
loading per cell.
[0147] In order to further examine the behavior of BAECs with
internalized magnetic particles, studies of calcium response under
shear simulation were performed side by side with controls of
unlabeled BAECs.
[0148] BAECs were routinely cultured as described above, seeded
onto 6 tissue culture treated cover slips (25 mm
diameter.times.0.25 mm thick), and cultured for 1 day to reach 90%
confluence. At 90% confluence, 3 cover slips were seeded with
magnetic particles at a 4.times.10.sup.3 particle to cell ratio,
using particle preparations as described above, the other 3 cover
slips.
[0149] Endothelial cells were loaded in the dark with fluo3 by
incubation with 5 uM Fluo3-acetoxymethyl ester (Molecular Probes,
Inc., OR) in Dulbecco's phosphate buffered saline (DPBS) (Sigma,
MO) at pH 7.4, for 40 minutes at room temperature. The cover slip
was then rinsed three times with DPBS before experiments were
performed.
[0150] For cell shearing experiments, a custom-built controlled
cell-shearing device based on a cone and plate configuration was
mounted on the microscope stage and used to apply precise
mechanical loading conditions to the endothelial cells. For each
experiment, a 25 mm diameter cover slip was placed in the circular
recess in the plate. Vacuum pressure was applied to hold the cover
slip and prevent motion during shear experiment. A volume of 1.5 ml
DPBS was added to the well to fill the gap between the cone and
plate. For the mechanical loading period, endothelial cells were
monitored for about 30 seconds under static conditions prior to the
onset of shear stress to establish the basal levels of calcium.
Then, the shear stress was ramped up linearly to 20 dyn/cm.sup.2
over 0.1 seconds and maintained at a steady level for 5
minutes.
[0151] Cell fluorescence was monitored and recorded using a Nikon
Diaphot TE300 Eclipse epifluorescent microscope (Optical Apparatus,
Inc, PA) with a 20.times. objective. Fluo3 was excited at 488 nm
and emitted fluorescence at wavelength of 515 nm upon binding
Ca.sup.2+. The illumination was controlled by means of an
electronic filter wheel (Lambda 10-2, Sutter Instruments Co., CA).
The emitted fluorescence passed through a barrier filter and was
detected by an intensified CCD digital camera unit (Vedio Scope
International, Ltd., VA). The rate of image acquisition was 2
seconds per frame. Prior to the each stimulus, images were recorded
for at least 30 seconds. Axon Workbench image acquisition software
(Axon Instruments, Inc., CA) was used to acquire fluorescence
images and to perform post-acquisition analysis.
[0152] Once it was determined that magnetic particles could be
delivered to the inside of BAECs, the logical next step was to
examine if these magnetic BAECs could be attracted to 316L mesh
electroplated with CoNi, under the influence of a magnetic field.
This was first demonstrated statically, in sterile cell culture
conditions, and then by high flow rate experiments using the
parallel plate flow chamber and methods.
[0153] For static capture experiments, using the protocol for
preparing mesh for cell culture experiments as described above, 5
mm punch-outs of mesh (3 magnetically plated, 3 unplated) were
sealed with a thin layer of PDMS onto the center of 25 mm diameter
cover slips, followed by cleaning, and coating with collagen. BAECs
were routinely cultured in 6 well plates as previously described,
and each well was loaded with magnetic particles at a
4.times.10.sup.3 particle per cell ratio. BAECs were allowed to
remain in culture for 24 hours for uptake of the magnetic
particles.
[0154] On the following day, two six-well plates were prepared, one
containing the 3 cover slips prepared with magnetic mesh discs, the
other containing 3 cover slips with unplated mesh discs. Neodymium
pieces, as described above, were mounted beneath each well, and
measured to apply an approximately 500 Gauss magnetic field at the
center of the mesh. Magnetic BAECs were routinely split and seeded
at a 1:2 split ratio to the cover slips and allowed to remain in
culture for 30 minutes under the applied field. At 30 minutes, the
magnets were removed, and the cover slips were imaged
fluorescently. Imaging was also performed at 6 hours, and 24 hours
after cell seeding.
[0155] For magnetic cell flow experiments, BAECs were routinely
cultured on a T75 flask. The flask was loaded with magnetic
particles by methods described above, at a seeding rate of
4.times.10.sup.3 particles per cell. BAECs were allowed to remain
in culture with the particles for 24 hours to internalize them into
the cells. After setting up the flow chamber and magnetic coil
under the microscope, the cells were trypsinized routinely, and
concentrated in 25 mL of culture medium. The 500 Gauss field was
turned on, and the particles were delivered past a 2.times.2 cm
piece of magnetically plated 316L mesh at 15 cm/s flow velocity,
followed by a 25 mL rinse with Earle's Balanced Saline Solution
(EBSS) (Sigma, MO) supplemented with 10% Fetal Bovine Serum (Sigma,
MO). The field was left on during rinsing. Based on AGM results
estimating an average loading success of approximately 10.sup.3
particles per cell, the experiments delivered an equivalent number
of particles as a concentration 0.25% by volume in the medium.
Fluorescent imaging was performed immediately after rinsing.
[0156] BAEC cultures were successfully grown atop of PDMS/collagen
coated magnetic mesh. The cultures grown on a magnetically plated
mesh and an unplated mesh were virtually indistinguishable.
[0157] The scarcity of commercially available superparamagnetic
particles in the sub-micron diameter scale has resulted in most
frequent use of much larger magnetic particles for external
labeling. From the performed cell culture studies, 350 nm
polystyrene magnetic particles coated with albumin were
compartmentalized and uptaken by BAEC cultures. Fluorescent images
were taken 24 hours after particle seeding, one hour after
re-seeding, and 3 days after re-seeding. Images of BAEC culture
with internalized fluorescent magnetic particles taken 24 hours
after particle seeding showed expected morphology upon inspection.
After imaging at 24 hours the cells were split at a 1 to 6 ratio.
Images taken one hour after re-seeding show clear definition of
internalized particles around the entire circumference, which
attached and spread to culture surfaces as expected from routine
cultures. Images taken three days later demonstrate that these
internalized particles are being distributed to many of but not all
daughter cells. These images indicated that BAECs can survive,
re-seed, and divide after the introduction of magnetic particles to
cultures.
[0158] FIG. 7A is an image of BAECs with internalized 350 nm
diameter nile red polystyrene magnetic particles, 1 hour after
being passed and re-plated at a 1:6 splitting ratio at 20.times.
magnification and FIG. 7B is at 40.times. magnification.
[0159] BAEC cultures with internalized magnetic particles (mBAECs)
were successfully delivered under static conditions with
specificity to the surfaces of a CoNi electroplated 316L mesh.
Clearly the mBAECs have been delivered to the areas directly over
the wires of the magnetic mesh, while for the unplated mesh sample,
the cells were very uniformly distributed across the entire
surface. In each case the magnet can influence the rate at which
the cells attach to the surface, but without a doubt the magnetic
coating of the experimental mesh in combination with the field
applied, caused those mBAECs to be captured by the device.
[0160] At 6 hours after mBAECs were magnetically delivered to the
magnetic mesh, it was observed that many mBAECs had begun to
migrate off of the wires into the surrounding area, indicating many
cells had survived the magnetic forces used to capture them to the
mesh surfaces. At 24 hours, a great many more of the mBAECs have
migrated from the area immediately over the wire into the
surrounding growth area, and have nearly reached confluence.
Clearly, there were still large congregations of cells at the wire
intersections, and it was postulated that if great numbers of cells
were brought down to these intersections, some cells may have died
from isolation from nutrients and proper gas exchange.
[0161] Cell flow chamber experiments at roughly 0.25% particle
concentration (based on a calculated average particle uptake per
cell from AGM analysis) by volume demonstrated the ability to
capture mBAECs at high flow velocity in a parallel plate flow
chamber. It was expected that these cells could be captured due to
an increased susceptibility to magnetic forces due to large
particle concentrations in each cell. This experiment indicates
that it may be possible to magnetically deliver cells of limited
availability, such as stem cells, with increased accuracy and site
specificity to a desired location.
[0162] The experiments demonstrated that not only do vascular
endothelial cells succeed in growth within microns of distance from
magnetically plated mesh under the influence of a magnetic field,
but can be labeled with magnetic particles, delivered by magnetic
force to the surface of the mesh and survive. Attempts by other
laboratories have failed at well dispersed and equal loading of
magnetic particles into cultures, mainly due to the use of larger
particles, but also from their use of applied fields during
particle seeding. By placing a permanent magnet underneath the
culture continuously during particle seeding to a culture, these
particles may have an increased tendency to form chains or large
aggregates too large for delivery to the inside of a cell, and may
therefore remain on top of the culture. These large aggregates may
even be pulled through intracellular space to the bottom of the
culture, beneath the cells, but if the field is left in place
throughout, one would expect minimal internalization for micron and
even large sub-micron particles.
[0163] From these experiments, it cannot be assumed that every cell
in the initial culture seeded with particles contained them after
the introduction to the culture, but it does seem quite likely that
many mBAECs distributed particles to daughter cells, and that most
cells without particles are daughter cells of the unlabeled cells
from the first culture. It was observed that certain cells do seem
to maintain the same density of fluorescence seen just after
initial particle introduction. In these cases, the question can be
raised on whether or not these mBAECs have undergone mitosis, or
were saturated with particles to the point of inhibition. In most
cases, by inspection, the culture had a significantly reduced
fluorescence per cell, indicating particle transfer during cell
division, or release of particles into the surrounding medium
during cell division, followed by eventual re-uptake.
[0164] Adaptation to other cell cultures, the use of smaller
particles, and applications can be determined by those skilled in
the art without undue experimentation provided with guidance of
theis disclosure. The ability to capture mBAECs under high flow
makes possible to deliver cells to implants within the body for the
purpose of in vivo tissue engineering or wound healing, magnetic
cells can also be used as vehicles for drug delivery. When
magnitizable particles are introduced into blood flow, they will
come in contact with numerous cells and substances, and are
particularly vulnerable to immune cell response. By loading cells
to saturation with magnetizable particles, the thus formed magnetic
cells will have extremely large magnetic susceptibilities, likely
raising the capture efficiency significantly while reducing
concerns about immune response and blocking of capillaries. In many
cases, autologous cell sources could be utilized with ease. If a
cell were used solely as a vehicle for drug-loaded particles, its
survival would be irrelevant as long as the carriers reached the
target site and could effectively transfer therapeutic agents upon
arrival. This presents a much more effective method for
magnetically targeted drug delivery, making intravenous injection a
preferred option, over the current "safe bets" of arterial puncture
or catheterization.
[0165] Further provided is a method of using the magnetic delivery
system for delivery of a therapeutic agent, the method comprising
providing the external source of the magnetic field, implanting the
magnetizable object in the body, wherein the magnetizable object
includes a plurality of segments distributed throughout the
magnetizable object and wherein the segments are configured to
provide a magnetic gradient for attracting a magnetizable particle
comprising a therapeutic agent, and providing an external magnetic
field by initializing the external source and thereby (i)
magnetizing the magnetizable particle and (ii) increasing the
degree of magnetization the magnetizable object and thereby
creating the magnetic gradient for attracting and advancing the
magnetizable particle toward the magnetizable object. Those skilled
in the art would appreciate that magnetizable material have a base
line degree of magnetization which is increased in the presence of
a magnetic field.
[0166] In certain embodiments, the method further comprises
administering the magnetizable particle comprising the therapeutic
agent to the body and attracting and advancing the magnetizable
particle toward the magnetizable object using the magnetic gradient
and thereby delivering the therapeutic agent to the location in the
body.
[0167] In certain embodiments of the method, a size of each segment
is selected to commensurate with a size of the magnetizable
particle such that at least one spatial dimension of each segment
does not exceed by more than about 10,000 times at least one
spatial dimension of the magnetizable particle. In certain
embodiments of the method, the magnetizable particle is in a form
of a superparamagnetic colloidal fluid.
[0168] In certain embodiments of the method, the magnetizable
particle has a diameter from about 10 nm to about 1000 nm.
Preferably, the magnetizable particle has a diameter from 10 nm to
500 nm.
[0169] In certain embodiments of the method, the magnetizable
object is a member selected from the group consisting of a stent, a
pacemaker, a catheter, a tube, a vascular graft, an artificial
joint, an artificial bone, a prostate seed, an aneurysm coil, a
surgical staple, and a suture.
[0170] In certain embodiments of the method, at least one of the
magnetizable object and the magnetizable particle is magnetized
only in the presence of the external magnetic field. In other
embodiments, at least one of the magnetizable object and the
magnetizable particle is permanently magnetized.
[0171] In certain embodiments of the method, the magnetizable
particle comprises a cell such that the magnetizable particle is
loaded within a cell and the therapeutic agent is associated with
the cell, the magnetizable particle or both.
[0172] In certain embodiments of the method, implanting the
magnetizable object is accomplished by administering a cluster of
gradient forming particles wherein a surface of each gradient
forming particle represents a segment of the magnetizable
object.
[0173] Further provided is a method of using the magnetic delivery
system for delivery of a cell to a body, the method comprising
providing the external source of the magnetic field, implanting the
magnetizable object in the body, wherein the magnetizable object
includes a plurality of segments distributed throughout the
magnetizable object and wherein the segments are configured to
provide a magnetic gradient for attracting a magnetizable particle
comprising a therapeutic agent, and administering the magnetizable
particle loaded within the cell to the body, providing an external
magnetic field by initializing the external source and thereby (i)
magnetizing the magnetizable particle and (ii) increasing the
degree of magnetization the magnetizable object and thereby
creating the magnetic gradient, and attracting and advancing the
magnetizable particle toward the magnetizable object using the
magnetic gradient and thereby delivering the cell to the location
in the body. In certain embodiments of the method, a size of each
segment is selected to commensurate with a size of the magnetizable
particle such that at least one spatial dimension of each segment
does not exceed by more than about 10,000 times at least one
spatial dimension (e.g., a width, a height or a diameter) of the
magnetizable particle.
[0174] Those skilled in the art would appreciate that methods of
administration of magnetizable particle or magnetic cells include
but not limited to systemic delivery (e.g., by injection,
catheterization, etc.).
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