Note: Descriptions are shown in the official language in which they were submitted.
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=
MAGNETICALLY CONTROLLABLE DRUG AND GENE DELIVERY STENTS
STATEMENT REGARDING FEDERALLY
SPONSORED RESEARCH OR DEVELOPMENT
This research was supported in part by U.S. Government funds (National Heart
Lung and
Blood Institute Grant No. HL59730 and NSF Grant No. 9984276), and the U.S.
Government
may therefore have certain rights in the invention.
BACKGROUND OF THE INVENTION
1. FIELD OF INVENTION
The present invention provides implantable devices for and methods of using
these
devices to capture therapeutic agents attached to, or encapsulated within,
magnetic carriers at
selected sites in a body or a subject. This invention further relates to
delivery of bioraaterial to a
cell or a tissue, and more particularly it relates to delivery of biomaterial
associated with
particles. This invention also relates to surface modifications and more
particularly to
immobilization ofmolecules to surfaces by photochemical coupling. Further,
this invention also
relates to a magnetizable implantable devices utilizing magnetizable or
magnetic particles acting
as carriers of biomaterial, biomolecules and cells.
2. DESCRIPTION OF RELATED ART
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.
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Stents comprise a tube made of metal or polymer, 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 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).
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
=
I opposed to the lengthy hospital stay required with bypass surgery
(Pricker, 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.
= 20 2001 59:227-48).
1
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).
= 25 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
. 30 mechanical scaffolding that almost completely eliminates
recoil and remodeling. However, neo-
I 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 checicpoint for
patient health post-surgery (Regar et al. Br. Med. Bull. 2001 59:227-48). Neo-
intima forms
2
=
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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).
Three major drug 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 aU
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.
Local drug delivery provides limited systemic release, thereby reducing the
risk of
systemic toxicity. Techniques for local drug delivery that have been described
include, but are
not limited to, 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;
Pricker, 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 ofthe
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).
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
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Magnetic Materials 1999 194:167-175). 'U.S. Patent 4,247,406 describes an
ititravascularly-
.
adm ini strabl e, 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 agentsto cancerpatients.
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.
Patent 5,129,877.
U.S. Patents 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, an adhesive and
preferably magnetic
= particles to control delivery of the mixture. In these methods, a
magnetic con 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.
= 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 implants with
patterned magnetic material to create high magnetic field gradients was
described (Forbes et al.
Abstract and Poster Presentation at the 6th Annual New Jersey Symposium on
Biomaterials,
=
October 17-18, 2002, Somerset, NJ). 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
(MEP Trans. Magn. 39(5): 3372-3377 (2003)).
= In general, methods and devices which have been proposed 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. These single magnetic fields for attracting magnetically
susceptible
=
= therapeutic agents can be applied by sources external to the body, or by
an internal implant as
described by Chen in U.S. Patent 5,921,244.
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
=
4
CA 02821214 2013-07-19
=
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, Cheri 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. 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.
1ln general, nanoparticles have been very ineffective vehicles for gene
delivery, with
= expression levels below those seen with naked DNA. Thus, there has been
relatively little
progress with DNA incorporation into biodegradable sustained release
particles. Also, problems
= encountered in gene therapy include slow accumulation and low
concentration of gene vector in
target tissues.
Nanoparticles formed from biodegradable polymers have been used to carry
active
molecules to sites in the body where the therapeutic effect is required (see
Quintanar-Guerrero et
al., Preparation techniques and mechanisms of formation of biodegradable
nanoparticles from
preformed polymers. Drug Dev Ind Pharm 1998; 24:1113-28; and Kumar MNR. Nano
and
microparticles as controlled drug delivery devices. J Pharrn Pharrnaceut Sci
2000; 3:234-58).
Quintanar-Guerrero et al. describe various techniques available to prepare
biodegradable
nanoparticles from polymers such as for example, emulsification-solvent
evaporation, solvent
displacement, salting-out, and emulsification diffusion. In general, such
nanoparticles have
limited loading capacity for most hydrophilic drugs and also are not efficient
in cases where
rapid accumulation of active molecules is required at their target sites.
Various studies were conducted to improve delivery of a biomaterial such as
viruses
. (e.g., adenovirus) and plasmid DNA by physical means such as an application
of a magnetic
field to a vector including magnetically responsive solid phases, which are
micro-to nanometer
sized particles or aggregates thereof (see Plank et al., Enhancing and
targeting nucleic acid
delivery by magnetic force. Expert Opin Biol Ther. 2003;3:745-58 (Plank I
thereafter); Plank et
al., The magmetofection method: using magnetic force to enhance gene delivery.
Biol Chem.
2003;384:737-47 (Plank 11 thereafter); Scherer et al. Magnetofection:
enhancing and targeting
gene delivery by magnetic force in vitro and in vivo. Gene Ther. 2002;9:102-
9).
5 ,
=
CA 02821214 2013-07-19
Ito et al. describe application of magnetic granules (0.1-0.5 microns)as
carriers for anti- =
cancer drugs administered orally in local targeting chemotherapy of esophageal
cancer ( see
= Magnetic Granules: A Novel System for Specific Drug Delivery to
Esophageal Mucosa in Oral
Administration. Infl. J. of Pharmaceutics, 61 (1990), pp. 109-117).
Compositions described by
Ito et al. were not made as colloidal particles, and magnetic granules used
therein were not
stabilized.
U.S. Patent No. 5,916,539 to Pilgrimm describes superparamagnetic particles
useful in
= medicine for destroying ttnnors, increasing immunity and diagnosing
conditions. The patent
describes aggregates of superparamagnetic single-domain particles bearing on
its surface
chemically bound organic substances for further binding of active substances
such as antigens,
antibodies, haptens, protein A, protein G. endotoxin-binding proteis, lectins,
and selectins.
Arias et al. describes an anionic polymerization procedure for preparing
colloidal
nanoparticles consisting of a magnetic core and a biodegradable polymeric
shell wherein the
polymerization medium was magnetite suspension in HC1 solution (see Synthesis
and
Characterization of Poly(ethy1-2-cyanoacrylate) Nanoparticles with a Magnetic
Core. J of
= Controlled Release 77 (2001),
pp. 309-321). =
GOmez-Lopem et al. describes preparation of colloidal particles formed by a
magnetite
nucleus and a biodegradable poly(DL-lactide) polymer coating by a double
emulsion method,
wherein aqueous suspension of magnetite particles was used to prepare an
emulsion with the
= 20 polymer (see Synthesis and Characterization of Spherical
Magnetite/Biodegradable Polymer
= Composite Particles. J. of Colloid and Interface Science 240, 40-47
(2001)). It is significant
= that the magnetite in the cited studies was not incorporated as an
organic suspension, resulting in
its poor incorporation in the particle.
. Plank et al. describe superparamagnetic iron oxide
nanoparticles manufactured with
polyelectrolyte surface coatings such as poly(ehylenimine) (PEI) and
polylysine further
associated with gene vectors by salt induced colloid aggregation
(Magnetofection: enhancing
and targeting gene delivery with superparamagnetic nanoparticles and magnetic
fields.
Liposome Res. 2003;13:29-32 (Plank In thereafter)). (See also Plank II,
Scherer et al.,
Magnetofection: enhancing and targeting gene delivery by magnetic force in
vitro and in vivo.
Gene Ther. 2002;9:102-9).
The same research group further used magnetic beads in combination with PEI
and
pDNA as a model of a non-viral vector mediated gene expression system for
transfection of cells
(see Krotz et al., Magnetofection potentiates gene delivery to cultured
endothelial cells. J Vase
6
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Res. 2003;40(5):425-434) and delivery of antisense oligonucleotides in a
catheter-based
= coronary angioplastic therapy for occlusive cardiovascular disease (see
Krotz et al.,
= Magnetofection-A highly efficient tool for antisense oligonucleotide
delivery in vitro and in
: = , vivo. Mol Ther. 2003;7:700-10). The magnetic beads used by
this research group lack the
concept =of sustained release and increased colloidal stability achievable
with biodegradable
polymer-based particles. Moreover, the reported results show a considerable
extent of cell
.=
=
toxicity caused by nanoparticulate formulations (see Plank II, supra). The PEI
coating stability
and a possible aggregation in biological fluids have not been examined, but
might potentially be
a concern in these formulations.
Muller et al. studied cytotoxicity of poly(lactide), poly(lactide-co-
glucolide),
=
poly(styrene) and solid lipid particles loaded with magnetite. No attempts to
incorporate
= magnetite as a stable organic dispersion were described (see Cytotoxicity
of Magnetite-Loaded
Polylactide, Polylactide/Glycolide Particles and Solid Lipid Nanopatticles.
Intl. J. of
Pharmaceutics 138 (1996) 85-94). Further, the possibility of loading the
particles with a drug has
= 15 not been examined.
Igartua et al. describes encapsulation of magnetite particles stabilized by
oleic acid in =
solid lipid nanoparticles (see Development and Characterization of Solid Lipid
Nanoparticies
= Loaded with Magnetite. Intl J. of Pharmaceutics 233 (2002) 149-157). The
authors did not
address using polymer as a matrix and presented no results on drug loading in
such particles.
De Cuyper et al. describes magnetoliposomes which are phospholipid bilayer
coated
magnetite particles prepared by adsorption of sonicated phospholipids onto
magnetite stabilized
by lauric acid in an aqueous solution (see Magnetoliposomes. Fomultion and
structural
characterization. Eur Biophys J 1988; 15:311-319). Such liposomes are too
small to be
=
effectively manipulated by magnetic field. Although, ability to bind drug have
not been studied,
. 25 the liposomes prepared by this method have limited capacity
for drug substances since they can
be loaded only by surface adsorption.
Messai et al. describe poly (lactic acid)-based particles ((PLA nanoparticles)
surface
modified by electrostatic adsorption of PEI, wherein PEI is associated with
DNA (see
=
Elaboration of Poly(ekleneimine) Coated Poly(D, L-Iactic acid) Particles.
Effect of Ionic
= 30 Strength on the Surface Properties and DNA Binding
Capabilities. Colloids and Surfaces B:
Bioninterfaces 32 (2003), pp. 293-305). PEI adsorbed onto PLA nanoparticles
does not provide
a stable coating and readily dissociates into the extemal medium upon
dilution.
7
=
CA 02821214 2013-07-19
3...1 L'.=(== VP' = ro,
I = N -
Sullivan et al. describe gene delivery scaffolds based on DNA plasmid
condensation
with colloidal gold/PEI conjugates (see Development of a Novel Gene Delivery
Scaffold
Utilizing Colloidal Gold-Polyethylenimine Conjugates for DNA Condensation.
Gene Therapy
(2003) 10, 1882-1890). Although, such conjugates when used as a vehicle for
gene delivery
exhibit improved size stability when compared to PEI alone, they can not be
targeted by
magnetic field and lack sustained release properties.'
= =
'Further, a common shortcoming of implantable medical devices or surfaces is
= recognition of these devices by an organism as foreign objects followed
by inflammation or even
=
a rejection of such devices. Surface modification science concentrates on
finding a better
=
10 interface between a living tissue and a solid matrix. It is known to use
various coatings to
==
impart desirable properties to implantable surfaces. Such coatings are based
on polymers and
may include biologically active materials. Challenges in preparing such
coatings include
attaching biologically active materials to inert surfaces.
One of the techniques for derivatizing inert surfaces is photochemical
coupling (see
Amos et al., Biomaterial surface modification using photochemical coupling
technology. In:
Wise D L, Trantolo D I, Altobelli D E, Yaszemski M J, Gresser J D, Schwartz E
R editors.
Encyclopedic handbook of biomaterials and bioengineering, Part A: materials.
New York:
Marcel Dekker Inc., 1995, p. 895-926). Photo-cross-linking chemistry based on
organic
solvents is well established (see Wetzels et al., Photoinunobilization of
poly(N-
= 20 vinylpyrrolidone) as a means to improve haemocompatibility of
polyurethane biomaterials,
Biomaterials 1999, 20, 1879-87; Mcaung et al., Lysine-derivatized polyurethane
as a clot
lysing surface: conversion of absorbed plasminogen to plasmin and clot lysis
in vitro,
Biomaterials 2001, 22, 1919-24; Aldenhoff et al, Photo-immobilization of
dipyridamole
= (PERSANTI" at the surface of polyurethane biomaterials: reduction of in
vihv
trombogenicity, Biomaterials 1997, 18, 167-72; Kuijpens et al., Immobilization
of theophylline
on medical-grade polyurethane inhibits surface-induced activation of blood
platelets, J. Am.
Chem. Soc. 1995, 117, 8691-8697). It is not known to use adsorption from
aqueous solutions
E3r application of photo-cross-tinkers onto a polymer surface.
Aryl ketones (e.g., benzophenone or acetophenone derivatives) and aryl azides
are
known as photo-activatable cross-linkers suitable for covalent binding to
virtually any type of
polymer surface as described by Amos et al.) Upon irradiation with long-wave
UV (at about
350 nm), benzophenone residues form energy-rich excited triplet species, which
then insert into
carbon-hydrogen bonds (C-H bonds) of a polymeric surface by abstraction of
hydrogen atoms
8
=
CA 02821214 2013-07-19
from C-H bonds and form new carbon-carbon bonds (C-C bonds), resulting in
covalent binding
of benzophenone residues onto the polymeric surface (see FIG. 1).
U.S. PatentNo. 5,071,909 to Pappin et al. discloses a method for immobilizing
proteins
or peptides onto a membrane by formation of a polymeric network, which entraps
the protein or
peptide.
U.S. Patent Nos. 3,959,078, 5,512,329 and 5,741,551 to Guire et al. disclose
covalent
bonding ofpolymeric molecules to surfaces through external activation. This
approach is used
to bind fibronectin peptide to a polystyrene surface by a photo-reaction
between the peptide and
a surface having a photo-activatable group.
U.S. Patent No. 5,637,460 to Swan et al. discloses attaching a target molecule
(synthetic
polymers, carbohydrates, proteins, lipids, nucleic acids, etc.) to a surface
by using photo-
, activatable groups.
. It is evident from the prior art discussed above that prior to the present
invention, photo-
cross-linkers were not used for the attachment of functional reactive groups
(other than the same
photo-activatable groups used in the initial step of photo-inunobilization) to
the surface in order
to activate it for further immobilization of biomolecules. =
Despite the foregoing developments, there is a need in the art for alternative
means of
delivery of biornaterial.
BRIEF SUMMARY OF THE INVENTION
IThe present invention provides a magnetizable implant, prefbrably a stent,
for targeting
of magnetic therapeutic agents to a selected site of implantation
ofthemagnelizable 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 magnetio 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.
An object of the present invention is to provide devices for implantation in a
body or a
subject and methods for use of these devices in delivering a therapeutic agent
encapsulated in or
dispersed in a magnetic carrier to the implanted device. The device of the
present invention
comprises a biocompatible metal or polyineric structural supporting implant
coated with or
comprising segments of a magnetizable compound. The device is specifically
designed to
produce strong magnetic fieki gradients through creation of magnetic or
magnetizable features
9
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_
1
distributed throughout the implant. An exemplary embodiment of a device 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 features. 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
= ofthe implant is etched into a pattern to produce strong local field
gradients at desired locations
I 0 monatth h
erie al-nopflathnt rfaesuimpoelan. In tcreye:teanth
s another im_demefibnoeddmeangnt,tle
structureeitizesegmenoftsa ninonan-magnetic
isseupunpoifrtionnng
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.
Optimization of the magnetic 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 tmifonn 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.
Ole present invention has the following features: adjustable particle size and
rapid cellular
uptake, surface or bulk binding of a biomaterial (e.g., DNA vectors),
biodegradable polymer matrix
of the carrier, the use of a polyelectrolyte (e.g., poly(ethyleneimine) (PEI))
for enhanced biomaterial
delivery and protection, wherein the polyelectrolyte is complexed with or
adducted to an
amphiphilic agent, co-incorporation of a magnetic field¨responsive agent in
association with an
amphiphilic agent to confer a magnetic targeting capability to particles, and
optional inclusion of
. , biocompatible surface-modifying agents to provide improved
colloidal stability and "stealth"
properties in vivo.
Accordingly, the invention provides a particle comprising a matrix-forming
agent; and a
= polyelectrolyte-amphiphilic agent adduct wherein the polyelectrolyte-
amphiphilic agent adduct is in
physical communication with the matrix-forming agent.
. -
I In certain embodiments, the polyelectrolyte-arnphiphilic agent
adduct has a C4-C24 hydrocarbon
=
CA 02821214 2013-07-19
= I
I I
chain. Preferably, the polyelectrolyte-amphiphilic agent adduct is
poly(ethyleneimine)
carboxylate. In certain embodiments, the polyelectrolyte-amphiphilic agent
adduct is formed by
an association of a polyelectrolyte with a first amphiphilic agent.
In certain embodiments, the particle further comprises a coated magnetic field-
responsive
agent having a magnetic field-responsive agent in communication with a second
amphiphilic
= agent, wherein the coated magnetic field-responsive agent is in
communication with the matrix-
forming agent. In one variant, the first amphiphilic agent and the second
amphiphilic agent are
the same substance.
In certain embodiments, the particle further comprises a biomaterial in
communication
I 0 with at least one ofthe polyelectrolyte-amphiphilic agent
adduct or the matrix-forming agent. In
certain embodiments, the particle fiirther comprises a biomaterial in
communication with at least
= one of the polyelectrolyte-amphiphilic agent adduct or the matrix-forming
agent, wherein the
particle is free of the magnetic field-responsive agent.
= In certain embodiments, the particle further comprises a stabilizer.
Further provided is a magnetic particle comprising a matrix-forming polymer;
and a
coated magnetic field-responsive agent comprising a magnetic field-responsive
agent and a
second amphiphilic agent, wherein the coated magnetic field-responsive agent
is in
communication with the matrix-forming polymer, provided that the magnetic
particle is free of a
= polyelectrolyte. In certain embodiments, the magnetic particle further
comprises a biomaterial
in communication with the matrix-forming polymer.
Further provided is a particle comprising a matrix-forming polymer, a
polyelectrolyte-
amphiphilic agent adduct comprising a first C12-C24 carboxylate group, wherein
the first C12-C24
= carboxylate group is in physical communication with the matrix-forming
polymer; a second
amphiphilic agent comprising a second C12-C24 carboxylate group in
communication with the
= 25 matrix-forming polymer; and a magnetic-field responsive
agent in communication with a second
C12-C24 carboxylate group. In certain embodiments, the particle further
comprises a stabilizer.
= In certain embodiments, the particle further comprises a biomaterial in
communication with the
polyelectrolyte-amphiphilic agent adduct and optionally with the matrix-
forming polymer.
Further provided is a method of making the particle of the invention, the
method
comprising providing the matrix-forming agent, providing a polyelectrolyte,
providing a first
amphiphilic agent, providing a first medium and a second medium, optionally
providing a
stabilizer, mixing at least the matrix-forming agent, the first medium, and
the second medium
and optionally the polyelectrolyte, the first amphiphilic agent, and/or the
stabilizer to give a first
11
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= mixture, emulsifying the first mixture to give a first emulsion, and
removing the first medium
and thereby forming the particle, on a condition that the polyelectrolyte, the
first amphiphilic
agent, and the stabilizer are provided to at least one of the first medium,
the second medium, the
first mixture, the first emulsion, or the particle such that the
polyelectrolyte and the first
amphiphilic agent form the polyelectrolyte-amphiphilic agent adduct. In
certain embodiments,
the method further comprises providing a biomaterial.
In another embodiment, the method further comprises providing a coated
magnetic field-
responsive agent to at least one of the first medium, the second medium,
and/or the first mixture. In
one variant of this embodiment, the method further comprises providing a
biomaterial, wherein the
= 10 biomaterial is provided to at least one of the first
mixture, the first emulsion and/or the particle.
Further provided is a method of making the magnetic particle of the invention,
the
method comprising providing the matrix-forming polymer, providing the coated
magnetic field-
responsive agent, providing a first medium and a second medium, optionally
providing a
= .
stabilizer, mixing at least the matrix-forming polymer, the first medium, and
the second medium
to give a second mixture, emulsifying the second mixture to give a second
emulsion; and
= removing the first medium and thereby forming the particle, on a
condition that the coated
magnetic field-responsive agent and optionally the stabilizer are provided to
at least one of the
first medium, the second medium, or the second mixture. One variant of this
embodiment
=
includes further providing a biomaterial. The biomaterial can be provided to
at least one of the
second mixture, the second emulsion or the magnetic particle.
The particles of the invention have high specific loading of magnetically
responsive
agent displaying a loading of about 20-30 % by weight) and biodegradablity
Toxicity is low
with these particles because they are made from biocompatiple material and
made by novel
(combination) methods; surface modified by new method (thio reactive); stable
in
=
presencealbumin (plasma) in similar conditions as in plasma.
Further provided is a method of making a particle, the method comprising
providing a
= first medium and a second medium, providing a coated magnetic field-
responsive agent,
optionally providing a stabilizer, providing a composition comprising a matrix-
forming agent, a
polyelectrolyte, a first amphiphilic agent and optionally the stabilizer,
dispersing the coated
magnetic field-responsive agent in the first medium to form a dispersion,
mixing the
= composition with the dispersion, adding the second medium to the
composition and the
dispersion to form a first mixture, emulsifying the first mixture to give a
first emulsion, and
,
=
removing the first medium and thereby forming the particle.
12
. .
CA 02821214 2013-07-19
1., ' '
Also provided is a particle made by the above method. In one variant, the
particle further= 1
comprises a biomaterial.
Also provided is a method of delivery of a biomaterial to a target cell or a
target tissue, 1
the method comprising administering the particle of the invention comprising
the matrix-
forming agent, polyelectrolyte-amphiphilic agent adduct, the coated magnetic
field-responsive
agent and the biomaterial, optionally providing a magnetic device associated
with the target cell
. = or the target tissue, applying a magnetic force to the
particle, and guiding the particle by the
magnetic force and thereby delivering the biornaterial to the target cell or
the target tissue.
Further provided is a method of delivery of a biomaterial to a target cell or
a target
tissue, the method comprising
administering the particle of the invention comprising the matrix-forming
agent, the
coated magnetic field-responsive agent, and the biomaterial, wherein the
particle is free of the
polyelectrolyte, providing a magnetic device associated with the target cell
or the target tissue,
applying a magnetic force to the particle, and guiding the particle toward the
magnetic device by
the magnetic force and thereby delivering the biomaterial to the target cell
or the target tissue.
Also provided is a method of delivery of a biomaterial to a cell or a tissue,
the method
comprising administering the particle of the invention comprising the matrix-
forming agent, the
polyelectrolyte-amphiphilic agent adduct, and the biomaterinl, wherein the
particle is free ofthe
magnetic field-responsive agent, and delivering the biomaterial to the cell or
tissue using the
particle as a carrier, wherein the cell is optionally contacted with a
transfection agent prior to
said delivering.'
'Further, the invention provides a water-soluble photo-activatable polymer
comprising:
(a) a photo-activatable group, wherein the photo-activatable group is adapted
to be
activated by an irradiation source and to form a covalent bond between the
water-soluble photo-
activatable polymer and a matrix having at least one carbon;
=
(b) a reactive group, wherein the reactive group is adapted to covalently
react with a
biomaterial;
(c) a hydrophilic group, wherein the hydrophilic group is present in an amount
sufficient
to make the water-soluble photo-activatable polymer soluble in water; and
=
3.0 (d) a polymer precursor.
In certain embodiments, the polymer precursor comprises at least one monomer
selected
from the group consisting of allylamine, vinylamine, acrylic acid, carboxylic
acid, alcohol,
ethylene oxide, and acyl hydrazine..
13
CA 02821214 2013-07-19
frf: _ _ - ;
1
In certain embodiments, the reactive group is a member selected from the group
consisting of an amino group, a thiol-reactive group, a carboxy group, a thiol
group, a protected
= thiol group, an acyl hydrazine group, an epoxy group, an aldehyde group,
and a hydroxy group.
In certain embodiments, the thiol-reactive group is a member selected from the
group
consisting of a 2-pyridyldithio group, a 3-carboxy-4-nitrophenyldithio group,
a maleiMide
group, an iodoacetamide group, and a vinylsulfonyl group.
=
In certain embodiments, the hydrophilic group is a member selected from the
group
consisting of an amino group and a carboxy group.
In certain embodiments, the photo-activatable group is a member selected from
the group
consisting of an aryl ketone and an aryl azide. In certain embodiments, the
aryl ketone is a
member selected from the group consisting of benzophenone and acetophenone.
In certain embodiments, the water-soluble polymer is represented by a formula:
(¨CH¨CH2-)n41¨(¨CH¨C112-)k
CH2 CH2
NH2 NH¨OC COPh
. wherein n is 50 to 2000 and k is 10 to 1000.
In certain embodiments, the water-soluble polymer is represented by a formula:
(¨CH¨CH2)n-k-rti¨t¨CH¨CH2-)m¨(¨CH¨CH2)k
1 1
C1H2 CH2 CH2
NH NH NH-OC COPh
0=C 0=C
1 N
CH2CH2COOH CH2CH2SS-0
wherein n is 50 to 2000, k is 10 to 1000, and m is 10 to 1000.
Further provided is a process for producing the water-soluble photo-
activatable polymer
of the invention, the process comprising:
providing a polymer precursor comprising a plurality ofreactive groups and a
plurality of
hydrophilic groups;
providing a photo-activatable reagent; and
reacting the polymer with the photo-activatable reagent to obtain the water-
soluble
photo-activatable polymer, wherein a first portion of the reactive groups
and/or hydrophilic
groups is modified with the photo-activatable group.
= In certain embodiments of the process, the first portion is from about 1%
to about 50%
of the reactive groups and/or hydrophilic groups.
14
CA 02821214 2013-07-19
`Lr=I
In certain embodiments, the first portion is about 20% of the reactive groups
and/or
hydrophilic groups.
=
In certain embodiments, the process for producing the water-soluble photo-
activatable
polymer of the invention further comprises:
= 5 providing a reagent comprising a thief-reactive
group; and
reacting unmodified reactive groups and/or hydrophilic groups with the reagent
to obtain
=
=
the water-soluble polymer, wherein a second portion of the reactive groups
and/or hydrophilic
= groups is modified with the thiol-reactive group.
In certain embodiments, a sum of the first portion and the second portion is
from about
60% to about 80%.
In certain embodiments, the thiol-reactive group is a member selected from the
group
=
consisting of a 2-pyridyldithio group, a 3-carboxy-4-nitrophenyldithio group,
a maleimide
group, an iodoacetamide group, and a vinylsulfonyl group.
Further provided is a composition of matter comprising a monomolecular layer
of the
water-soluble photo-activatable polymer of the invention and a matrix having
at least one
carbon, wherein the monomolecular layer is covalently attached to the matrix
by a covalent bond
between the photo-activatable group and the at least one carbon.
In certain embodiments, the composition further comprises a biomaterial having
a
plurality of active groups, wherein the bioinaterial is covalently attached to
the monomolecular
layer by covalent bonding between the active groups and reactive groups.
In certain embodiments of the composition, at least one of the active groups
is a member
selected from the group consisting of amine, carboxyl, hydroxyl, thiol,
phenol, imidazole, and
indole. Prefembly, the at least one of the active groups comprise thiol.
In certain embodiments of the composition, the biomaterial is a member
selected from
the group consisting of an antibody, a viral vector, a growth factor, a
bioactive polypeptide, a =
= polynucleotide coding for the bioactive polypeptide, a cell regulatory
small molecule, a peptide,
a protein, an oligonucleotide, a gene therapy agent, a gene transfection
vector, a receptor, a cell,
a drug, a drug delivering agent, nitric oxide, an antimicrobial agent, an
antibiotic, an anthnitotic,
dimethyl sulfoxide, an antisecretory agent, an anti-cancer chemotherapeutic
agent, steroidal arid
non-steroidal anti-inflammatories, hormones, an extracellular matrix, a free
radical scavenger,
an iron chelator, an antioxidant, an imaging agent, and a radiotherapeutic
agent. Preferably, the
biomaterial is an anti-knob antibody, an adenovirus, a D1 domain ofthe
Coxsackie-adenovirus
=
CA 02821214 2013-07-19
. . _f
_ _
receptor, insulin, an angiogenic peptide, an antiangiogenic peptide, avidin,
biotin, IgG, protein
A, transferrin, and a receptor for transfenin.
In certain embodiments of the composition, the matrix is a member selected
from a
group consisting of polyurethane, polyester, polylactic acid, polyglycolic
acid, poly(lactide-co-
glycolide), poly(s-caprolactone), polyethyleneimine, polystyrene, polyamide,
rubber, silicone
rubber, polyacrylonitrile, polyacrylate, and polymetacrylate, poly(alpha-
hydroxy acid),
poly(dioxanone), poly(orthoester), poly(ether-ester), poly(lactone),
polytetrafluoroethylene,
organosilane, mixtures thereof and copolymers thereof.
In certain embodiments of the composition, the matrix further comprises a
superparamagnetic agent. Preferably, the superparamagnetic agent is a member
selected from
= the group consisting of magnetite and maghemite nanocrystals as such, as
aggregates or as
= dispersion in polymer from the list above.
In certain embodiments of the composition, the matrix is an implantable
device.
Preferably, the implantable device comprises at least one member selected from
the group
consisting of polyurethane, polyester, polylactic acid, poly(lactide-co-
glycolide),- poly(s-
caprolactone), polyethyleneimine, polystyrene, polyamide, rubber, silicone
rubber,
polyacrylonitrile, polyacrylate, polymetacrylate, polytetrafluoroethylene,
organosilane, mixtures
thereof and copolymers thereof.
In certain embodiments of the composition, the matrix is a particle having a
diameter of
about 5 nm to about 10 microns. Preferably, the particle comprises at least
one member selected
from the group consisting of polylactic acid, poly(lactide-co-glycolide),
poly(s-caprolactone),
polyethyleneirnine, mixtures thereof and copolymers thereof.
.
Further provided is a method of making the composition of the invention, the
method
comprising:
providing the matrix having at least one carbon;
= providing an aqueous solution ofthe water-soluble photo-activatable
polymer having the
photo-activatablegroup and the reactive group;
contacting the matrix with the aqueous solution; and
= photo-activating the photo-activatable group by irradiation to covalently
attach the water-
soluble polymer via the photo-activatable group to the matrix and thereby
forming the
= monomolecular layer of the composition.
16
CA 02821214 2013-07-19
In certain embodiments of the method of making the composition of the
invention, the
irradiation is performed at a wavelength from about 190 to about 900 nm.
Preferably, the
irradiation is performed at a wavelength of 280 to 360 nm.
Additionally, certain embodiments of the method further comprise providing a
biomaterial having a plurality of active groups and reacting the plurality of
active groups with
the water-soluble photo-activatable polymer to covalently attach the
biomaterial to the matrix.
Also provided is a process for delivery of a biontaterial, the process
comprising:
providing the composition of the invention as a monomolecular layer and a
matrix
having at least one carbon, wherein the monomolecular layer is covalently
attached to the matrix
by a covalent bond between the photo-activatable group and the at least one
carbon;
providing a biomaterial having a plurality of active groups, wherein the
biomaterial is
covalently attached to the monomolecular layer by covalent bonding between the
active groups
and the reactive groups; and
administering the matrix to the cell and thereby delivering the biomak-riall
I Further, the invention provides an implantable device comprising:
a surface having a magnetizable compound and
a magnetic carrier comprising at least one of a superparamagnetic agent,
magnetic-field
responsive agent, and a coated magnetic-field responsive agent , wherein the
magnetic carrier is
attracted to the magnetizable compound by a magnetic force, provided that the
magnetic carrier
is at least one of (a) a surface modified particle comprising a water-soluble
photo-activated
polymer, and (b) a magnetic particle comprising an amphiphilic agent.
In certain embodiments, the magnetizable compound comprises at least one of
cobalt,
iron, nickel, oxides and alloys thereof. In certain embodiments, the
magnetizable compotmd
comprises magnetic particles such as for example, ferromagnetic particle
provided that they are
biodegradable and biocompatible.
In certain embodiments, the surface modified particle comprises a water-
soluble photo-
activated polymer and a biomolecule covalently attached to the water-soluble
photo-activated
= polymer. In certain embodiments, the magnetic particle comprises a matrix-
forming polymer; a
= polyelectrolyte-amphiphilic agent adduct wherein the polyelectrolyte-
amphiphilic agent adduct
is in physical communication with the matrix-forming agent; a coated magnetic
field-responsive
_ _
agent; and the biomolecule in communication with the matrix-forming polymer.
In certain embodiments, the magnetic particle comprises: a matrix-forming
polymer; a
coated magnetic field-responsive agent comprising a magnetic field-responsive
agent and a
1 17
CA 02821214 2013-07-19
second amphiphilic agent, wherein the coated magnetic field-responsive agent
is in
communication with the matrix-forming polymer, provided that the magnetic
particle is free of a
polyelectrolyte; and the biomolecule in communication with the matrix-forming
polymer.
= Further provided is a method for delivery of a biomaterial to a cell, a
body or a subject,
the method comprising: providing the device comprising a first magnetic
carrier having a first
biomolecule at a first concentration; administering the device to the cell,
the body or the subject;
and administering a second magnetic carrier having a second biomolecule at a
second
concentration, and thereby delivering the first biomaterial and the second
biomaterial. This
describes a reloading of a stent in situ in a blood vessel with magnetic
particles, thereby
changing medications or dosages.
= A method for delivery of a biomaterial to a cell, a body or a subject,
the method
comprising: providing the device for implantation in a body comprising a
biocompatible metal
or polymer structural supporting implant having a plurality of magnetizable
features which
attract a therapeutic agent delivered in a magnetic carrier to the device upon
magnetization of
= 15 = the magnetic carrier by application of a far-penetrating magnetic
field to the subject;
= administering the device to the cell, the body or the subject;
administering a first magnetic
= carrier having a first biomolecule at a first concentration so that the
first magnetic carrier is
attracted to the device and thereby delivering the first biomaterial;
administering a second
magnetic carrier having a second biomolecule at a second concentration, and
thereby delivering
the second biomaterial.
BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS
;
The invention will be described in conjunction with the following drawings in
which like
reference numerals designate like elements and wherein:
Figs. 1A and 1B are diagrams of an exemplary embodiment of a device of the
present
!invention implanted in an artery. In this embodiment, the device comprises a
magnetizable stent
. =
=
= wherein segments of magnetizable compound have been electro-deposited as
a mesh-like
=
structure on some of the struts of the stent. Darkened bars depict the
segments of the
magnetizable compound which has been deposited on the stent. As shown in
Fig.1A, blood
flow through the artery brings a therapeutic agent in a magnetic carrier in
proximity with the
implanted stent As shown in Fig.1B, as blood flows through the stent, the
therapeutic agent in
the magnetic carrier is attracted to the segments of the stent with the
magnetizable compound.
Figs. 2A and 28 are scanning electron microscopy images of bead capture
obtained using =
a wire mesh electroplated with the magnetizable compound Cobalt Nickel alloy.
Fig. 2A shows
=
18
=
=
CA 02821214 2013-07-19
the mesh before exposure to the magnetic beads while Fig. 2B shows the mesh
after exposure to
2.8-micron magnetic beads.
Fig. 3 is a schematic illustration of the magnetically targeted delivery.
Fig. 4A is a schematic illustration of a top view of micro-channels having
walls seeded
with magnetic particles.
Fig. 4B is a chart depicting a flow of micro-particle solutions through micro-
channels
shown in Fig. 4A.
J
Fig. SA is a bar graph shovving transfection efficacy of magnetic
nanoparticles examined
as a function of magnetic field exposure in the absence of magnetic field
exposure.
Fig. 5B is a bar graph showing transfection efficacy of magnetic nanopartieles
examined
as a function of magnetic field exposure in the presence of magnetic field
exposure.' '
Fig. 6 is a graph showing cell toxicity of magnetic NP determined in BAEC
cells by
WST-1 assay following 4 hour incubation at 37 C as a function of the
concentration ofparticle¨
associated PEI or free PEI used as a control.
Fig. 7 is a graph showing the effect of the organic phase 'composition on the
size of
freshly prepared particles and size stability observed 7 days after particles'
preparation.
Fig 8 is a graph showing the extent and stability of PEI association with
magnetic NP as
a function of the organic phase composition.
Fig. 9 is a scheme showing a preferred method of making the particle of the
invention
comprising the matrix-forming agent, polyelectrolyte-amphiphilic agent adduct,
the coated
magnetic field-responsive agent and the biomaterial.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides devices and methods for using these devices in
the
targeted delivery of therapeutic agents in a body or a subject.
By the term "therapeutic agent" as used herein is meant to include, but is not
iimitici to,
drugs, imaging agents such as radionuclides, cells, gene therapy agents such
as vectors and
viruses, and proteins and peptides. Therapeutical agent can be biomolecule as
defined below.
19
CA 02821214 2013-07-19
Devices of the present invention comprise a structural supporting implant of
biocompatible metal or polymer. Implants of the present invention further
comprise a
magnetizable compound. In one embodiment, the magnetizable compound is
segmented on the
thstrue cimtupralanl stn. pipon planteinbotdoi mp
d e ther emg iaouin se toi of ah bi
einldpgoruanddieinstsundiifsotrrmibulytedcoathteroduogInlot inlet
=
ent
high
cfio
rta ni no iemr
structural supporting implant, and the magnetized state of the coating is
either locally recorded
to provide regions of high field gradients distributed throughout the implant
or unifonnly
magnetized. bi yet another embodiment, the implant itself comprises the
magnetizable
compound.
By magnetizable compound, as used herein, it is meant a material that conducts
magnetic
= flux strongly. Examples of magnetizable compounds useful in the implants
of the present
= invention include, but are not limited to, cobalt, iron, iron oxides,
nickel, and rare earth magnetic
materials and various alloys. In one embodiment, the magnetizable compound is
magnetized
=
=
only in the presence of externally applied fields. Examples of these types of
magnetizable
= 15 compound include, but are not limited to, superpararnagnets and
soft ferromagnets. In other
embodiment, magnetizable compounds known as ferromagnets, which can be
permanently
magnetized, are used.
= =
In one embodiment, the structurally supporting implant itself is made entirely
from .
alloys of magnetizable compounds.
= = 20 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
25 extending over the implant and a mesh-like structure surrounding the
implant such as depicted
in Figures 1 and 2.
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.
=
=
30 Magnetic carriers 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. Thus, several means of patterning the magnetic coating to
control domain
patterns in the devices of the present invention can be used.
CA 02821214 2013-07-19
, vt
One such means 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 ofthe 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 dieleetrophoresis. 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-1MHz) electric field
creates strong, high
= frequency electric field gradients on the implant that attracts the
nanoparticles.
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
= 15 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.
In one 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, niclsel, 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
= 25 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 intimal
thickening,
respectively.
The segments of magnetizable compound coated on the stent provide the stent
with the
capability of attracting arterially injected magnetic carriers including, but
not limited to
magnetic particles, magnetic liposomes and ferrofl aids encapsulating or
attached to the
21
CA 02821214 2013-07-19
. _
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.
In an alternative embodiment of the device of the present= invention, the
implant
comprises a plural ity 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 ofthe 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 oftreatment so that the therapeutic agent
is dispersed over the
treatment site and possible clogging of the artery by deposited agent is
decreased.
= 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.
= 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.
= 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.
22
CA 02821214 2013-07-19
j = .4 4.1:::A 1
=
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
fermfluid.
There are numerous other potential sites of implantation envisioned for a
magnetizable/magnetic device ofthe present invention. It is important to note
that in all ofthese
cases, a magnetic carrier such as !magnetic particles, magnetic liposomes or a
ferroflu id 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.
In addition, devices of the present invention surgically placed within the
brain for local
1
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 awl treatments may
limit healthy
neural tissue exposure to chemotherapy while maximizing dosage levels.
The therapeutic agent to be delivered is encapsulated in, attached to, or
dispersed in a
magnetic carrier. 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 feffofluid. 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.
It is also preferred that magnetic carriers such as magnetic particles or
magnetic
liposomes comprise magnetite. Magnetite is a member of the spinet group with
the standard
formula Fe203 or Fe304. Magnetite particles to be incorporated in the magnetic
particles or
23
CA 02821214 2013-07-19
magnetic liposomes enoapiulating the therapeutic agent are preferably around
10 nm in diameter
and are dispersed within the magnetic particle or magnetic Iiposome to account
for 10-50% of
sphere volume.
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) ?LOA, 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 elastomerie
poly(ester-
arnide), 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.
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 ferrofiuid
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 ofthe
present invention.
Some examples of such therapeutic agents include, but are not limited to,
antiplatelet agents
such as AspirinTm, glycoprotein receptor antagonists, and cilostazol for
prevention of thrombus
formation by interferingwith 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;
Rapamunee); anti-inflammatory agents; and anti-proliferation agents such as
Actinomycin D
(Cosmegen0), Estrogen (Estrodiole), and Paclitaxel (Taxo10). Thus, for PTCA
procedures
and tumor treatments using a magnetizable stent ofthe 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-
= 24
CA 02821214 2013-07-19
:=
1 : . ¨ = n"),
:.RE "1=`;'h'
intimal growth.
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.
In some embodiments, the device of the present invention and/or magnetic
Carrier ofthe
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.
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
= Figure IA 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
embodiinent, 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.
1
, 30
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.
CA 02821214 2013-07-19
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 ofthe radial position at which the sphere
enters the tube (Segre,
G. and Silberberg, A. 1. Fluid Mech. 1962 14136). Further, it has been shown
that the
=
trajectories ofthe particles are portions of one master trajectory, and that
the origin ofthe forces
causing the radial displacements is in the inertia of the moving fluid (Sege,
G. and Silberberg, =
A. I, Fluid Mech. 1962 14:136).
=
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 FT., 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.
Ecl= 1 = = PhYi = 0/-
Eq. 2
= +3
In Equations 1 and 2, 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 x 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 tenninates on the site of the implant:
=
Eq. 3 Az =
= 6n-qavõ
1
=
Using values ranging from extreme to moderate to account for varying inducible
magnetic field
26
=
CA 02821214 2013-07-19
strength, particle size, and blood flow velocity, simulations have shown that
for a variety of
different physiologically significant parameters, F. 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.
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.
= 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
micrpspheres aggregated on the magnetized embedded beads.1
1Experiments 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 (50x50, 100x100, 500x500 pm cross-section) were fabricated in
poIydimethyl
siloxane (PDMS). The walls of the micro-channels were seeded with
superparamagneticilatex
composite microparticles (5tim diameter) (See Forbes ZG, Yellen BB, Barbee KA,
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 Figure 2. Various
concentration solutions of
commercially available magnetic particles of various sizes (1 p.m, 3 pm, 5
pin, 7 gra) were
= observed to flow in the channels while applying external relatively
uniform magnetic field
varied up to 300 Oe (0.03 Testa). 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 (10s 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 observwed.
The possibility of making stents that could attract magnetic particle carriers
under similar
= 27
CA 02821214 2013-07-19
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 gm and 5 gm diameter). A basic set-up for electroplating
these stents was
developed and stents were coated with 1 gm thicic 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..I
Explanation of Basic Principles of the Proposed Method
The magnetic force dragging isolated magnetic carriers toward the stent is:
fr (.11-6' = 03- (1)
where the magnetic moment Jí===1/1:i3 of the drug carrying particle is
approximately proportional
to the total field -h up until saturation which occurs for most nanoparticles
in the range of about
= 600 Oe (0.06 Tesla). The total field + 13 õ 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
1
(V
0). The stent, on the other hand, produces very large gradients near itself
because of
the presence ofvery small magnetized features on it. Thus, from (1) and from
above arguments,
the force on magnetic drug carriers can be approximately written as
= VYken, (2)
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. 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 gm in
diameter and about 200-500 nm in thickness.
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
.=
isuch coatings are hard to control. This invention provides several means of
patterning the
magnetic coating to control domain patterns..
One involve laser assisted electrodeposition of alloys of magnetic metals such
as Co, Ni
= 28
CA 02821214 2013-07-19
[ [
.
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 sots that are exposed to a focused laser beam.
Another approach to stent patterning will involve 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 (1001(11z-1MEz) electric field will
create strong high
frequency electric field gradients on the stent that will attract the
nanoparticles.
Another approach will involve 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 thermomagnetie 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.
Polylactide-based nanoparticles (NP), sized about 160nm or about 360nm with
narrow
size distribution formed by a modified emulsification-solvent diffusion or
emulsification-solvent
evaporation methods, respectively. The NP surface modification with an anionic
thiol-reactive
= derivative of polyallyl amine is accomplished photochemically by a brief
exposure to long¨wave
UV light. This permits attachment of affinity adaptors for binding of gene
vectors to the surface
of these particles. After separation from unreacted polymer, NP can be
bioreversibly coated
with, for example, a thiolated form of the DI domain of the Coxsackie-
adenovirus (AdV)
receptor, to permit delivery of adenoviral gene vectors. Magnetic NP (for use
in either gene or
drug delivery) are formed by inclusion of iron oxide (preferred from a
biocompatibility stand
point) nanocrystals in the polymeric matrix. The particle core and the virus
were stained with
green BODIPY and red Cy3 dyes, respectively, for fluorimetric studies. In
addition to binding
to the nanoparticle's outer surface via polymeric modification, the
therapeutic substance (e.g., a
biomolecule) can be incorporated into the particle's core. AdV-NP binding
efficacy can be
.= determined. by measuring the residual fluorescence of unbound
virus following magnetic
=
separation of AdV-NP composite.
29
CA 02821214 2013-07-19
r vitti '
Cell culture experiments demonstrating magnetic targeting ofcells and loading
of these
magnetic nanoparticle containing cells onto a magnetized surface were
performed. In these
studies fluorescent labeled (polylactic polymer was covalently modified with
BOD1PY 5641570
and used for labeling)
Magnetic nanoparticles comprising the water-soluble polymer as described below
(PAA-
BzPh or PDT-BzPh) were loaded into cells (arterial smooth muscle cells (A10)
in culture)
under the influence of a magnetic field. These cells were then grown to
confluence, trypsinized
and suspended. The suspension was then placed in contact with a cobalt
magnetized stainless
steel mesh prepared as described above, which demonstrated adhesion of the
cells to the
magnetized tip of the mesh thereby providing proof of concept for magnetic
mediated
nanoparticle driven cell delivery. Bright field channel Light Microscopic
image of magnetic
nanoparticle loaded cells was observed as well as a fluorescent image. In was
observed that
magnetic nanoprticles were distributed in cells.
'The invention was also driven by the desire to develop particles capable of
carrying and
delivering biomaterial. In certain embodiments, the particles are capable of
targeted delivery of
biomaterial being guided by a magnetic, force, wherein the particles further
comprise a coated
magnetic field-responsive agent.
Accordingly, the invention provides a particle comprising a matrix-forming
agent and a
polyelectrolyte-amphiphilic agent adduct, wherein the polyelectrolyte-
amphiphilic agent adduct
. .
is in physical communication with the matrix-forming agent. In certain
embodiments, the
particle further comprises a biomaterial in communication with at least one of
the
polyelectolyte-amphiphilic agent adduct or the matrix-forming agent.
=
Advantageously, the particle of the invention possesses two compartments for
= incorporation of a biomaterial, the polyelectrolyte and the matrix-
forming agent, wherein one of
the compartments or both can be utilized. The particles of the invention
provide an improved
loading of a biomaterial such as, for example, a plasmid DNA by utilizing an
ionic binding with
a polyelectrolyte (e.g., poly(ethyleneimine) (PEI)) complexed with or adducted
to an
amphiphilic agent to form a polyelectrolyte-amphiphilic agent adduct. The
polyelectrolyte-
,
amphiphilic agent adduct has a strong association with the surface of
particles due to its
,
possessing a lipophilic and hydrophilic domains with high affinities to
respective media used in
=
the emulsification step of the particle preparation. The polyelectrolyte
useful in this invention
can be cationic (e.g., PEI) or anionic (e.g., dextran sulfate).
A non-limiting example of the particle comprising an anionic polyelectrolyte
is PLA (the
CA 02821214 2013-07-19
= .?..=
..7r
matrix-forming polymer)/stearylamine (the amphiphilic agent)/dextran sulfate
(polyanion)/PDGF (cationic biomaterial). A non-limiting example of the
particle comprising a
cationic polyelectrolyte is PLA/oleic acid (the amphiphilic agent)/PEI (the
amphiphilic
agent)/DNA (anionic biomaterial)..
In addition, the biomaterial can be incorporated in the particles of the
invention by being adsorbed,
entangled with or entrapped by the matrix-forming polymer.
Further, the particles of the invention may possess magnetic targeting
capability and thus
make possible the enhanced targeted delivery of the biomaterial to specific
cells or organs either in
cell culture or in vivo, wherein a magnetic field gradient is induced by a
magnetic field. The
I 0 particles of the invention interface well with, for example, a
nanoscale controllable magnetic
patterned device surface (the magnetic device) for bindingõ release, and
repeated loading of
drug,/gene delivery systems. Thus, in certain embodiments, the particle
further comprises a coated
magnetic field-responsive agent having a magnetic field-responsive agent in
communication with an
amphiphilic agent, wherein the coated magnetic field-responsive agent is in
conununication with the
matrix-forming agent. In certain embodiments, such particle further comprises
a biomaterial in
communication with at least one of the polyelectrolyte-amphiphilic agent
adduct or the matrix-
forming agent.
In certain embodiments, the particle of the invention comprises a matrix-
forming polymer, a
polyelectrolyte-amphiphilic agent adduct comprising a first C12-C24
carboxylate group in physical
communication with the matrix-forming polymer, a second arnphiphilic agent
comprising a second
C12-C24 carboxylate group in communication with the matrix-forming polymer;
and a magnetic-field
responsive agent in communication with a second C12-C24 carboxylate group. In
certain
embodiments, the particle further comprises a stabilizer. In certain
embodiments, the particle
further comprises a biomaterial in communication with the polyelectrolyte-
amphiphilie agent adduct
and optionally with the matrix-forming polymer.
In a preferred embodiment, the particle of the invention comprises poly(lactic
acid) (PLA),
PEI-oleic 'acid adduct, Pluronicl" F-68, magnetite coated with oleic acid and
DNA, wherein PIA,
PEI-oleic acid adduct, and PluronicT" F-68 were premixed, mixed with a
dispersion of mapetite/oleic
acid in organic solvent, and emulsified in water, followed by removal of
organic solvent. =
Further, the particle of the invention can be a magnetic particle comprising a
matrix-forming
=
polymer; and a coated magnetic field-responsive agent comprising a magnetic
field-responsive agent
and a second amphiphilic agent, wherein the coated magnetic field-responsive
agent is in
communication with the matrix-forming polymer, provided that the magnetic
particle is free of a
31 =
CA 02821214 2013-07-19
?.= =e,
=
polyelectrolyte. A used herein, the term "magnetic particle" denotes a
particle possessing magnetic
capabilities conferred by the coated magnetic field-responsive agent but in
the absence of the
polyelectrolyte to differentiate from a particle possessing magnetic
capabilities and comprising the
polyelectrolyte. In certain embodiments, the magnetic particle further
comprises a biomaterial in
communication with the matrix-forming polymer.
The particle of the invention can be used for delivery of biornaterial
entrapped in or adsorbed
on the matrix-forming polymer and/or associated with the particle surface via
the polyelectrolyte for
various applications such as, for example, drug therapy, chemotherapy,
chemoembolization,
hyperthermic cancer treatment, diagnostics, and radiotherapy.
' 10 One of the applications for this formulation is a targeted gene or
drug delivery to the heart.
In one embodiment of the invention, a cardiac catheterization is carried out
with positioning of a
powerful magnet in the part of the heart where gene delivery is desired, for
example, in the right
atrium (a targeted tissue). Next, the particles of the invention (e.g., the
biodegradable PEI-modified
magnetic nanoparticles loaded with DNA) are administered to the coronary
artery (e.g., by injection)
and the myocardial circulation together with the magnetic field localizes the
bulk of the particles in
The desired area, the right atrial myocardium. A similar approach could be
applied to virtually any
target cell or organ region by providing the particles of the invention and a
magnetic device
associated with the targeted cell orthe targeted tissue.
The particles of the invention can be used also for the prevention,
diagnostic, or
treatment of various conditions or disorders such as, for example, tumors,
gastro-intestinal
disease, pulmonary and bronchial disorders by delivering the appropriate
biomaterial. The
particles of the invention can also be used in hormonal therapy and anesthetic
medication.
Also; the particles of the invention can be used as an analytical tool, for
example for
screening.
Delivery of biomaterial under influence of magnetic force includes reversible
movement
of particles from one target site to another.
In certain embodiments, the particle further comprises a biomaterial in
communication
with at least one of the polyelectrolyte-amphiphilic agent adduct or the
matrix-forming agent,
wherein the particle is free of the magnetic field-responsive agent.
The particle ofthe invention, its components, and methods of malcing the
particle will be
described in detail below.
PARTICLE-
=
32
CA 02821214 2013-07-19
-4
.1
=)"-K"." ",t-t
The term "particle" as used herein denotes a solid colloidal particle having a
diameter of
about 5 nm to about 10 microns. The particle can be a sphere or a capsule,
wherein the sphere is
composed of a solid matrix, while the capsule has an oil-based or water-based
core surrounded
by the matrix-forming polymer. An example of a magnetic particle is shown in
Fig. 9. The
.= 5 particle can have one or more coated magnetic field-responsive
agents incorporated within the
= matrix-forming polymer. Preferably, multiple coated magnetic nanocrystals
are incorporated in
= the core of the particle.
MATRIX-FORMING AGENT
The matrix-forming agent used in the invention includes synthetic or natural
polymers
and non-polymeric substances.
The term "matrix-forming polymer" as used herein denotes a synthetic or a
natural
.
polymer, which forms the core of the particle. The matrix-forming polymer can
be
biodegradable, non-biodegradable, biocompatible, and is water-insoluble. The
release kinetics
1 of biomaterial from nanoparticles can be determined by the rate
of the matrix-forming polymer's
= 15 degradation, diffusion or dissolution of material
entrapped in the particle or desorption of
= surface-bound substances.
Non-limiting examples of natural polymers are proteins, polysaccharides and
lipids as
described by Quintanar-Guerrero et al., supra and Kumar, supra. Non-limiting
examples of
synthetic polymers are poly(ester)s, poly(urethane)s,
poly(allcylcyanoacrylate)s,
poly(anhydride)s, poly(ethylenevinyl acetate), poly(lactone)s, poly(styrene)s,
poly(amide)s,
poly(acrylonitrile)s, poly(acrylate)s, poly(metacrylate)s, poly(orthoester)s,
poly(ether-ester)s,
poly(tetrafluoroethylene)s, mixtures thereof and copolymers thereof. In
certain embodiments,
the poly(ester) is a member selected from the group consisting
ofpoly(lactide), poly(glycolide),
poly(lactide-co-glycolide), poly(g-caprolactone), poly(dioxanone),
poly(hydroxybutyrate), and
poly(ethylene terephthalate).
= Non-limiting examples of non-polymeric substances are solid lipids such
as glycerides
and fatty acids.
AMPH1PHILIC AGENT
The term "anaphiphilic agent" as used herein denotes a biocompatible agent
having a
molecule comprising a reactive group and a lipophilic group. Non-limiting
examples of
amphiphilic agents are fatty acids and lipids as well as salts thereof such as
carboxylates,
phosphonates, bisphosphonates, phosphates, sulfonates, and sulfates. In
certain embodiments,
fatty acids are C12-C24 carboxylic acids and salts or esters thereof; the
preferred fatty acid is oleic
33
CA 02821214 2013-07-19
= _
acid. In certain embodiments, lipids are phospholipids such as
phosphatidylglycerol and
phosphatidylinositoI. Amphiphilic agent can also be cationic, such as
stearylamine, 30-(N-
[dimethylamine ethane] carbamoyl) cholesterol (DC-Chol), N11-(2,3-
dioleoyloxy)propyll-
N,N,N-trimethylammonium chloride (DOTAP).
One of the functions of the amphiphilic agent is to provide a connection
between the
matrix-forming agent and the polyelectrolyte.
The reactive group of the amphiphilic agent is a polar chemical group such as,
for
example, a carboxylate group, a phosphonate group, a bisphosphonate group, a
phosphate group,
a sulfonate group, and a sulfate group_ The association between the
amphiphilic agent and the
polyelectrolyte may be ionic (e.g., an ammonium carboxylate ion-pair) or
covalent (e.g. via
amide, imine, urethane, urea, alkyl bond, etc.).
The lipophilic group of the amphiphilic agent is a hydrocarbon chain with low
water
affinity, preferably a C4-C24 chain having all saturated bonds (e.g., lauric
acid and palmitic acid)
or at least one unsaturated bond (e.g., oleic acid). The hydrocarbon chain can
present a cyclic
structure (e.g. cholesterol or cholic acid). The hydrocarbon chain can further
have one or more
hydrogen atoms substituted with for example an aromatic group. The hydrocarbon
chain can
further have one of more carbon atoms substituted with heteroatorns (e.g.,
nitrogen, sulfur, and
oxygen).
The adduct of the amphiphilic agent and the polyelectrolyte can be preformed
or made in
situ in the course of the particle preparation.
POLYELECTROLYTE
The polyelectrolyte of the invention provides a connection between the matrix-
forming
polymer and the biomaterial, wherein the polyelectrolyte is associated with
the matrix-forming
polymer through the amphiphilic agent. The polyelectrolyte can be cationic or
anionic.
In one embodiment, the polyelectrolyte and the biomaterial are ionically
associated. For
example, the polyelectrolyte is cationic (e.g., poly(ethyleneimine)) and the
biomaterial is anionic
(e.g., DNA) or the polyelectrolyte is anionic (e.g., dextran sulfate) and the
biomaterial is cationic
(e.g., PDGF).
Association between the biomaterial and the polyelectrolyte-amphiphilic agent
adduct
can also be covalent, wherein reactive groups of the biomaterial chemically
react with reactive
groups of the polyelectrolyte-amphiphilic agent.
34
CA 02821214 2013-07-19
:-t$ I
In certain embodiments, the polyelectrolyte is a member selected from the
group
consisting of poly(ethyleneirnine), poly(propyleneimine), poly[N-ethyl-4-vinyl
pyridinium
bromide], polyamidoamine dendrimer, poly(allylamine) and derivatives thereof.
preferably about
used lienstshteo iennvheannticoenithsaeslaimminoaletciounl weightafir.ome
obfodabyoourti2s5aKDbioda,eagranddmaobrle
e
th
derivative thereof.
PEI with the molecular weight of at least 25 KDa is known to be toxic to cells
while
decreasing the size of PEI reduces the toxicity but it also reduces the
efficacy of gene transfer.
(See Forrest et al., Gosselin et al., ).
Examples of biodegradable PEI derivatives useful in the invention can be found
in
Gosselin et al., Efficient gene transfer using reversibly cross-linked low
molecular weight
polyethylenimine, Bioconjug Chem. 2001 Nov-Dec;12(6):989-94, and expected to
result in
further reduction of toxic effects. PEI derivatives can be prepared by cross-
linking 800 Da PEI
with dithiobis(succinimidylpropionate) (DSP) and/or dimethyl 3,3 '-
dithiobispropionimidate
21IC1 (DTBP).
Forrest et al., A degradable polyethylenimine derivative with low toxicity for
highly
efficient gene delivery, Bioconjug Chem. 2003 Sep-Oct;14(5):934-40); disclose
highly branched
14-30 KDa polycations that are biodegradable analogs of 25 KDa PEI produced by
addition of =
amino groups on 800 Da PEI to diacrylates such as, for example, 1,3-butanediol
diacrylate of
varying spacer length. These or similar derivatives can also be useful in the
invention.
MAGNETIC FIELD-RESPONSIVE AGENT
A magnetic field-responsive agent as used herein is a paramagnetic,
superparamagnetic, ,
or ferromagnetic substance capable of moving under influence of a magnetic
force. In certain
embodiments, the magnetic field-responsive agent is a member selected from the
group
consisting of iron, cobalt or nickel, alloys thereof, oxides thereof and mixed
oxides/hydroxides
ofFe(11) and/or Fe(III) with at least one of Co(Il), Mn(11), Cu(II), Ni (II),
Cr(M), Gd(I11), Dy(M),
and Sin(111). Preferably, the magnetic field-responsive agent is at least one
of Fe304, gamma-
Fe203, or a mixture thereof. Preferably, the magnetic field-responsive agent
is iron oxide in a
shape of nanocrystals.
The magnetic field-responsive agent can be prepared by methods known in the
art in
various shapes and sizes (see Hyeon T., Chemical Synthesis of Magnetic
Nanoparticles. The
Royal Society of Chemistry 2003, Chem. Commun., 2003, 927-934). In certain
embodiments,
iron oxide nanocrystals were obtained by precipitation of mixed iron chlorides
in the presence of
CA 02821214 2013-07-19
a base in aqueous medium (see Khalafalla SE. Magnetic fluids, Chemtech 1975,
Sept.: 540-
547).
The term "coated magnetic field-responsive agent" as used herein denotes a
magnetic
field-responsive agent in communication with an amphiphilic agent (an
amphiphilic agent can
be defined as "first" or "second" to indicate that these agents can be
different substances,
however embodiments in which these agents are the same are contemplated as
well). In the
particle of the invention, the coated magnetic field-responsive agent is in
communication with
the matrix-forming agent.
STABILIZING AGENT
Optionally, the particle of the invention includes a stabilizing agent. Non-
limiting
examples of stabilizing agents include poly(sotbate) (e.g., TWEENTm (Merck and
Co. Inc.,
Whitehouse Station New Jersey, USA)), sorbitan ester, ethylene oxide-propylene
oxide block
copolymers (e.g., poloxamer), poloxamine, poly(ethylene glycol) (PEG), alkyl
polyethylene
glycol ether, PEG-based non-ionic surfactants (e.g., fatty acid polyethylene
glycol ester and
mixtures thereof). Examples of the functions of such stabilizing agents
include providing steric
protection, contributing to the stability in high ionic strength media in
vitro and in serum in
vivo, preventing rapid sequestration of the nanoparticles by the
reticuloendothelial system
following an IV injection.
B1OMATERIAL
The biomaterial of the present invention can be any molecule or macromolecule
having a
therapeutical utility. In certain embodiments fele composition, the
biomaterial is a member
selected from the group consisting of a nucleic acid, a protein, a peptide, an
oligonucleotide, an
antibody, an antigen, a viral vector, a bioactive polypeptide, a
polynucleotide coding for the
bioactive polypeptide, a cell regulatory small molecule, a gene therapy agent,
a gene transfection
vector, a receptor, a cell, a drug, a drug delivering agent, an antimicrobial
agent, an antibiotic, an
antimitotic, an antisecretory agent, an anti-cancer chemotherapeutic agent,
steroidal and non-
steroidal anti-inflammatories, a hormone, a proteoglycan, a glycosaminoglycan,
a free radical
scavenger, an iron chelator, an antioxidant, an imaging agent, and a
radiotherapeutic agent.
In certain embodiments of the composition, the biomaterial is any molecule or
macromolecule to which a suitable ionizable reactive group, such as, for
example, a carboxy (-
COOT) group or an amino (-NH2) group is attached.
The biomaterial can associate with the particle in several ways. In one
embodiment, the
biomaterial is in ionic association with the charged groups of the
polyelectrolyte-amphiphilic
36
CA 02821214 2013-07-19
agent adduct (e.g., the carboxylate group) while the lipophilic part of the
polyelectrolyte-
.
amphiphilic agent adduct is in association (i.e., anchored in the matrix or
chemicallybound to it)
with the matrix-forming polymer of the particle. Association between the
biomaterial and the
polyelectrolyte-amphiphilic agent adduct can also be covalent, wherein
reactive groups of the
biomaterial chemically react with reactive groups of the polyelectrolyte-
amphiphilic agent.
In certain embodiments, the biomaterial can be in physical association (i.e.,
entrapped or
adsorbed) with the matrix-forming polymer.
In certain embodiments, physical interactions as well as ionic and/or covalent
and are
utilized.
Suitable biomaterial include pharmaceuticals, nucleic acid sequences, such as
transposons, signaling proteins that facilitate wound healing, such as TGF-0,
FGF, PDGF, IGF
and GH proteins that regulate cell survival and apoptosis, such as 13c1-1
family members and
caspases; tumor suppressor proteins, such as the retinoblastoma, p53, PAC,
DCC. NF I, NF2,
RET, V1:11, and WT-1 gene products; extracellular matrix proteins, such as
laminins,
fibronectins and integrins; cell adhesion molecules such as cadherins, N-CAMs,
selectins and
immunoglobulins; anti-inflammatory proteins such as Thymosin beta-4, IL-10 and
IL-12.
In certain embodiments, the biomaterial includes at least one of: heparin,
covalent
heparin, or another thrombin inhibitor, hirudin, hirulog, argatroban, D-
phenylalanyl-L-poly-L-
arginyl chloromethyl ketone, or another antithrombogenic agent, or mixtures
thereof; urokinase,
streptokinase, a tissue plasminogen activator, or another thrombolytic agent,
or mixtures thereof;
a fibrinolytic agent, a vasospasm inhibitor; a calcium channel blocker, a
nitrate, nitric oxide, a
nitric oxide promoter or another vasodilator; an antimicrobial agent or
antibiotic; aspirin,
ticlopidine, a glycoprotein 11b/Illa inhibitor or another inhibitor of surface
glycoprotein
receptors, or another antiplatelet agent; colchicine or another antimitotic,
or another microtubule
inhibitor, dimethyl sulfoxide (DMSO), a retinoid or another antisecretory
agent; cytochalasin or
another actin inhibitor; a remodeling inhibitor; deoxyribonucleic acid, an
antisense nucleotide or
= another agent for molecular genetic intervention; methotrexate or another
antimetabolite or
antiproliferative agent; tamoxifen citrate, TaxolTm or derivatives thereof, or
other anti-cancer
chemotherapeutic agents; dexamethasone, dexamethasone sodium phosphate,
dexamethasone
acetate or another dexamethasone derivative, or another anti-inflammatory
steroid or a non-
steroidal anti-inflammatory agent; cyclosporin or another immunosuppressive
agent; trapidal (a
PDGF antagonist), angiogenin, angiopeptin (a growth hormone antagonist), a
growth factor or
=
an anti-growth factor antibody, or another growth factor antagonist; dopamine,
bromocriptine
37
=
CA 02821214 2013-07-19
_
mesylate, pergolide mesylate or another dopamine agonist; radiotherapeutic
agent; iodine-
containing compounds, barium-containing compounds, gold, tantalum, platinum,
tungsten or
another heavy metal functioning as a radiopaque agent; a cellular component;
captopril,
1
enalapril or another angiotensin converting enzyme (ACE) inhibitor; ascorbic
acid, alpha
tocopherol, superoxide dismutase, deferoxamine, a 21-amino steroid (lasaroid)
or another free
radical scavenger, iron chelator or antioxidant; a I4C-, 3H-, 32P- or 36S-
radiolabelled form or
other radiolabelled form of any of the foregoing; a hormone; estrogen or
another sex hormone;
= AZT or other antipolymerases; acyclovir, famciclovir, rimantadine
hydrochloride, ganciclovir
sodium or other antiviral agents; 5-aminolevulinic acid, meta-
tetrahydroxyphenylchlorin,
hexadecafluoro zinc phthalocyanine, tetramethyl hematoporphyrin, rhodamine 123
or other
photodynamic therapy agents; an IgG2 Kappa antibody against Pseudomonas
aeruginosa
exotoxin A and reactive with A431 epidermoid carcinoma cells, monoclonal
antibody against
the noradrenergic enzyme dopamine beta-hydroxylase conjugated to saporin or
other antibody
targeted therapy agents; gene therapy agents; and enalapril and other
prodrugs, or a mixture of
any of these.
Additionally, the biomaterial can be a component of any affinity-ligand pair.
Examples
of such affinity ligand pairs include avidin-biotin and IgG-protein A.
Furthermore, the
biomaterial can be either component of any receptor-ligand pair. One example
is transferrin and
its receptor. Other affinity ligand pairs include powerful hydrogen bonding or
ionic bonding
entities such as chemical complexes. Examples of the latter include metallo-
amine complexes.
Other such attractive complexes include nucleic acid base pairs, via
immobilizing
oligonucleotides of a specific sequence, especially antisense. Nucleic acid
decoys or synthetic
analogues can also be used as pairing agents to bind a designed gene vector
with attractive sites.
Furthermore, DNA binding proteins can also be considered as specific affinity
agents; these
include such entities as histones, transcription factors, and receptors such
as the gluco-corticoid
receptor.
In one preferred embodiment, the biomaterial is an anti-nucleic acid antibody.
The
antibody can therefore specifically bind a nucleic acid, which encodes a
product (or the
precursor of a product) that decreases cell proliferation or induces cell
death, thereby mitigating
the problem of restenosis in arteries and other vessels. The nucleic acid that
is tethered to a
support via the antibody can efficiently transfect/transducer cells. In
general terms, the field of
"gene therapy" involves delivering into target cells some polynucleotide, such
as an antisense
DNA or RNA, a ribozyme, a viral fragment, or a functionally active gene, that
has a therapeutic
38
CA 02821214 2013-07-19
== r-M
,k
El
= .
=
or prophylactic effect on the cell or the organism containing it. The antibody
of the composition
=
can be a full-length (i.e., naturally occurring or formed by normal immuno-
globulin gene
fragment recombinatorial processes) immunoglobulin molecule (e , g, , an IgG
antibody, or IgM or
. any antibody subtype) or an immunologically active (i.e.,
specifically binding) portion of an
=
immunoglobulin molecule. The antibody comprises one or more sites, which
specifically bind
with a nucleic acid (i.e., which does not substantially bind other types of
molecules). The
binding site can be one that binds specifically with a nucleic acid of a
desired type without
= regard to the nucleotide sequence of the nucleic acid. The binding site
can, alternatively, be one
which binds specifically only with a nucleic acid comprising a desired
nucleotide sequence.
The complex formed between a polynucleotide and a cognate antibody can be
immobilized on a variety of surfaces such that, when the surface is exposed to
a physiological
environment in situ, the attached polynucleotide is released, over time, in a
manner that
enhances delivery of the polynucleotide to cells in the proximity. DNA
transfer by way of
immunospecific tethering has previously been shown to maintain the nucleic
acid in regions
that are subject to gene therapy. ,
Examples of suitable antibodies include Fv, F(ab), and F(ab")2 fragments,
which can be
generated is conventional fashion, as by treating an antibody with pepsin or
another proteolytic
enzyme. The nucleic acid-binding antibody useful in the present invention can
be polyclonal
antibody or a monoclonal antibody. A "monoclonal" antibody comprises only one
type of
antigen binding site that specifically binds with the nucleic acid, A
"polyclonal" antibody can
comprise multiple antigen binding sites that specifically bind the nucleic
acid. An antibody
employed in this invention preferably is a full-length antibody or a fragment
of an antibody, such
as F(ab)2, that possesses the desired binding properties.
A nucleic acid for use in the present invention can be any polynucleotide that
one desires
=
= 25 to transport to the interior of a cell. In this context,
a "therapeutic polynucleotide" is a polymer
= , of nucleotides that, when provided to or expressed in a
cell, alleviates, inhibits, or prevents a
= disease or adverse condition, such as inflammation and/or promotes tissue
healing and repair
(e.g., wound healing). The nucleic acid can be composed of
deoxyribonucleosides or
ribonucleosides, and can have phosphodi ester linkages or modified linkages,
such as those
=
=
= . 30 described below. The phrase "nucleic acid" also
encompasses polynucleotides composed of
bases other than the five that are typical of bioicigical systems: adenine,
guanine, thymine,
cytosine and uracil.
39
CA 02821214 2013-07-19
' '."1",:f.-4", 113. _ 4
A suitable nucleic acid can be DNA or RNA, linear or circular and can be
single-or-
double-stranded. The "DNA" category in this regard includes: cDNA; genemie
DNA; triple
=
helical, supercoiled, Z-DNA and other unusual forms of DNA; polynucleotide
analogs; an
expression construct that comprises a DNA segment coding for a protein,
including a therapeutic
protein; so-called "antisense" constructs that, upon transcription, yield a
ribozyme or an
antisense RNA; viral genome fragments, such as viral DNA; plasmids and
cosmids; and a gene
or gene fragment.
The nucleic acid also can be RNA, for example, antisense RNA, catalytic RNA,
catalytic
RNA/protein complex (i.e., a "ribozyme"), and expression construct comprised
of RNA that can
be translated directly, generating a protein, or that can be reverse
transcribed and either
transcribed or transcribed and then translated, generating an RNA or protein
product,
respectively; transcribable constructs comprising RNA that embodies the
promoter/regulatory
sequence(s) necessary for the generation of DNA by reverse transcription;
viral RNA; and RNA
that codes for a therapeutic protein, inter alict. A suitable nucleic acid can
be selected on the
basis of a known, anticipated, or expected biological activity that the
nucleic acid will exhibit
upon delivery to the interior of a target cell or its nucleus.
The length of the nucleic acid is not critical to the invention. The nucleic
acid can be
linear or circular double-stranded DNA molecule having a length from about 100
to 10,000 base
pairs in length, although both longer and shorter nucleic acids can be used.
The nucleic acid can be a therapeutic agent, such as an antisense DNA molecule
that
inhibits rnRNA translation. Alternatively, the nucleic acid can encode a
therapeutic agent, such
as a transcription or translation product which, when expressed by a target
cell to which the
nucleic acid-containing composition is delivered; has a therapeutic effect on
the cell or on a host
organism that includes the cell. Examples of therapeutic transcription
products include proteins
(e.g., antibodies, enzymes, receptors-binding ligands, wound-healing proteins,
anti-restenotic
proteins, anti-oncogenic proteins, and transcriptional or translational
regulatory proteins),
antisense RNA molecules, ribozymes, viral genome fragments, and the like. The
nucleic acid
likewise can encode a product that functions as a marker for cells that have
been transformed,
using the composition. Illustrative markers include proteins that have
identifiable spectroscopic
properties, such as green fluorescent protein (GFP) and proteins that are
expressed on cell
surfaces (i.e., can be detected by contacting the target cell with an agent
which specifically binds
the protein): Also, the nucleic acid can be a prophylactic agent useful in the
prevention of
disease.
=
CA 02821214 2013-07-19
1
A nucleic-acid category useful in the present invention encompasses
polynucleotides that
encode proteins that affect wound-healing. For example, the genes egf; tgf; ke
hb-egf; pdgf, igf
fel , fgf-2, vegf; other growth factors and thelr receptors, play a
considerable role in wound
repair.
Another category of polynucleotides, coding for factors that modulate or
counteract
inflammatory processes, also is useful for the present invention. Also
relevant are genes that
encode an anti-inflammatory agent such as MSH, a cytokine such as 1L-10, or a
receptor
antagonist that diminishes the inflammatory response.
Suitable polynucleotides can code for an expression product that induces cell
death or,
alternatively, promotes cell survival, depending on the nucleic acid. These
polynucleotides are
= useful not only for treating tumorigenic and other abnormal cells but
also for inducing apoptosis
in normal cells. Accordingly, another notable nucleic-acid category for the
present invention
. relates to polynucleotides that, upon expression, encode an anti-
oncogenic protein or, upon
transcription, yield an anti-oncogenie antisense oligonucleotide. In this
context, the phrases
"anti-oncogenic protein" and "anti-oncogenic antisense oligonucleotide"
respectively denote a
protein or an antisense oligonucleotide that, when provided to any region
where cell death is
desired, or the site of a cancerous or precancerous lesion in a subject,
prevents, inhibits, reverses
abnormal and normal cellular growth at the site or induces apoptosis of cells.
Delivery of such a
polynucleotide to cells, pursuant to the present invention, can inhibit
cellular growth,
differentiation, or migration to prevent movement or unwanted expansion
oftissue at or near the
= site of transfer. Illustrative of this anti-oncogenic category are
polynucleotides that code for one
of the known anti-oncogenic proteins. Such a polynucleotide would include, for
example, a
nucleotide sequence taken or derived from one or more of the following genes:
obi, akt2, apc,
bc12-alpha, bc12-beta, bcI3, bc13, bc1-x, bad, bcr, brcal, brca2, cbl, ccndl,
cdk4, crk-II, csflr/frns,
dbl, dcc, dpc4/smad4, e-cad, e2flfrbap, egfr/erbb-1, elkl, elk3, eph, erg,
etsl, ets2,ferõfgr/src2,
=
fos, fps/fes, fral, fi-a2, fyn, hck hek, her2/erbb-2/neu, her3/erbb-3,
her4/erbb-4, hrasl , hst2,
hsYl, ink4a, ink4b, int2ffgf3, jZ17; junk fund, kip2, kit, kras2a, kras2b, ck,
lyn, rnas, max, mcc,
= met, nzlhl, mos, msla, msh3, msh6, myb, myba, mybb, myc, mycll , mycn,
nil, j2, nras, p53,
pdgjb, piml , pmsl , pms2, ptc, pten, raft, rb I , rel, ret, rosl , ski, srcl,
tall, tgfbr2, thral , thrb,
= 30 tiaml, trk vav, vhl, waft ,wnt 1 , wnt2,1411 and yes] .
By the same token, oligonucleotides that
inhibit expression of one of these genes can be used as anti-oncogenic
antisense
oligonucleotides.
=
41
=
CA 02821214 2013-07-19
Nucleic acids having modified inter-nucleoside linkages also can be used in
composition
according to the present invention. For example, nucleic acids can be employed
that contain
= modified internucleoside linkages, which exhibit increased nuclease
stability. Such
polynuclotides include, for example, those that contain one or more
phosphonate,
phosphorothioate, phosphorodithioate, phosph oramidate methoxyethyl
phosphoramidate,
formacetalithioformacetal, diisopropylsilyl, acetamidate, carbamate,
dimethylene-sulfide (-CH2-
S-CH2-), dimethylene-sulfoxide (-CH2-SO-CH2-), dimethylenesulfone (-Cf12-S02-
CH2), 2'.O-
alkyl, and 2'-deoxy-2'-fluoro-phosphorothioate intemucleoside linkages.
For present purposes, a nucleic acid can be prepared or isolated by any
conventional
1 10 means typically used to prepare or isolate nucleic acids. For
example, DNA and RNA can be
= chemically synthesized using commercially available reagents and
synthesizers by known
methods. RNA molecules also can be produced in high yield via in vitro
transcription
techniques, using plasmids such as SP65, available from Promega Corporation
(Madison, W1).
The nucleic acid can be purified by any suitable means. For exarnple, the
nucleic acid can be
purified by reverse-phase or ion exchange HPLC, size exclusion chromatography,
or gel
electrophoresis. Of course, the skilled artisan will recognize that the method
of purification will
depend in part on the se of the DNA to be purified. The nucleic acid also can
be prepared via
any of the innumerable recombinant techniques that are known or that are
developed hereafter.
A suitable nucleic acid can be engineered into a variety of known host vector
systems
that provide for replication of the nucleic acid on a scale suitable for the
preparation of an
inventive composition.
= Vector systems useful in the present invention can be viral or non-viral.
Particular
examples of viral vector systems include adenovirus, retrovirus, adeno-
associated virus and
=
herpes simplex virus. Preferably, an adenovirus vector is used. A non-viral
vector system
includes a plasmid, a circular, double-stranded DNA molecule. Viral and
nonviml vector
systems can be designed, using known methods, to contain the elements
necessary for directing
transcription, translation, or both, of the nucleic acid in a cell to which is
delivered. Methods
known to the skilled artisan can be used to construct expression constructs
having the protein
= coding sequence operably linked with appropriate
transcriptional/translational control signals.
These methods include in vitro recombinant DNA techniques and synthetic
techniques. For
instance, see Sambrook et al., 1989, MOLECULAR CLONING: A LABORATORY MANUAL
(Cold Spring Harbor Laboratory, New York).
=
!
; 42
=
CA 02821214 2013-07-19
,
=-
.
=
A nucleic acid encoding one or more proteins of interest can be operatively
associated
with a variety of different promoter/regulator sequences. The
promoter/regulator sequences can
include a constitutive or inducible promoter, and can be used under the
appropriate conditions to
direct high level or regulated expression of the gene of interest. Particular
examples of
promoter/regulatory regions that can be used include the cytomegalovirus (CMV)
promoter/regulatory region and the promoter/regulatory regions associated with
the SV40 early
genes or the SV40 late genes.
It also is within the scope of the present invention that the employed nucleic
acid
contains a plurality of protein-coding regions, combined on a single genetic
construct under
control of one or more promoters. The two or more protein-coding regions can
be under the
transcriptional control of a single promoter, and the transcript of the
nucleic acid can comprise
one or more internal ribosome entry sites interposed between the protein-
coding regions. Thus,
a myriad of different genes and genetic constructs can be utilized.
= METHODS OF MAKING PARTICLES
Further provided is a method of making the particle of the invention, the
method comprising:
(i) providing (a) the matrix-forming agent, (b) a polyelectrolyte, (c) a first
amphiphilic agent, (d) a
first medium, (e) a second medium, and optionally providing a stabilizer; (ii)
mixing at least the
matrix-forming agent, the first medium, and the second medium and optionally
the polyelectrolyte,
the first amphiphilic agent, and/or the stabilizer to give a first mixture;
(iii) emulsifying the first
mixture to give a first emulsion; and (iv) removing the first medium and
thereby forming the
particle, on a condition that the polyelectrolyte, the first amphiphilic
agent, and the stabilizer are
provided to at least one of the first medium, the second medium, the first
mixture, the first emulsion,
= or the particle such that the polyelectrolyte and the first amphiphilic
agent form the polyelectrolyte-
= amphiphilic agent adduct.
In certain embodiments of the method, the polyelectrolyte, the first
amphiphilic agent,
and optionally the stabilizer are combined with the matrix-forming agent prior
to mixing with
the first medium or the second medium.
The term "first medium" as used herein denotes a solution, a micellar
solution, an
emulsion, or a suspension. In certain embodiments of the method, the first
medium comprises
an organic solvent. In certain embodiments of the method, the first medium
comprises the
organic solvent selected from the group consisting of chloroform,
dichloromethane,
tetrahydrofiiran, acetone, ethanol, hexane, heptane, methylethylketone,
propylene carbonate,
ethyl acetate, acetylacetone, acetic anhydride, dimethylsulfoxide,
dimethylformamide,
43
CA 02821214 2013-07-19
-==-1-.7.ftl
_
acetonitrile and mixtures thereof. in certain embodiments, the organic solvent
is provided as a
ratio of at least two different organic solvents wherein the ratio influences
a size of the particle.
In one variant, the least two different organic solvents are tetrahydrofuran
and chloroform
provided at the ratio of about 0.1 to about 3 and the particle's diameter is
about 370 nm to about
100 nm.
Emulsification can be performed by methods known in the art such as
ultrasonication,
high pressure homogenization, and microfluidization as described by Quintanar-
Guerrero et al.,
supra.
Methods of preparing particles of the invention avoid using harsh conditions
(e.g.
I 0 extreme pH, elevated temperatures), which can cause degradation
of the matrix-forming
= polymer and/or some biomaterials. Room temperature is preferred for steps
involving addition
= of the biomaterial.
= = Preparations of coated magnetic field-responsive agent can be conducted
at elevated
temperatures in the absence of biomaterial.
In certain embodiments of the method, the second medium is a member selected
from
the group consisting of water, alcohols, and liquid hydrocarbons.
In certain embodiments, the method finther comprises providing a biomaterial.
In certain embodiments of the method, the biomaterial is provided to at least
one of the
first mixture, the first emulsion and/or the particle. It may be added as is
or in a co-solvent (for
=
example, methotrexate (the biomaterial) can be added in dimethylsulfoxide (the
co-solvent) to
the first medium. Further, it can be incorporated in either component of the
first medium, i.e. as
= a part of a solution, a micellar solution, an emulsion, or a suspension
depending on solubility of
the biomaterial in particular substances used.
= In another embodiment, the method further comprises providing a coated
magnetic field-
responsive agent to at least one of the first medium, the second medium,
and/or the first mixture.
In certain embodiments, the coated magnetic field-responsive agent is provided
by combining a
= magnetic field-responsive agent and a second amphiphilic agent in the
presence of the first
medium, the second medium, or the first mixture. Preferably, the coated
magnetic field-
.
= responsive agent is dispersed in the first medium and mixed with the
matrix-forming agent, the
= 30 polyelectrolyte, the first amphiphilic agent, and the
stabilizer prior to mixing with the second
medium as shown in Fig. 5. In one variant of this embodiment, the method
further comprises
=
providing a biomaterial, wherein the biomaterial is provided to at least one
of the first mixture,
the first emulsion and/or the particle. Preferably, the biomaterial is
provided to the particle, in
44
CA 02821214 2013-07-19
such a manner that the biomaterial is in communication with the
polyelectrolyte-first
amphiphilic agent adduct, provided that the polyelectrolyte, the first
amphiphilie agent, and the
stabilizer are combined with the matrix-forming agent before mixing with the
first medium.
Further provided is a method of making the magnetic particle of the invention,
the method
comprising: (i) providing the matrix-forming polymer, the coated magnetic
field-responsive agent, a
first medium and a second medium, and optionally providing a stabilizer; (ii)
mixing at least the
matrix-forming polymer, the first medium, and the second medium to give a
second mixture; (iii)
emulsifying the second mixture to give a second emulsion; and (iv) removing
the first medium and
thereby forming the particle, on a condition that the coated magnetic field-
responsive agent and
optionally the stabilizer are provided to at least one ofthe first medium, the
second medium, or the
second mixture. Preferably, the coated magnetic field-responsive agent is
provided by combining
the magnetic field-responsive agent and the second amphiphilic agent in a
presence of the first
medium, the second medium, or the second mixture. One variant of this
embodiment includes
further providing a biomaterial. The bioinaterial can be provided to at least
one .of the second
mixture, the second emulsion or the magnetic particle.
METHODS OF DELIVERY OF BIOMATERIAL TO TARGET CELT OR TARGET SSUE
Also provided is a method of delivery of a biomaterial to a target cell or a
target tissue, the
method comprising: administering the particle of the invention comprising the
matrix-forming
agent, polyeleetrolyte-amphiphilic agent adduct, the coated magnetic field-
responsive agent and the
biomaterial; optionally providing a magnetic device associated with the target
cell or the target
tissue; applying a magnetic force to the particle; and guiding the particle by
the magnetic force and
thereby delivering the biomaterial to the target e,ell or the target tissue.
The source of magnetic field can be any source known in the art, e.g., an
electromagnet.
Magnetic field can be applied internally by placing a magnetic device inside a
body iti need of
delivery of biomaterial; the magnetic field can be applied externally or as a
combination of both.
Further provided is a method of delivery of a biomaterial to a target cell or
a target
tissue, the method comprising: administering the particle of the invention
comprising the
matrix-forming agent, the coated magnetic field-responsive agent, and the
biomaterial, wherein
the particle is free of the polyelectrolyte; providing a magnetic device
associated with the target
cell or the target tissue; applying a magnetic force to the particle; and
guiding the particle by the
magnetic force and thereby delivering the biomaterial to the target cell or
the target tissue.
Also provided is a method of delivery ofr biomaterial to a cell or a tissue,
the method
comprising administering the particle ofthe invention comprising the matrix-
forming agent, the
CA 02821214 2013-07-19
-
polyelectrolyte-amphiphilic agent adduct, and the biomaterial, wherein the
particle is free ofthe =
magnetic field-responsive agent; and delivering the biomaterial to the cell or
tissue using the
particle as a carrier, wherein the cell is optionally contacted with a
transfection agent prior to
said delivering.
=
Administering of the particle can be done, for example, by layering, spraying,
pouring,
injection, inhalation, or ingestion or a combination of any of the abovel
1 The
invention was also driven by the desire to develop a composition and a method
for
surface modification of inert surfaces useful for implantation, which would
permit attachment of
molecular therapeutics such as proteins, genes, vectors, cr cells and avoid
using organic solvents
that can potentially damage both the surface and molecular therapeutics.
Moreover, utilizing
therapeutic potential of site-specific therapy (ssr) with custom synthesized
stent surfaces and
heart valve leaflets, the present photochemical approach will permit surface
activation of a broad
range of existing medical device configurations.
The inventors have discovered that biomaterial can be covalently attached to
surfaces
having at least one carbon by utilizing a water-soluble photo-activatable
polymer of the
invention which functions as a multipoint polymeric cross-linker, wherein one
function of the
cross-linker is to photoimmobilize the water-soluble polymer to a desired
surface and another
function is to attach the desired biomaterial.
The water-soluble photo-activatable polymer of the invention comprises:
(a) a photo-activatable group, wherein the photo-activatable group is adapted
to be
activated by an irradiation source and to form a covalent bond between the
water-soluble photo-
activatable polymer and a matrix having at least one carbon;
(b) a reactive group, wherein the reaetNe group is adapted to covalently react
with a
biomaterial;
(c) a hydrophilic group, wherein the hydrophilic group is present in an amount
sufficient
to make the water-soluble photo-activatable polymer soluble in water; and
(d) a polymer precursor.
The specific chemistry used herein has distinct advantages since it involves:
1) aqueous
based exposures, thus removing any risk of damaging surfaces that could be
susceptible to
organic solvent damage, and 2) addition of reactive groups, e.g., PDT groups,
thus enabling
sulfhydryl chemistry approaches for attaching linking proteins and peptides,
such as antibodies
or receptor fragments.
=
46
CA 02821214 2013-07-19
=:e = 1
-
A reaction between a thiol-reactive group (2-pyridyklithio, maleimide, etc.)
attached to
one protein molecule with a thiol group of another protein molecule (or other
biomolecule) is a
widely used for preparation of protein conjugates (See Greg T. Hermanson,
Bioconjugate
Techniques, Academic Press, San Diego 1996). Reaction of a thiol group with
most of thiol-
reactive groups (particularly 2-pyridyldithio group) is very selective and
fast in aqueous media at
mild conditions. Proteins can be thiolated using a partial reduction of
disulfide bridges or via
thiolation of lysine residues with a variety of reagents (see Hermanson, pp.
57-70). The reaction
of thiolated proteins with polymeric surfaces containing thiol-reactive groups
would be ideal for
the immobilization of proteins on polymeric supports. At the same time, there
is no method for
providing polymeric surfaces with such thiol-reactive groups. The present
invention can
efficiently solve this problem.
One of the reasons the surface-attachment of 2-pyridyldithio-groups (PDT-
groups) to
polymers via benzophenone photo-cross-linkers is not obvious is that PDT-
groups are rather
unstable and may not survive conditions of photo-immobilization wherein
active, energy-rich
species appear.
The term "photo-activatable group" used herein denotes chemical groups capable
of
generating active species such as free radicals, nitrenes, carbenes and
excited states of ketons
upon absorption of external electromagnetic or kinetic (thermal) energy. These
groups may be
chosen to be responsive to various portions of the electromagnetic spectrum,
i.e., the groups
responsive to ultraviolet, visible and infrared portions of the spectrum. The
preferred photo-
activatable groups of the invention are benzophenones, acetophenones and aryl
azides. Upon
excitation, photo-activatable groups are capable of covalent attachment to
surfaces comprising at
least one carbon such as polymers.
The water-soluble photo-activatable polymer of the invention may have one or
more
photo-activatable groups. In certain embodiments, the water-soluble photo-
activatable polymers
have at least one photo-activatable group per molecule. Preferably, the water-
soluble photo-
activatable polymers have a plurality of photo-activatable groups per
molecule. More
preferably, photo-activatable groups modify at least 0.1% of monomeric units
of a polymer
precursor, even more preferably at least 1%, and most preferably from about 20
to about 50 %.
, 30 The irradiation source can be any source known in the art
capable of emitting the light
having a wavelength absorbable by the photo-activatable group of the
invention. A-UV-lamp is
preferred when the benzophenone is used as the photo-activatable group.
47
CA 02821214 2013-07-19
The term "water-soluble polymer" as used in this disclosure means that the
water-soluble
photo-activatable polymer of the invention can be diluted 'with water to at
least 1 wt% and
preferably to at least 0.1 wt% to form a single phase at a temperature of 20
C, provided that
water is essentially free of an organic co-solvent.
The terms "surface" or "matrix" as used interchangeably herein mean a surface
having at
least one carbon. In a preferred embodiment of the invention, the surface is a
polymeric matrix. =
Other surfaces such as organosylated materials (i.e., organosylated metals)
can also be used in
= the present invention.
= The surface contemplated by the present invention can have any shape or
form suitable
for variety of purposes such as for example a delivery of a biomaterial to an
organism. In that,
the surface can be an existing medical implant such as a stent or a
cardiovascular valve, which
can be covered with the composition of the invention. Also, the surface can be
first modified
with either the water-soluble polymer of the invention or the composition of
the invention and
then molded into the desired shape. Moreover, the surface can be in a form of
polymeric
particles.
Medical devices appropriate for the gene delivery system in the present
invention
include, but are not limited to, heart valves, wire sutures, temporary joint
repracements and
urinary dilators. Other suitable medical devices for this invention include
orthopedic implants
such as joint prostheses, screws, nails, nuts, boltsõ plates, rods, pins,
wires, inserters, osteoports,
halo systems and other orthopedic devices used far stabilization or fixation
of spinal and long
bone fractures or disarticulations. Other devices may include non-orthopedic
devices, temporary
placements and permanent implants, such as tracheostomy devices, jejunostomy
and
gastrostomy tubes, intraurethral and other genitourinary implants, stylets,
dilators, stents,
vascular clips and filters, pacemakers, wire guides and access ports of
subcutaneously implanted
vascular catheters.
The polymeric matrix of the invention can be biodegradable and non-
biodegradable.
Non-limiting examples of the polymeric matrix used in the invention are
poly(urethane),
poly(ester), poly(lactic acid), poly(glycolic acid), poly(lactide-co-
glycolide), poly(s-
caprolactone), poly(ethyleneimine), poly(styrene), poly(amide), rubber,
silicone rubber,
poly(acrylonitrile), poly(acrylate), poly(metacrylate), poly(alpha-hydroxy
acid),
po1y(dioxartone), poly(orthoester), poly(ether-ester), poly(lactone), mixtures
thereof and
copolymers thereof.
In certain embodiments of the compos'tion, the matrix further comprises a
48
CA 02821214 2013-07-19
-1
A....,
. = .
=
=
superparamagnetic agent. Preferably, the superparaimagnetic agent is a member
selected from
the group consisting of magnetite and maghernite nanocrystals.
The water-soluble photo-activatable polymer of the invention comprises a
polymer
= precursor and the following groups covalently attached to the polymeric
precursor: the photo-
.
activatable group as described above, a reactive group, and a hydrophilic
group.
The polymeric precursor of the water-soluble photo-activatable polymer of the
invention
can be prepared by using methods known in the art from a polymer
(biodegradable or a non-
biodegradable) comprising reactive groups, and hydrophilic groups, which is
then modified to
contain photo-activatable groups. Also, it can be prepared by polymerization
of monomeric
blocks containing the above groups. In certain embodiments of the invention,
the polymeric
precursor comprises at least one monomer selected from the group consisting of
allylamine,
=
vinylamine, acrylic acid, carboxylic acid, alcohol, ethylene oxide, and acyl
hydrazine.
Preferably, the polymer precursor is polyallylamine. In certain embodiments of
the invention,
the polyanylarnine has a molecular weight of about 200 KDa to about 5 KDa. In
the preferred
embodiment, the molecular weight is from '70 KDa to 15 KDa.
The reactive group of the water-soluble photo-activatable polymer of the
invention is a
=
chemical group adapted to covalently react with a biomaterial. Non-limiting
examples of the
reactive group are an amino group (primary or secondary), a thiol-reactive
group, a carboxy
group, a thiol group, a protected thiol group, an acyl hydrazine group, an
epoxy group, an
aldehyde group, and a hydroxy group. Preferably, the thiol-reactive group is
selected from the
group consisting of a 2-pyridyldithio group, a 3-carboxy-4-nitrophenyldithio
group, a maleimide
group, an iodoacetamide group, and a vinyisulfonyl group.
The hydrophilic group ofthe water-soluble photo-activatable polymer of the
invention is
present in an amount sufficient to make the water-soluble photo-activatable
polymer soluble in
water. In certain embodiments of the invention, the hydrophilic group is a
member selected
from the group consisting of an amino group and a carboxy group.
The reactive group and the hydrophilic group of the water-soluble photo-
activatable
polymer of the invention can be identical or different. In one embodiment
ofthe invention, both
the reactive group and the hydrophilic group are arnino groups. In another
embodiment of the
invention, the reactive group is the 2-pyridyldithio group, and the
hydrophilic group is the
carboxy group.
In certain embodiments of the inventien, the photo-activatable group is a
member
selected from the group consisting of an aryl ketone and an aryl azide.
Preferably, the aryl
=
49
=
CA 02821214 2013-07-19
I ..i.; , = -
. . . _ .
' .
'
,
ketone is a member selected from the group consisting of benzophenone and
acetophenone.
,
In one embodhnent of the invention, the water-soluble polymer is
polyallylamine based
. benzophenone (PAA-BzPh) and is represented by a formula:
I(¨Y-1--c1-12-.)a-k¨(--c4--CH2-)k
CH2 CH2
I 1
NH2 N H-0C-1j¨COPh
,
. .
. 5
,
. wherein n is 50 to 2000 and k is1. 0 to 1000.
,
. In another embodiment of the inveriticn, the water-soluble polymer
is=polyallylarnine
based benzophenone further modified to DC)11131:. 2- pyridyldithio groups (PDT-
BzPh) and is
represented by a formula:
(¨CH¨CH2-)roc-nil¨CH¨CF12-)m--(¨CH¨C1-12-)k
I I I
CH2 CH2 CH2
I
=
. NH NIH 1I\11-1-0C-0¨COPh
0=C 0=C
. I I N \
,
,
CH2CH2COOH CH2CH2SS--c_) ,
=
wherein n is 50 to 2000, k is 10 to 1000, and m is 10 to 1000.
. The water-soluble polymer the invention can be prepared by using
radical polymerization '
.
. . of a mixture of three types of monomers (e.g., acryIamide-
based), each containing only one of
the groups, as shown below:
. Radical
initiator
; CH:CH2 + CH:CH2 + CH:CH2 __ -
, X
,
I I I I t t
. Y Z X Y Z
1 i i i I I
A B C A B C
= X,Y and Z are suitable spacers
A = photo-actiyatable group, 13 .1., hydrophilic group, C = reactive group
The ratio between these groups in the -line product can be controlled by
changing the
ratio of monomers. Conditions for polymerization are known to persons skilled
in the art.
Additionally, each monomer can bear two, or all the three types of the groups.
Other types of
i . polymerization or polycondensation known in the art can also be
used. '
,
Upon excitation of photo-activatable groups, the water-soluble polymer ofthe
invention
=
. is covalently bonded to the surface by forming a.
menomelecular layer on the surface. .
. .
i The term "layer" used herein means a contiguous and non-
contiguous deposit formed
=
. .
.
.
f Ci
CA 02821214 2013-07-19
= =
by a covalent bonding ofpolymers of the invention to the surface. Preferably,
the layer is highly
homogeneous and high purity in that it consists essentially of the water-
soluble polymer of the
invention.
Further provided is a composition of matter comprising a monomolecular layer
of the
water-soluble photo-activalable polymer of the invention and a matrix having
at least one
carbon, wherein the monomolecular layer is covalently attached to the matrix
by a covalent bond
between the photo-activatable group and the at least one carbon.
In certain embodiments, the composition further comprises a biomaterial having
a
plurality of active groups, wherein the biomaterial is covalently attached to
the monomolecular
layer by covalent bonding between the active groups and reactive groups.
In certain embodiments of the composition, at least one of the active groups
is a member
selected from the group consisting of amine, carboxyl, hydroxyl, thiol,
phenol, imidazole, and
indole. Preferably, the at least one of the active groups comprise thiol.
Another embodiment of the invention is a method for delivery of a biomaterial
to a cell,
the process comprising: providing the composition of the invention comprising
the
monomolecular layer ofthe water-soluble photo-activatable polymer covalently
attached to the
matrix; providing a biomaterial having a plurality of active groups, wherein
the biomaterial is
covalently attached to the monornolecular layer by covalent bonding between
the active groups
and the reactive groups; and administering the matrix to the cell and thereby
delivering the
biomaterial. Preferably, the biomaterial is a protein, a D1 domain of the
Coxsackie-adenovirus
receptor or an antibody specifically bound to a nucleic acid, wherein said
antibody is bound to
the matrix. Still more preferred, the protein, the DI domain of the Coxsackie-
adenovirus
receptor, and the antibody are a thiol modified. The composition can then be
used to deliver the
biomaterial to the interior of a cell in need of, îr example, gene therapy.
Antibodies specific for non-viral vectors or nucleic acid may require use of a
transfection
agent to enhance administration of nucleic acid. The transfection agent is a
cationic
macromolecule that is positively charged, comprises two or more art-recognized
modular units
(e.g., amino acid residues, fatty acid moieties, or polymer repeating units),
and is preferably
capable of forming supermolecular structures (e.g, aggregates, liposomes or
micelles) at high
concentration in aqueous solution or suspens,on. Among the types of cationic
macromolecules
that can be used are cationic lipid and polycationic polypeptides.
The amount of the transfection agent to iv:- used when transfecting cells can
be calculated
based on nucleic acid content of the composition. The capacity cf the medium
comprising or
51
CA 02821214 2013-07-19
containing the transfection agent can also affect the amount of transfection
agent to be used.
When the antibody of the transfection agent is immobilized on a matrix, the
amount of cationic
macromolecule and DNA that can be complexed with the antibody can be limited
by the
physical requirements of the metal support. For example, rigidity, flexibility
and chemical
5. reactivity may influence the amount of transfection agent used.
Such vectors have included
retroviral, adenovirus, adeno-associated viral vectors and herpes viral
vectors. Cells can be
infected with viral vectors by known methods.
Further provided is a method of making the composition of the invention, the
method
comprising: providing the matrix having at least one carbon; providing an
aqueous solution of
.==
=
the water-soluble photo-activatable polymer having the photo-activatable group
and the reactive
group; and photo-activating the photo-activatable group by irradiation to
covalently attach the
= water-soluble polymer via the photo-activatable group to the matrix and
thereby forming the
monomolecular layer of the composition.
In certain embodiments of the method of making the composition of the
invention, the
. - 15 irradiation is performed at a wavelength from about 190 to
about 900 nm. Preferably, the
irradiation is performed at a wavelength of MO to 360 nm.
= Additionally, certain embodiments of the method further comprise
providing a
biomaterial having a plurality of active groups and reacting the plurality of
active groups with
the water-soluble photo-activatable polymer to zovalently attach the
biomaterial to the matrix.
PHOTOCHEMICAL MODIFICATION OF MICRO-AND NANOPARTICLES
The water-soluble photo-activatable polymers of the invention obtained as
described
above can be bound to the surface of pre-formed micro- or nanoparticles (MP
and NP,
= respectively) or during the particle formation process to form modified
particles capable of
reacting with the biomaterial.
Advantageously, in the preparation of biodegradable and non-biodegradable
polymeric
micro- and nanoparticles, the water-soluble pboto-activatable polymer of the
invention (e.g.,
BzPh-PDT) not onlymakes the surface reactive towards the thiol-containing
biomolecules, but
also prevents the aggregation of the particles and thus stabilizes the
particle suspension in the
suspending medium.
Non-limiting examples of uses of particles modified with PDT-BzPh are various
=
applications for delivery oftherapeutic.als to tile body. For example,
injectable nanoparticles can
be used to dither provide an intravenously applied sustained delivery system
for proteins and
peptides potentially targeted to a site of interest, or if injected into a
spexific locatition such as a
52
CA 02821214 2013-07-19
4 -
tumor, or the myocardium to provide, for example, sustained local presence of
therapeutic
peptides and proteins. This approach would be desirable in a broad range of
applications
covering virtually every conceivable disease process.
Depending on the specific application of the particulate formulation stating
demands on
its properties such as particle size and composition, the particles can be
prepared by one of the
existing methods (See Couvreur P et al., Nanoparticles: preparation and
characterization. In:
Benita S, Editor, Microencapsulation. Methods and industrial applications.
vol. 73. New York:
Marcel Dekker, 1996. pp. 183-211; Kumar MNR. Nano- and microparticles as
controlled drug
delivery devices. J Pharm Pharmaceut Sci 2000; 3:234-53).
Generally, particle formation may be accomplished by a number of methods of
which the
most widely used are emulsification-polymerization and polymer precipitation
techniques. The
former may be accomplished by in situ polymerization of monomers either in
aqueous solution
or emulsified in aqueous phase (namely, emulsification-polymerization).
Alternatively, methods
exploiting pre-formed biocompatible polymers, usually of poly(ester) and
poly(anhydrid)
families, can be used to form particles by polymer precipitation methods. The
most popular
methods are emulsification-solvent evaporation, emulsification-diffusion and
nanoprecipitation
methods (See Quintanar-Guerrero et al., Preparation techniques and mechanisms
of formation of
biodegradable nanopartioles from preformed polymers. Drug Dev Ind Pharm 1998;
24:1113-
28). The latter methods are based on emulsifying an organic solution of a
polymer with or
without drug in an aqueous phase in presence of a stabilizer substance (e.g.
Poloxamer 188,
polyvinyl alcohol, etc.) achieved either by external energy input or through
spontaneous
diffusion of water-miscible solvents with subseqvent solvent elimination to
forrn solid particle
dispersion.
= While both emuIsification-polymerizaf on and polymer precipitation are
applicable" for
preparing matrix-type particles (spheres), some of these methods with
appropriate modifications
can be used for producing core-shell type vesicles (capsules). The drug
substance can either be
encapsulated (dissolved or dispersed in the polymeric matrix of a sphere or
dissolved in the
= liquid core of a capsule) or adsorbed/chemically bound to the particle
surface. The latter
approach where a substance is attached to the surface of a preformed particle
has the advantage
s 30 of avoiding harsh conditions (extreme pH, exposure to organic
solvents or elevated
temperatures) employed for the particle formulation. Covalent association
ofthe drug with the
particle surface employing biodegradable chemical bonds (e.g. by disulfide
linking) provides an
alternative that achieves both controlled and site Fine Mc release of the
drug.
= 5`3
=
CA 02821214 2013-07-19
I =:.=
An additional degree of site-specificity may be achieved by rendering the
particles
magnetic by inclusion of magnetite/maghemite nanocrystals in the polymeric
matrix (e.g. Ito R,
Machida Y, Sannan T, Nagai T1.I-hwscsaBTW-13-Gddaaca. Magnetic granules: a
novel system
for specific drug delivery to esophageal mucosa in oral administration. Int J
Pharm 1990; =
61:109-117). This modification allows for concentrating the particles at their
target tissue using
magnetic field, thereby increasing their therapeutic efficacy and minimizing
the potential= =
formulation toxicity.
Also provided is a process for delivery of a biomaterial to a cell or an
organism, the
process comprising (1) providing the composition of the invention as a
monomolecular layer
and a matrix having at least one carbon, wherein the rnoriornolecular layer is
covalently attached
to the matrix by a covalent bond between the photo-activatable group and the
at least one
carbon, (2) providing a biomaterial having a plurality of active groups,
wherein the biomaterial
is covalently attached to the monomolecular layer by covalent bonding between
the active
=
groups and the reactive groups; and (3) administering the matrix to the cell
or an organism. =
Amounts of the biomaterial may vary depending on the purpose of delivery,
e.g.,
= prophylactic, diagnostic, therapeutic, etc. and on the nature of the
biomaterial involved.
In certain embodiments, the biomaterial delivered by this method is at least
one of,a low
. molecular weight therapeutical, a protein, a nucleic acid or a
therapeutic virus, of which the
latter three can be bound either directly, or through an affinity ligand, such
as specific antibody
or D1 domain of the Coxsackie-adenovirus receptor in the case of the
adenovirus.
The invention will be illustrated in more detail with reference to the
following Examples,
= but it should be understood that the present invention is not deemed to
be limited thereto.
EXAMPLES
EXAMPLE 1
PREPARATION OF A COATED MA.G'NETIC-FTEI .n RESPONSIVE AGENT'
The formulation of superparamagnetic biodegradable nanoparticles involves two
main
= steps: forming a long-chain carboxylic acid stabilized iron oxide
nanocrystals and incorporation
of the iron oxide nanocrystals in the biodegradable polymeric matrix of PLA-
based
nanoparticles formulated with PEI. The first step involves co-precipitation of
ferric and ferrous
=
= 30
chlorides in the presence of aqueous base solution (NaOH, 0.1N)
with subsequent coating with =
oleic acid, thus rendering the nanocrystal surface lipophilic (see De Cuyper,
supra; and
Khalafalla supra). The obtained stabilized iron oxide naeocrystals were
separated by extraction
= or centrifugation/magnetic sedimentation with subsequent resuspension in
chloroform or a
54
CA 02821214 2013-07-19
tt4 : z A1.0
=
mixture of chlorofom and tetrahydrofuran.
1 ml of an aqueous solution containing 65 mg of FeCl3 hexahydrate, 32 mg of
FeCl2
tetrahydrate and 50 mg of Pluronic F-68 (as an optional stabilizer) (BASF
Corp.) were rapidly
mixed with 10 ml aqueous solution of NaOH (0.i M. Next, 200 mg of oleic acid
were added
dropwise, and the mixture was degassed in argon. The mixture was heated to 90
C in a water
bath for 5 min and cooled to room temperature. The mixture in a form of a
suspension was
vortexed with 5 ml of CHC13 to extract thus formed iron oxide nanocrystals,
and the bottom
layer was further used to prepare nanoparticles.
PREPARATION OF NANOPARTICEES
Biodegradable matrix-fonning polymer (poly cticle.) and PEI were incorporated
into the iron
oxide dispersion in the organic medium, and the orga.-lic phase thus obtained
is emulsified in water
followed by the organic solvent's evaporation (emuisification-solvent
evaporation or the
emulsification solvent-diffusion method) and nanopaiticles filtration as in
the example below.
200 mg of PLA (DX-PLA (70-120 K.Da) Sigzia), 50 mg of Pluronic F-68 (BASF
Corp.),
100 mg of PEI (25 KDa; Aldridge) were dissolved in 5 mi of iron oxide
suspension in CHC13 as
described above. The organic solution was added to 15 ml of distilled water
pre-cooled to 0 C, and
the mixture was emulsified by Sonication on the ict, bath. CliC13 was removed
by rotavaporation at
= 32 C. The particles were filtered through 6uni WhatmanTm paper filter.
The size of thenanoparticles
was found to be 302 nm and remained stable for at least one week. The iron
content in the
nanoparticle suspension was found to be 744 with only 2.4% localized outside
the nanoparticles
indicating a high yield of the applied magnetite entrapment procedure. The
total arnotmt of PEI =
initially taken was determined in the formu1ati6n with only 13.5% localized
outside the
nanoparticles; the escape of PEI from the nanopanicles was slow resulting in
about ao% localized
outside the nanoparticles after 7 days as opposed to sirnilarly prepared non-
magnetic nanoparticles
which did not include oleic acid and iron oxide in their composition.
EXAMPLE 2
PREPARATION OF NANOPARTICLES COMPRISMOBIOMATERIAL
Nanoparticles were prepared as described in Example I. DNA encoding for green
fluorescent protein was used as a biomaterial. DNA in 5% glucose aqueous
solution was added to
nanoparticles suspended in 5% aqUeous glucose scAlon, incubated at RT for 30
min; and gently
mixed by up and down pipetting.
The nanoparticles were shown to completely bind DNA at theoretical
nitrogen:phosphorus
=
CA 02821214 2013-07-19
I
ratios above 10 and 5 for glucose 5% solution and MES 0.1 M (p1-1.5) buffer,
respectively, used as
complexation media.
Next particles were studied for transfection of cells in culture with and
without use of
external magnetic field.
Three kinds of particles were prepared at three different PEEDNA theoretic
ratios (5:1; 10:1;
and 151).
One kind of particles denoted in Figs 5 A and 5B as "magnetic NP" included
magnetic
nanoparticles comprising PLA, PEI, oleic acid, iron oxide coated with oleic
acid and DNA (shown
in Fig. 1 as magnetic NP). Serving as controls, "PEI" included particles
comprising PEI and DNA
and "PEl+blankNP" were particles consisting of free PEI mixed with DNA in the
presence of blank
PLA particles.
Next, particles were added to BAEC cells and transfection was measured in the
absence and
presence of external magnetic field as shown in Figs 5A and 58 respectively.
EXANIPLE 3
TRANSFECTION EVHCACY AND TOXICITY
Bovine aortic endothelial cells (BAEC) were seeded on day-1 (2 x 104/well, on
four 24-
=
well plate). The cells were washed 2 times (2 x 1 hr) with the unsupplemented
medium
(DMEM) on day prior to transfection. DNA stock solution (0.55 ml) was slowly
added to 0.55
ml of complexants diluted to provide 0.25 g DNA per well complexed at
predetermined
theoretical charge ratios and left for 30 min. 0.125 ml of the unsupplemented
medium was
added to each well followed by 0.125 ml preparation; the cells were incubated
at 37 C, while
one plate was placed at a time on the magnet (15 min) (the other kept at a
distance from it). The
medium was then replaced with fresh pre-warrned medium supplemented with 10%
FCS. The
= cells were observed for transfection after 24 hr.
The nanoparticles and PEI showed comparable transfection of BAEC cells in
culture with
DNA encoding for green fluorescent protein when applied without use of
external magnetic field
(Fig 5 A), whereas the transfection efficacy of the magnetic nanoparticles
applied in the presence of
a permanent magnet (Fig. 5B) was substantially inermsed as compared to both
control formulations
and the magnetic nanoparticles applied in the absence of the magnet.
A similar effect of the magnetic field on the transfection efficacy was also
observed in A10 =
cells in culture.
== Notably, the magnetic NP were able to effectively transfe,ct cells
in presence of 109'o and
80% serum apparently due to their protective effect against DNA enzymatic
degradation, whereas
.=
=
56
CA 02821214 2013-07-19
practically no transfection was found when DNA:PEI complex was added to the
cells in the
presence of serum for the same time period.
Magnetic nanoparticles were found superior to PEI with regards to cell
toxicity when
applied to BAEC cells in equivalent amounts as shown in Fig_ 6. Cell toxicity
was measured by
WST-1 assay following 4-hour incubation at 37 C.
EXAMPLE 4
USE OF SOLVENT TO CONTROL SIZE OF PARTICLE
Use of tetrahydrofuran (TILF), a water-miscible solvent, in a mixture with
chloforrn to form
the organic phase allows to considerably decrease the size of the resultant
nanoparticles, thus
= making possible to optimize the nanoparticle uptake by a given cell type
without affecting the
= amounts of the structural components in the formulation (i.e. FLA, PEI,
Fe304, oleic acid) and the
colloidal stability of the dispersion. Therefore, one ciao independently
control size and surface
charge, which is unachievable in complexes prepared by simply combining DNA
and PEI. The
= effect of varying the volume of THF and chlorofean (the organic phase
composition) on. particle
size and size stability (67 mg ferric chloride: 33 mg ferrous chloride, 45 mg
oleic acid) was
observed Within 24 hours of preparation (day 0) and on the seventh day as
shown is shownin Fig. 7.
The total volume of the tetrahydrofuranichlorofcrrn mixture was kept constant
and equal to 6.0 ml.
The extent and stability of PEI association with magnetic NP as a function of
the organic
phase composition is shown in Fig. 8.1
EXAKPLE 5
Preparation of water-soluble photo-activatable polymers based on
poly(allylamine)
Synthesis of PAA-13zPh
Synthesis of PAA-BzPh is demonstrated in Seleine 1. Poly(ally-lamine)(PAA)
base was
prepared from PAA hydrochloride (Sigma-Aldrich St. Louis, MO, MW 70 KDa) by
treatment
in aqueous medium with a strong anionite DO \ ex116 0-55 followed by
replacement of water by 2-
propanol. A 5.1% solution of PAA base in 2-propanol (4,06 g, containing 3.65
rnmol of amino
goups) was diluted with CH2Cl2 (7 ml) and cooled on an ice bath. Succinoyl 4-
benzoylbenzoate (Sigma-Aldrich, 236 mg, 0.7:i nimol) in CH2C12 (12 ml) was
added over a 10
-
min period. The mixture was stirred near 0 C for 10 min, then warmed to room
temperature and
acidified with concentrated HCI (024 ml, 2.9 mrnol). The resulting suspension
was dried in
vacuo, resuspended in CH2C12, and the precipitate was filtered off. After
washing with CH2C12
and pentane, 0.544 g of PAA-BzPh hydrochloride were obtained. A 1H NMR study
of this
polymer (utilizing D70) indicated that 20% of polymer's amino groups were
modified with 4 =
-
57
=
CA 02821214 2013-07-19
benzoylbenzoic residues (broad signal at 6.9-8.0 ppm). Analogously, using the
calculated
amount of fluorescein isothiocyanate (fen C) (Sigma-Aldrich, St. Louis, MO,)
simultaneously
with succinoyl 4-benzoylbenzoate in the reaction with :PAA base, FITC-labeled
PAA-BzPh
having about 20% of 4-benzoylbenzoic residues and about 2% of the P1TC label
was prepared.
Synthesis of PDT-BzPh
Synthesis of PDT-BzPh is shown in Scheme 1. A 5.1% solution of PAA base in 2-
propanol
(2.671 g, containing 2.40 mmoI of amino groups) was diluted with CH2C12 (5 ml)
and cooled on
ice. Succinoyl 4-benzoylbenzoate (145 mg, 0.45 mmol) and SPOT> (Pierce
Biotechnology hie,
Rockford, IL, 281 mg, 0.90 mmol) were simult., neously dissolved in CH2C12 (8
ml) and
introduced over a 5-min period. The mixture was stirred near 0 C for 15 min,
and succinie
anhydride (130 mg, 1.30 mrnol) was added at once. The stirring at 0 C was
continued for 0.5 h,
the mixture was dried in vacuo and extracted first with ethyl acetate and then
with water. The
polymeric residue was dissolved in water (15 ml) with addition of IGIC03
(0.3g, 3.0nunol):
The solution was filtered and acidified with H3PO4 to pH of 3.5. The
precipitate was filtered
of washed with water, and air-dried PDT-BzPh (488 mg) was obtained. A1H NKR.
study of
this polymer (utilizing D20 and K2CO3 at pH 9) indicated that about 40% of 2-
pyridyldithio
groups and about 20% of 4-benzoylbenzoic residues were attached to the PAA
backbone. The
rest of amino groups was modified with 3-carboxyNopionyl residues resulting
from succinic
anhydride.
EXAMPLE 6
Preparation of water-soluble photo-activatable polymers based on poly(acrylic
acid)
Poly(actylic acid) can be coupled in aqueous solutions simultaneously with
amino-
derivatized benzophenone compound and a compound aIso amino-derivatized)
containing one
of the reactive groups described above, particularly 2-(2-
pyridyldithio)ethylamine. Water-
soluble carbodinnide (EDC) can be used for such coupling (see Scheme below).
=
58
CA 02821214 2013-07-19
H2N-Y COPh
H2N-CH2CH2SS-{_4)
(¨CH¨ CH2k_m-(¨CH¨CH2--)m¨(¨CH¨C1-12-)k
EDC
COOH COOH CO CO
1
Poiyacrylic NH NH¨Y * COPh
acid
C 2H
H 2
= -
Y = any suitable spacer
EDC==-= 1-ethy1-3-dimethylaminopropylcarbodlimide
EXAMPLE 7
= Photo-Immobilization of Polymeric Modifiers onto Matrix:
Surface amination of polymeric matrix with PAA-BzPh.
An aqueous solution (2 mg/m1) of PAA-BzPh or its FITC-labeled variant was
mixed
with an equal volume fa buffer containing 0.1M NH40Ac and 0115M NH3. PU
Tecothane Tr-
= 1074A films or polyester (PE) fibers were immersed into the mixture for 5-
60 min, rinsed with
a I% solution of NH3 and dried on a filter paper. The polymers were irradiated
under an UV-
.
I 0 lamp (UVGL-25, long wave) for 15¨ 30 min to achieve the covalent
binding ofmodifiers to the
polymer surface. Finally, the surface modified polymers were thoroughly washed
with diluted
(2%) HCI and water.
Modification of polymeric matrix with PDT-BzPh.
PDT-BzPh (30 mg) was dissolved in water (30 ml) by addition of KHCO3 (20 mg)
and
acidified with a 20% solution of KH2PO4 (1 rn1). PU films and PE fibers were
soaked in the
resulting mixture for 5 ¨ 40 min., rinsed with 0.1% acetic acid, dried and
irradiated as above.
Finally, the polymers were exhaustively washed with 0.1M KHCO3 arid water.
EXAMPLE 8
Fluorescent Labeled PAA-BzPh Studies Demonstrating Attachment of Biomolecules
Fluorescence microscopy ofPU films and PE fibers surface modified with FITC-
labeled
PAA-BzPh was conducted and confirmed the presence of the modifier bound to the
polymer
surfaces.
=
EXAMPLE 9
Cell Culture Data Demonstrating Antibody Linkage of Cy3 labeled GFP-Adenovirus
- 25 PU Matrix
Surface-aminated PU films (group NI-1,-A) were reacted with LC-sulfo-SPDP
dissolved
59
=
CA 02821214 2013-07-19
= =-==
= -
in PBS (9 mg/m1; 1ml; 90 min). Then, the films were extensively washed in PBS
and reacted in
5%BSA with anti-knob Ab (0.66 mg/m1) reduced with l .5 mg of 2-
mercaptoethylamine for 90
= min at 37 C. Prior to conjugation, Ab was purified by gel filtration
using a desalting column
equilibrated with degassed PBS containing 10 rnMED'TA. The conjugation was
allowed to run
for 38 hours at room temperature (RT) under mild shaking. Next, the films were
washed in
PBSx3 and immersed in the suspension of3013 particles of Cy3-labeled
adenovirus(Cy3-AdV-
GFP) in 1.5 ml of5% BSA/PBS. Surface aminated films that were not modified by
antilcnob Ab
(Nliz-B) served as controls. Irnmunoconjugation was carried out for 12 hours
at RT under mild
shaking. Finally, the films were washed in PBS and examined under fluorescent
microscope to
= 10 assess tethering of Cy3-labeled adenoviruses. A uniform
virus coverage of the surface was
observed for the films conjugated with antiknob Ab, while the control films
were virtually non-
fluorescent.
PDT-BzPh-modified PU Tecothane TT-1074A films were directly modified with the
reduced antilcnob Ab. After washing the films (PDT-A) along with the control
samples, the
PDT-BzPh-modified PU samples that were not conjugated with the antiknob Ab
(PDT-B), both
PDT-A and PDT-B were incubated with Cy3-iabeled GFP-AdV. Antibody reduction,
= purification and conjugation, and virus tethering were carried out
according the procedures
outlined above. Similar to the results obtained for the surface-aminated PU
samples, a uniform
fluorescent AdV layer was observed for the Ab-mediated AdV tethering, while no
Cy-3-labeled
AdV was bound to the surface of control films.
= PE Matrix
PDT-BzPh-modified PE fibers were reacted with 2 mg of antiknob antibody
reduced
= with 10 mg of 2-mercaptoethylamine at 37 C for 1 hour. Prior to
utilization, the reduced Ab
was purified using a desalting column equilibrated with degassed PBS/10 mM
EDTA. The
conjugation was carried out in 5% BSA/PBS for 20 hours at room temperature
under mild
shaking.
= After PBS x 3 washing, the Ab-coupled fibers were immersed into the
suspension of 5
= x1011 particles of Cy3-labeled adenovirus in 1.5 ml of 5% BSA/PBS. Immune
conjugation was
carried out for 14 hours at RT under mild shaking. Immobilization of Cy3-AdV
on the surface
of PDT-BzPh-modified PE fibers was confirnted by fluorescent microscopy.
EXAMPLE 10
=- Preparation of nanoparticles (NPs) Surface-Modified with PDT-BzPh
CA 02821214 2015-05-13
In the following example, an aqueous dispersion ofNPs was prepared by
emulsification-
:
solvent evaporation and subsequently surface-modified with PDT-BzPh
D,L-polylactide (D,L-PLA, Sigma), branched poly(ethyleneimine) (PEI 25K,
Aldrich)
= and Poloxamer 188 (Pluronierm F-68, Sigma) were dissolved in 5 ml DCM
HPLC grade in
= 5 amounts of 300 mg, 100 mg and 50 mg, respectively.
Polylactide is a nanoparticle-forming polymer (i.e. constitutes the polymeric
matrix of
the nanoparticle). Poly(ethyleneimine) makes the nanoparticles cationic,
capable of binding
anionic substances and contributes to nanoparticles'stability during the
emulsification step of
their preparation; is not a requisite and can be omitted. Poloxamer 188 is a
non-ionic stabilizer,
which is required for nanoparticles' stability, it provides sterical
stabilization.
The organic solution was emulsified in 15 mi of distilled water on ice-bath at
0 C using
sonication. The solvent was removed by rotary evaporation at 35 C. The NPs
were filtered
through Whatman paper filter (2.5 um cut-off). The NP size was determined by
Photon
Correlation Spectroscopy and was found to be 600 nm. Next, the NPs were
dialyzed to remove
unbound stabilizers (300 K.Da cut-offMW) in distilled water at 4 C for 48 hr
with several water
replacements.
2 ml of preformed PLA/PEI nanoparticles were mixed with 1 ml of 0.1% aqueous
solution of PDT-BzPh. 50 ul of aqueous solution of 15% KH2PO4 were added to
the reaction
mixture to adjust the pH to 5.5. At this pH, PDT-BzPh separates from the
solution, presumably
associating with the lipophilic surface of the NPs. The total amount of PDT-
I3zPh in the
formulation (1 mg) was calculated to have a 4-fold excess over the estimated
amount of the
PDT-BzPh needed to establish a monolayer on the surface of PLA/PEI
nanoparticles. The
reaction mixture was transferred to a round-bottom flask. The flask was
rotated (at 100 rpm)
causing thin layer distribution of the reaction volume over the flask's walls.
The hand-held UV
lamp (UVGL-25, UVP) was approximated to the rotating flask, and the reaction
mixture was
irradiated at about 350 nm from the distance 1-2 cm for 30 min at room
temperature.
According to another methOd of preparing particles, the BzPh-PDT polymer is
included during the particle formation stage. In this variant, the BzPh-PDT
acts as a particle
= surface stabilizer without additional colloidal stabilizers as in the
previous protocol.
The organic phase obtained by dissolving 200 mg poly(D,L-lactide) (Mn =32,000)
in 5
.1 ml chloroform, added to 15 ml of aqueous phase comprising 10 mg
BzPh-PDT and 6.7 mg
KHCO3 and pre-cooled to 0 C. Next, the mixture was emulsified by sonication
on ice bath.
Chloroform was removed by rotavaporation at 30 C. The particles were filtered
through a 1.0
61
CA 02821214 2013-07-19
gm glass fiber filter, UV-irradiated in a Petri dish for 5 min with several
stirrings on a
preparative UV-lamp and separated from the free BzPh-PDT by gel filtration
using agarose gel
= with a fractionation range 25,000-2,400,000 and an exclusion limit of
4,000,000. The thiol-
reactive particles thus obtained could further be used for surface
modification with thiol-
containing proteins, such as cysteinated DI or reduced IgG immunoglobul ins.
EXAMPLE 11
Tethering of Biomolecu les to NPs Modified with DPT-BzPli -
PDT-BzPh -modified NPs were separated from the excess of PDT-BzPh bythe
dialysis
against double distilled water (DDW) through the 300 kDa cut-off membrane (24
hours, 3
changes of DDW)the BzPh-PDT polymer is included during the particle formation
stage, the
= dialysis is not necessary.A thiolated (cysteinated) form of DI domain of
the Coxsackie-
Adenovirus receptor (produced by inventors according to the procedure
described in Nyanguile
et al, Gene Ther., 2003; 10:1362-1369) was conjugated to the thiol-reactive
nanoparticles to
= impart adenovirus-tethering properties.
2 mg of DI was column-desalted in2,.7 mi of degassed MES (0.01M, pH 6.5)
supplemented with 10 mM EDTA and mixed with 3.3 ml ofPDT-BzPh-modified
nanoparticles.
The coupling reaction was carried out for 14 hours at room temperature under
moderate
= shaking. Dl-derivatized nanoparticles were separated from the excess ofD1
by by gel filtration
using agarose gel with a fractionation range 25,000-2,400,000 and an exclusion
limit of
4,000,000. Finally, 2 ml of the eluate was mixed with 125 pi of Ad-GFP (3.75 x
1011 particles).
BSA was added to final 5% concentration. Irnmune conjugation was carried out
at room
temperature under mild shaking for 12 hours. 2 ml of diluted (1:3) non-
modified PLA/PEINPs
=
= were mixed with the same amount of BSA and Cy3-Ad V-GFP to serve as
control. Tethering of
= fluorescent Cy3-labeled adenovirus (AdV) to NPs was documented by
fluorescent microscopy.
In another variant of this protocol, 500 ILA of DI solution (ca 4 mg/ml) were
gel filtered
through a desalting column (PIERCE 43243) equilibrated with degassed 0.01 M
MES/10 mM
= EDTA (pH=6.5). The fraction between 2.5 and 4.5 nil of the eluent was
collected. One ml
= aliquots of the desalted thiolated DI were mi:xed with 1 ml of the
nanoparticles. The
conjugation was allowed to run for 20 hours at room temperature under argon
with shaking.
= 30
Dl-derivatized nanoparticles were separaed from the excess of DI
by gel filtration using =
agarose gel with a fractionation range 25,090-2,400,000 and an exclusion limit
of 4,000,000.
= One ml of the nanoparticles was mixed with I ml ofa dnovicus 5spension
containing 1.48 x1011
52
CA 02821214 2013-07-19
=
=
virus particles. The nanoparticle-virus conjugation was carried cut for 2.5
hours at room =
ternperature in presence of 5%. bovine seaun albumin under mild shaking.
EXAMPLE 12
Preparation of Magnetic Particles
= Magnetically-
responsive pasticles can be prepared similarly to the procedure described in -
Example 6 using ultrasmall magnetite/magherninanocrystals dispersion in
chloroform instead
of pure chloroform as stated in the procedure abcve. Such dispersion can be
obtained by
precipitation of aqueous ferrous chloride or its co-preeipitation with ferric
chloride in an alkaline
= =
aqueous solution and further stabilized with a fatty acid (See De Cuyper M,
Soniau M.
helagnetoliposomes. Formation and structural chaeactetizatior.. Eur Biophys
.11988; 15:311-9;
Khalafalla SE. Magnetic fluids, Chemtech 1975, Sept,: 540-7) that also imparts
a degree of
lipophilicity to the nanocrystal surface depending on the hydrocarbon chain
length and the
=
coating density. Such coated nanocrystals can forther be extracted into or re-
suspended in
chloroform as well as other organic solvents, such as clielilorom ethane,
tetrahydrofuran, etc.,
which can be used for polymer-based particle foimulation by the methods
mentioned above as in
=
the following example:
= One ml of an
aqueous solution contairing 65 ing FeC13 hexahydrate, 32 mg FeCi2 .
tetrahydrate and 10 mg PLURONIC F-68 was rapidly mixed with 10 ml aqueous
solution of
NaOH (0.1 M. Oleic acid (100 nig) was added dropwise, and the mixture was
degassed in
= argon. The contents were mixed and heated in a evater bath for 5 min at 90
C and cooled to
room temperature. The suspension was sedimented on a Ne-B-Fe magnet for 5 min,
the liquid
=
above the precipitate was carefully aspirated, and the precipitate was mixed
with 5 ml CHC13.
The obtained organic dispersion of oleic acid surIce-medified neagnetite was
added to 200 mg.
. poly(D,L-
lactide) (Mn 32,000) and vortexed to completely dissolve the polymer, and
further =
used as an organic phase as described in Examples 10 and HI
. .
.
=
While the invention has been described in de-eail and with referenceto
specific examples
thereof, it will be apparent to one skilled in the art that various changes
and rnodiflcations can be .
made therein, The scope of the claims
=
..
= ..
,should not be limited by the preferred embodiments set forth in the examples,
but should be
give the broadest interpretation consistent itI the description as a whole_
=
63 =
,
=
, .
=
CA 02821214 2013-07-19
,
. Scheme 1 illustrates binding of benzophenone cross-linkers to polymers
haying C-H
bonds.
H
I
R R ..---...-C....."--.. R R
0 14.. 0 __Ps irfaYmceer
_/,...
co 00* Ph-C-OH Ph--OH
1 I 1
Ph Ph ....---.....-C-------
,
Excited
Benzophenone Rad =cal cross-linker
= cross-linker triplet pair .
Immobilized
state
Scheme 2 illustrates synthesis of polymeric multi-pcint benzophenone
modifiers.
o o
6-o-8-0-coph
i
(-CH-CH2-), o, _____________________________________
L'Hz C1H2 I
CH2 .
I I I
14H2 NH2 1µ,1H-0C-0-COPh
' .....
Polyallylamine= PAA-12aPh
(PM) .
(-01-1-CH2-)(-CH-0112)m-(-CH-CH2-)k
= 1 I 1
0 0 CH2 CH2 CH2
N-0-8-0-COPh i
NH 1
NH I
mi-oc # 0,11, '
I I
0
= 12 0=0 00
=
I I
0 0 CHICH2COOH 01120H2SS*-0 )
*1-078-014201-12SS <i) 0
PDT-BzPh
0 SPDP
Cl&C) //
0/
(-CH. -0H2-)1,44411-(-CH-CH2-)m-(-CH-CH21k
61-12 1
CH2 C1
H2
I
NI
WH2 H NH-100-n)-COPh
.
I v._,./
0=C
412CH2S8-10
_
22211128.1 64
CA 02821214 2013-07-19
1 4,
=
Scheme 3 depicts immobilization of thiol-containing proteins on a polymeric
surface.
Polymer Polymer
surface surface
=-u
o
,-2 74 .
S' t13
N
61 If =13
w
S--I PROTEIN
s-e 1 ______
NH 2 HS¨ PROTEIN] S
LC-sulfo-SPDP ----- ____________ .4,.. ...-------1----
----,
----...),...,-----
Aminated Surface activated Immobilized
surface with thiol-reactive protein
2-pyridyldithio groups
I ,,¨,,,..---,,,A. )\ ---,/-s-O3Na
' '1µ1S'S-----irNH
0--N j
0 )1
. 0
LC-sulfo-SPDP
22211128.1 65
