U.S. patent application number 13/334913 was filed with the patent office on 2012-06-07 for polymer nanoparticles coated by magnetic metal oxide and uses thereof.
This patent application is currently assigned to HENRY FORD HOSPITAL. Invention is credited to Chaya Brodie, Shlomo Margel, Tom Mikkelsen, Benny Perlstein.
Application Number | 20120141380 13/334913 |
Document ID | / |
Family ID | 40282352 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120141380 |
Kind Code |
A1 |
Margel; Shlomo ; et
al. |
June 7, 2012 |
POLYMER NANOPARTICLES COATED BY MAGNETIC METAL OXIDE AND USES
THEREOF
Abstract
The invention provides nanoparticles consisting of a polymer
which is a metal chelating agent coated with a magnetic metal
oxide, wherein at least one active agent is covalently bound to the
polymer, said nanoparticles may optionally further comprise at
least one active agent physically or covalently bound to the outer
surface of the magnetic metal oxide. Pharmaceutical compositions
comprising these nanoparticles may be used, inter alia, for
detection and treatment of tumors and inflammations.
Inventors: |
Margel; Shlomo; (Rehovot,
IL) ; Perlstein; Benny; (Raanana, IL) ;
Brodie; Chaya; (Southfield, MI) ; Mikkelsen; Tom;
(Detroit, MI) |
Assignee: |
HENRY FORD HOSPITAL
DETROIT
MI
BAR ILAN UNIVERSITY
RAMAT GAN
|
Family ID: |
40282352 |
Appl. No.: |
13/334913 |
Filed: |
December 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12232818 |
Sep 24, 2008 |
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13334913 |
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60960270 |
Sep 24, 2007 |
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Current U.S.
Class: |
424/9.34 ;
424/133.1; 424/85.1; 424/85.2; 424/9.4; 424/9.6; 424/94.1; 514/249;
514/34; 514/9.3; 514/9.7; 977/773; 977/906; 977/915; 977/927;
977/928; 977/930 |
Current CPC
Class: |
A61K 49/1878 20130101;
A61P 35/00 20180101; A61K 47/62 20170801; B82Y 5/00 20130101; A61K
47/6921 20170801; A61K 41/0052 20130101; A61P 29/00 20180101; A61P
35/02 20180101; A61K 47/6923 20170801; A61K 47/6935 20170801; A61P
1/14 20180101; A61P 3/04 20180101; A61K 49/1866 20130101; A61P 3/10
20180101 |
Class at
Publication: |
424/9.34 ;
424/9.6; 514/9.3; 424/85.1; 424/85.2; 514/249; 514/34; 424/94.1;
424/133.1; 514/9.7; 424/9.4; 977/773; 977/906; 977/915; 977/928;
977/930; 977/927 |
International
Class: |
A61K 49/06 20060101
A61K049/06; A61K 38/39 20060101 A61K038/39; A61K 38/19 20060101
A61K038/19; A61K 38/20 20060101 A61K038/20; A61K 31/519 20060101
A61K031/519; A61K 38/43 20060101 A61K038/43; A61K 39/395 20060101
A61K039/395; A61K 38/22 20060101 A61K038/22; A61K 49/04 20060101
A61K049/04; A61P 29/00 20060101 A61P029/00; A61P 35/00 20060101
A61P035/00; A61K 49/00 20060101 A61K049/00; A61K 31/704 20060101
A61K031/704 |
Claims
1.-64. (canceled)
65. A nanoparticle comprising a metal chelating polymer, a magnetic
metal oxide, and at least one active agent covalently bound to said
polymer.
66. The nanoparticle of claim 65, wherein said metal chelating
polymer is gelatin, polymethylenimine, chitosan or polylysine.
67. The nanoparticle of claim 65, wherein said magnetic metal oxide
is an iron oxide or a ferrite derived from an iron oxide.
68. The nanoparticle of claim 67, wherein said iron oxide is
magnetite, maghemite or a mixture thereof.
69. The nanoparticle of claim 65, wherein said at least one active
agent is a fluorescent dye, a contrast agent, a peptide or
peptidomimetic, a polypeptide, an antifolate drug, an antibiotic,
an anti-inflammatory agent, an anthracycline chemotherapeutic
agent, or any combination thereof.
70. The nanoparticle of claim 69, wherein said fluorescent dye is
rhodamine or fluorescein.
71. The nanoparticle of claim 69, wherein said peptide or
peptidomimetic is a cyclic RGD (cRGD) or an acyclic RGD.
72. The nanoparticle of claim 69, wherein said polypeptide is
TNF-related apoptosis-inducing ligand (TRAIL) or interleukin-12 (IL
12).
73. The nanoparticle of claim 69, wherein said antifolate drug is
methotrexate.
74. The nanoparticle of claim 69, wherein said anthracycline
chemotherapeutic agent is doxorubicine.
75. The nanoparticle of claim 65, further comprising at least one
additional active agent bound to an outer surface of said
nanoparticle.
76. The nanoparticle of claim 75, wherein said at least one
additional active agent is covalently bound to the outer surface of
the nanoparticle via a linker.
77. The nanoparticle of claim 76, wherein said linker is derived
from a polysaccharide, protein, peptide, polyamine,
polyethyleneglycol, acryloyl chloride, divinyl sulfone (DVS),
dicarbonyl imidazole, ethylene
glycolbis(sulfosuccinimidylsuccinate), m-maleimidobenzoic acid
N-hydroxysulfosuccinimide ester or any combination thereof
78. The nanoparticle of claim 75, wherein said at least one
additional active agent bound to the outer surface of the
nanoparticle is a fluorescent dye, a contrast agent, a peptide or a
peptidomimetic, a polypeptide, an antifolate drug, an antibiotic,
an anti-inflammatory agent, an anthracycline chemotherapeutic agent
or any combination thereof
79. The nanoparticle of claim 78, wherein said peptide or
peptidomimetic is a cyclic RGD (cRGD) or an acyclic RGD.
80. The nanoparticle of claim 78, wherein said polypeptide is a
cytokine, an enzyme, an antibody or a hormone.
81. The nanoparticle of claim 80, wherein said antibody is
Bevacizumab (trade name: avastin) or Infliximab (trade name:
remicade).
82. The nanoparticle of claim 78, wherein said polypeptide is
TNF-related apoptosis-inducing ligand (TRAIL) or interleukin-12
(IL12).
83. A pharmaceutical composition comprising a nanoparticle of claim
65 and a pharmaceutically acceptable carrier.
84. A method for detecting a tumor in an individual comprising
administering to said individual a nanoparticle according to claim
65, wherein TRAIL, cRGD peptide, IL-12, doxorubicin, methotrexate
or any combination thereof is bound to the outer surface of the
nanoparticle, and a contrast agent or a fluorescent dye is
covalently bound to said polymer.
85. A method for inducing apoptosis, autophagy, or both in a cancer
cell, comprising the step of contacting said cell with a
nanoparticle of claim 75, wherein said at least one active agent is
TRAIL, cRGD peptide, IL-12, doxorubicin, methotrexate, bevacizumab
or any combination thereof, and wherein said cancer cell is a
glioma cell, a cancer stem cell, a cervical carcinoma cell, a
bladder carcinoma cell, a breast cancer cell, an ovarian cancer
cell, or a lung cancer cell.
86. The method according to claim 85, wherein said inducing
apoptosis, autophagy, or both in a cancer cell is treating a
patient afflicted with glioma, cervical carcinoma, bladder
carcinoma, breast cancer, ovarian cancer or a lung cancer.
87. The method of claim 85, further comprising the step of
contacting said cell with a proteasome inhibitor.
88. The method according to claim 85, further comprising the step
of Gamma irradiating said cell.
89. A method for employing X-ray imaging or magnetic resonance
imaging (MRI) in a subject, comprising administering to said
subject a nanoparticle according to claim 65, wherein said active
agent is a contrast agent.
90. A method for detecting inflammation in a subject comprising
administering to said subject a nanoparticle according to claim 65,
wherein said magnetic metal oxide is iron oxide, and said at least
one active agent is a fluorescent dye or a contrast agent.
91. A method for treating inflammation in a subject comprising
administering to said subject a nanoparticle according to claim 65,
wherein said magnetic metal oxide is iron oxide, and said at least
one active agent is an anti-inflammatory agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority as a continuation
application to U.S. patent application Ser. No. 12/232,818, filed
on Sep. 24, 2008; which claims the benefit of U.S. Provisional
Patent Application No. 60/960,270, filed on Sep. 24, 2007, the
entire contents of which being herewith incorporated by reference
in its entirety as if fully disclosed herein.
FIELD OF THE INVENTION
[0002] The present invention provides nanoparticles consisting of a
polymer which is a metal chelating agent coated with a magnetic
metal oxide, wherein at least one active agent is covalently bound
to the polymer, as well as pharmaceutical compositions and uses
thereof.
BACKGROUND OF THE INVENTION
[0003] Nanoparticles are spherical particles in sizes ranging from
a few nanometers up to 0.1 .mu.m. Polymeric nano-scaled particles
of narrow size distribution are commonly formed by controlled
precipitation methods or heterogeneous polymerization techniques,
e.g., by optimal emulsion or inverse emulsion polymerization
methods. Properties of solid materials undergo drastic changes when
their dimensions are reduced to the nanometer size regime. It is
important to keep in mind that the smaller the particles are, the
larger portion of their constituent atoms is located at the
surface. Nanoparticles, particularly in sizes below ca. 20 nm,
predominantly exhibit surface and interface phenomena that are not
observed in bulk materials, e.g., lower melting and boiling points,
lower sintering temperature and reduced flow resistance.
[0004] In view of their spherical shape and high surface area,
nano-scaled particles may provide neat solutions to a variety of
problems in materials science, such as composite materials,
catalysis, three dimensional structures and photonic uses, and can
further be used in biomedical applications such as specific cell
labeling and separation, cell growth, affinity chromatography,
diagnostics, specific blood purification by hemoperfusion, drug
delivery and controlled release (Bockstaller et al., 2003; Hergt et
al., 2004; Margel et al., 1999). Each application requires
polymeric nanoparticles of different optimal physical and chemical
properties. The synthesis and use of numerous types of nano-scaled
particles of different surface chemistry, e.g., variety of surface
functional groups such as hydroxyl, carboxyl, pyridine, amide,
aldehyde and phenyl chloromethyl, have already been described
(Margel et al., 1999). Such nanoparticles have been designed for
various industrial and medical applications, e.g., enzyme
immobilization, oligonucleotide and peptide synthesis, drug
delivery, specific cell labeling and separation, medical imaging,
biological glues and flame retardant polymers (Bunker et al., 1994;
Szymonifka and Chapman, 1995; Margel et al., 1999; WO 2004/045494;
Galperin et al., 2007).
[0005] Of particular interest are particles with magnetic
properties, which are usually used for separation of the particles
and/or their conjugates from undesired compounds via a magnetic
field. Due to their magnetic properties, these particles have
several additional significant applications such as magnetic
recording, magnetic sealing, electromagnetic shielding and
biomedical applications. Magnetic iron oxide, i.e., magnetite and
maghemite, nanoparticles are the main particles that have been
investigated for biomedical applications, e.g., magnetic
hyperthermia, magnetic drug targeting, magnetic cell separation and
as MRI contrast agents (Lacoste et al., 1993; Green-Sadan et al.,
2005; Leemputten and Horisberger, 1974; Hergt et al., 2004).
Magnetic iron oxides nanoparticles are non-toxic and biodegradable,
and have already been approved for clinical use as MRI contrast
agents. These nanoparticles are usually prepared by adding to an
aqueous solution containing stoichiometric concentrations of
ferrous and ferric ions, and a polymeric stabilizer such as
dextran, wherein a base, e.g., NaOH or ammonia, is added until
basic pH (usually above 8.0) is reached. The obtained coated
magnetic iron oxide nanoparticles are than washed by different
ways, e.g., by magnetic columns or dialysis. Extensive efforts to
synthesize efficient iron oxide magnetic nanoparticles have been
carried out in the last several years; however, most of these
nanoparticles suffer from major disadvantages such as broad size
distribution that is considered to be toxic for in vivo medical
applications, iron ions leaching and instability towards
agglutination processes.
[0006] WO 99/062079 and corresponding EP 1088315B1 of the same
Applicant, herewith incorporated by reference in their entirety as
if fully disclosed herein, disclose new uniform magnetic
gelatin/iron oxide composite nanoparticles, formed by controlled
nucleation of iron oxide onto an iron ion chelating polymer, e.g.,
gelatin, dissolved in an aqueous solution, followed by stepwise
growth of thin layers of iron oxide films onto the gelatin/iron
oxide nuclei. These magnetic nanoparticles can be prepared in a
very narrow size distribution and in sizes ranging from about 10 nm
up to 100 nm.
SUMMARY OF THE INVENTION
[0007] In one aspect, the present invention provides a nanoparticle
consisting of a polymer which is a metal chelating agent coated
with a magnetic metal oxide, wherein at least one active agent is
covalently bound to the polymer.
[0008] In another aspects, the present invention provides
pharmaceutical compositions comprising nanoparticles as defined
above and a pharmaceutically acceptable carrier, as well as various
methods of use.
[0009] The pharmaceutical compositions of the present invention may
be used, inter alis, for detection of a tumor; reducing or
inhibiting the growth of a tumor or for reducing or inhibiting the
growth of tumor cells left at a site in a patient from which a
tumor has been surgically removed; reducing or inhibiting the
growth of a tumor and monitoring the size thereof; and evaluating
responsiveness of tumor cells to treatment with a candidate
compound. In addition, these compositions may be used for detection
of a site of inflammation and treatment of said inflammation, as
well as for treatment of type 2 diabetes, obesity and anorexia.
[0010] In a further aspect, the present invention provides a
nanoparticle consisting of a polymer which is a metal chelating
agent coated with a magnetic metal oxide, wherein at least one
agent having an anti-tumor activity selected from a peptide, a
peptidomimetic, a polypeptide or a small molecule is bound to the
outer surface of the magnetic metal oxide. The present invention
further provides pharmaceutical compositions comprising these
nanoparticles and a pharmaceutically acceptable carrier, for use in
reducing or inhibiting the growth of a tumor, as well as various
methods of use.
BRIEF DESCRIPTION OF THE FIGURES
[0011] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0012] FIGS. 1A-1D show transmission electron microscopy (TEM)
micrographs of gelatin/iron oxide magnetic composite nanoparticles
of increased average diameter, prepared as described in Example 1,
by repeating the thin magnetic coating process during the growth
step 4, 5, 6 and 7 times (1A, 1B, 1C and 1D), respectively.
[0013] FIG. 2 shows a histogram of the diameter of gelatin/iron
oxide composite nanoparticles prepared as described in Example 1
and dispersed in water.
[0014] FIGS. 3A-3B show high resolution TEM (HTEM) (3A) and
electron diffraction (ED) (3B) picture of gelatin/iron oxide
magnetic composite nanoparticles prepared as described in Example
1.
[0015] FIG. 4 shows X-ray diffraction (XRD) pattern of gelatin/iron
oxide magnetic composite nanoparticles prepared as described in
Example 1.
[0016] FIG. 5 shows mossbauer spectrum of gelatin/iron oxide
magnetic composite nanoparticles prepared as described in Example
1.
[0017] FIG. 6 shows room temperature magnetization (VSM)-loop
obtained for gelatin/iron oxide magnetic composite nanoparticles
prepared as described in Example 1.
[0018] FIG. 7 illustrates the nucleation step of the preparation of
fluorescent dye-labeled gelatin/iron oxide magnetic composite
nanoparticles, as described in Example 2.
[0019] FIG. 8 shows the stability of free tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL) vs. TRAIL
conjugated to gelatin/iron oxide magnetic composite nanoparticles
(NP-TRAIL), at 10.degree. C. during 35 days.
[0020] FIGS. 9A-9B show the apoptosis in human A 172 glioma cells
(9A) and in glioma spheres established from primary HF2020 tumors
(9B) induced by gelatin/iron oxide magnetic composite nanoparticles
(NP), free TRAIL (100 ng/ml) and TRAIL-conjugated gelatin/iron
oxide magnetic composite nanoparticles (NP-TRAIL, 10 ng/ml).
[0021] FIG. 10 shows the effect of free TRAIL and TRAIL-conjugated
gelatin/iron oxide magnetic composite nanoparticles (NP-TRAIL) on
the apoptosis of glioma spheroids established from the human glioma
specimens HF1254, HF1308 and HF2020. Spheroids were plated in
24-well plates and were treated with medium (Control), TRAIL (100
ng/ml), gelatin/iron oxide magnetic composite nanoparticles (NP)
and TRAIL-conjugated gelatin/iron oxide magnetic composite
nanoparticles (NP-TRAIL, 10 ng/ml). Cell death was determined after
24 h of treatment using LDH assay. The results are the mean.+-.SE
of triplicates in two different experiments.
[0022] FIG. 11 shows the cytotoxic effect of free TRAIL (100 ng/ml)
and TRAIL-conjugated gelatin/iron oxide magnetic composite
nanoparticles (NP-TRAIL, 10 ng TRAIL/ml) on glioma cells U87, A172
and U251, as well as on primary cultures of glioma cells HF1308,
HF1254 and HF1316. Gelatin/iron oxide magnetic composite
nanoparticles (NP) alone or PBS (Control) served as controls. Cell
death was determined after 24 h of treatment by FACS analysis.
[0023] FIG. 12 shows the cytotoxic effect of TRAIL-conjugated to
non-fluorescent or fluorescent dye-labeled gelatin/iron oxide
magnetic composite nanoparticles on A172 cells. A172 cells were
incubated for 5 h with control nanoparticles (NP-Control),
TRAIL-conjugated non-fluorescent nanoparticles (NP-TRAIL), control
rhodamine-labeled nanoparticles (NPR-Control) or TRAIL-conjugated
rhodamine-labeled nanoparticles (NPR-TRAIL). Cell apoptosis was
determined using propidium iodide staining and FACS analysis, and
the results are the mean.+-.SE of three different experiments.
[0024] FIG. 13 shows the specific internalization of
TRAIL-conjugated rhodamine-labeled gelatin/iron oxide magnetic
composite nanoparticles (NPR-TRAIL) into glioma cells as compared
to normal astrocytes. NPR-TRAIL were incubated with A172 cells and
with normal astrocytes for 30 min, and the immunofluorescence of
the cells was determined using a confocal microscopy. The results
represent one out of three experiments which gave similar
results.
[0025] FIG. 14 shows synergistic effect of .gamma.-irradiation and
TRAIL-conjugated gelatin/iron oxide magnetic composite
nanoparticles (NP-TRAIL) on U87, A172, U251 and LN-18 glioma cell
lines. Cells were incubated with NP-TRAIL (5 ng TRAIL/ml) or with
gelatin/iron oxide magnetic composite nanoparticle (NP) alone for
24 hr, or .gamma.-irradiated (10 Gy for 2 h) and then treated with
NP-TRAIL (NP-TRAIL+Rad, 5 ng TRAIL/ml) or with NP alone (NP-Rad)
for 24 h. Cell apoptosis was determined by FACS analysis.
[0026] FIG. 15 shows that both cRGD peptide (cRGD) and cRGD
peptide-conjugated gelatin/iron oxide magnetic composite
nanoparticles (NP-cRGD) induce autophagy, i.e., an increase of
punctuated staining, in glioma U251 cells, compared with control or
gelatin/iron oxide magnetic composite nanoparticles (NP)
only-treated cells.
[0027] FIG. 16 shows the effect of TRAIL-conjugated gelatin/iron
oxide magnetic composite nanoparticles (NT-TRAIL) on apoptosis of
cervical carcinoma cell line HeLa, breast cancer cell line MCF-7
and lung cancer cells A549. The various cell lines were incubated
with TRAIL (100 ng/ml), NP-TRAIL (50 ng TRAIL/ml), nanoparticles
(NP) alone or PBS (Control) for 24 h, and data is shown as
mean.+-.SE.
[0028] FIGS. 17A-17B show synergistic effect of .gamma.-irradiation
and TRAIL-conjugated gelatin/iron oxide magnetic composite
nanoparticles (NP-TRAIL) on glioma stem cells spheroids established
from the tumor specimens 2355 (17A) and 2303 (17B). Cells were
treated with either TRAIL (100 ng/ml), the nanoparticles
[0029] (NP) alone (Control) or NP-TRAIL (50 ng/ml) for 24 h, the
supernatants were then collected and LDH analysis was performed. In
order to evaluate the combined effect of irradiation (IR) and
TRAIL, cells were first irradiated with 5 Gy radiation and after 4
h were treated either with TRAIL or NP-TRAIL for additional 24 h.
Cell death was determined using LDH levels in culture supernatants.
The results represent one experiment that was done in triplicate
out of three similar experiments.
[0030] FIG. 18 shows the effect of IL-12 and IL-12-conjugated
gelatin/iron oxide magnetic composite nanoparticles (NP-IL-12) on
the apoptosis of ovarian cancer cells. Ovarian cancer cells were
treated with medium alone, 1L-12 (50 ng/ml), the nanoparticles (NP,
50 .mu.g/ml) alone or NP-IL-12 (50 ng bound IL-12/ml), incubated
for 24 h and were then analyzed for cell death using the LDH
assay.
[0031] FIG. 19 shows tumor sections from rats treated with either
TRAIL-conjugated gelatin/iron oxide magnetic composite
nanoparticles (NP-TRAIL) or the nanoparticles (NP) alone. Human
U251 glioma cells were employed as xenografts in rat brains and
tumors were allowed to develop for 7 days at which time either NP
or NP-TRAIL were intracranially injected at the site of the tumor.
The degree of cell apoptosis in tumors of control rats treated with
PBS (not shown), NP or NP-TRAIL were examined 7 days after
treatment, and cell apoptosis was determined using TUNEL staining
(brown staining), which specifically detects apoptotic cells.
[0032] FIG. 20 shows the effect of TRAIL-conjugated gelatin/iron
oxide magnetic composite nanoparticles (NP-TRAIL) on survival of
human U251 implanted rats. The nanoparticles (NP) alone, NP-TRAIL
or PBS were injected directly into the tumor 7 days post tumor cell
implantation, and animals were observed for signs of distress
and/or morbidity and were euthanized at that time.
[0033] FIGS. 21A-21C show the effect of TRAIL-conjugated
gelatin/iron oxide magnetic composite nanoparticles (NP-TRAIL)
(21C) vs. the nanoparticles (NP) alone (21B) or PBS (21A)
administration on tumor volume at time of morbidity. Rats were
perfused with formalin at time of euthanasia, brains were harvested
and H&E staining was performed. Representative tumor volume
slices are shown. Number above series of pictures represents the
day of euthanasia.
[0034] FIG. 22 shows that TRAIL-conjugated gelatin/iron oxide
magnetic composite nanoparticles (NP-TRAIL) administration leads to
a decrease in overall tumor burden as compared with the
nanoparticles (NP) alone or PBS administration. Rats were
intracranially implanted with human U251 cells and after 7 days,
NP-TRAIL, NP or PBS were administered. Animals were euthanized at
day 21, and brain tissue was harvested and sectioned for volume
determination. Slides were photodoc-umented with identical settings
for all slides used in this determination. Volume was determined by
measuring greatest width and length of tumor for every 15.sup.th 5
.mu.m slice. Volume for each slice is determined and multiplied by
the number of slices to the next measured slice until edge of tumor
is achieved.
[0035] FIG. 23 shows the ability of gelatin/iron oxide magnetic
composite nanoparticles (NP) to track to sites of tumor growth.
Rhodamine-labeled NP (NPR) were implanted in the contralateral
hemisphere of rat brains 7 days after U251 tumor cell implantation.
Four days later, animals were euthanized, and brains were harvested
and snap frozen for sectioning and imaging. Panels A, D and G show
images of NPR in the corpus collosum part close to the site of the
NPR injection; panels B, E and H shows the site of NPR
implantation; and panels C, F and I show images of NPR in the
corpus collosum part close to the site of tumor cells.
Magnification 4.times. (panels A, B, C); 10.times. (panels D, E,
F); and 20.times. (panels G, H, I).
[0036] FIG. 24 shows that TRAIL-conjugated gelatin/iron oxide
magnetic composite nanoparticles (NP-TRAIL) administration leads to
large areas of tissue destruction, specifically demonstrated in
panels b, d and f, which is not seen neither with nanoparticles
(NP) alone nor with PBS administration. Nude rats were implanted
with U251n tumors on day 0; NP-TRAIL, NP alone or PBS were
intraneoplastically administered on day 7; and animals were
euthanized and tissue was harvested on day 14: NP-TRAIL (panels
a-f); NP (panels g-l); PBS (panels m-r). Magnification 1.times.
(panels a, c, e, g, i, k, m, o, q); 10.times. (panels b, d, f, h,
j, l, n, r). Arrows indicate the lower part of the tumor mass.
[0037] FIG. 25 shows increased tumor cell destruction in rats
receiving TRAIL-conjugated gelatin/iron oxide magnetic composite
nanoparticles (NP-TRAIL) vs. the nanoparticles (NP) or PBS only.
Animals were implanted with human U25 ln cells on day 0 and were
administered with PBS, NP or NP-TRAIL on day 7. Animals were
euthanized on day 14 and brain tissue processed and hemotoxylin and
eosin stained. PBS (panels a-b); NP (panels c-e); NP-TRAIL (panels
f-h). Magnification 40.times..
[0038] FIGS. 26A-26B show that TRAIL-conjugated gelatin/iron oxide
magnetic composite nanoparticles (NP-TRAIL) (26B) induce lower
signal intensity both at the margin and inside the tumor, compared
with the nanoparticles (NP) alone (26A), as shown by MRI,
indicating that NP-TRAIL arrive at the borders and inside human
glioma xenograft implanted within nude rats. U251n human glioma
cells were implanted in nude rats, and NP or NP-TRAIL were
intracranially injected in the contra lateral side of the tumor 11
days later. MR images were obtained 8 days later. Each figure shows
4 consecutive sections of MRI, each section has 4 images with
different echo time (TE). Longer TE (right lower image in small
box). More signal intensity loss (black) due to iron. R-right;
L-left; IS-injection site (left); Tm-Tumor (right); LV-lateral
ventricle; and 3V-3.sup.rd ventricle.
[0039] FIG. 27 shows the ability of TRAIL-conjugated
rhodamine-labeled gelatin/iron oxide magnetic composite
nanoparticles (NPR-TRAIL) to be taken up by tumor cells in vivo.
Rhodamine-labeled gelatin/iron oxide magnetic composite
nanoparticles (NPR) alone or NPR-TRAIL were implanted directly
within the tumor mass 7 days after GFP-U251 tumor cells
implantation. Four days later, animals were euthanized, and brains
were harvested and snap frozen for sectioning and imaging (red
color and green color indicate the presence of NPR and GFP-U251
tumor cells, respectively). As shown in the panel G,H and I,
NPR-TRAIL are not only found in areas of tumor mass but also
colocalized with tumor cells (panel I). However, NPR alone are
found in areas of tumor mass but are not colocalized with tumor
cells (panels D, E and particularly F). Control animals were
treated with PBS.
[0040] FIG. 28 shows that cRGD peptide-conjugated rhodamine-labeled
gelatin/iron oxide magnetic composite nanoparticles (NPR-cRGD)
migrate towards tumor cells in vivo. NPR-cRGD (10 .mu.l containing
0.05 mg nanoparticles bound to about 2 .mu.g cRGD peptide) were
injected to the contra-lateral side of the nude rat brains 7 days
after GFP-U251 tumor cells implantation. Four days later animals
were euthanized, and brains were harvested and snap frozen for
sectioning and imaging (red color and green color indicate the
presence of rhodamine-labeled gelitin/iron oxide magnetic composite
nanoparticles (NPR) and GFP-U251 tumor cells, respectively).
[0041] FIG. 29 shows that both cRGD peptide-conjugated
rhodamine-labeled gelatin/iron oxide magnetic composite
nanoparticles (NPR-cRGD) and TRAIL-conjugated rhodamine-labeled
gelatin/iron oxide magnetic composite nanoparticles (NPR-TRAIL)
migrate towards injury site in vivo. Injury was induced by needle
injection of PBS at the left side of the brain, and following 4
days, 5 .mu.g (25 .mu.g) of rhodamine-labeled gelatin/iron oxide
magnetic composite nanoparticles (NPR) alone, NPR-TRAIL (50 ng
bound TRAIL) and NPR-cRGD were injected to the contra-lateral side
of the brain. After 4 days, the animals were sacrificed and the
fluorescence of the NPR was visualized under fluorescent
microscope. As shown, few NPR were present at the other side of the
brain but their distribution was abundant. NPR-TRAIL were localized
mainly along the site of injury. In contrast, the NPR-cRGD were
distributed all over the side of the injured brain and also along
the corpus callosum.
[0042] FIGS. 30A-30B show the effect of TRAIL (10-100 ng/ml),
gelatin/iron oxide magnetic composite nanoparticlds (NP) and
TRAIL-conjugated gelatin/iron-oxide magnetic composite
nanoparticles (NP-TRAIL, 10-40 ng bound to TRAIL/ml), both in the
absence or presence of proteasome inhibitor (PS, 5 mM), on the
bladder carcinoma tumor cells TSU-PR1 (30A), as well as on both the
breast cancer cells MDA-MB and the normal breast cells MCF 10A
(30B). Cell death was determined after 24 h using LDH assay. 100%
cell death was determined in Triton X-100-treated cells and data
normalized.
DETAILED DESCRIPTION OF THE INVENTION
[0043] In one aspect, the present invention provides a nanopartidle
consisting of a polymer which is a metal chelating agent coated
with a magnetic metal oxide, wherein at least one active agent is
covalently bound to the polymer.
[0044] The magnetic polymer/metal oxide composite nanoparticles of
the present invention are based on the magnetic polymer/metal oxide
composite nanoparticles disclosed in WO 99/062079, herewith
incorporated by reference in their entirety as if fully disclosed
herein; however, further comprising at least one active agent that
is covalently bound to the polymer inside the nanoparticle. Such
nanoparticles may be prepared by any suitable method known in the
art, e.g., the process described in detail in Examples 1-3
hereinafter, namely, by controlled nucleation of a magnetic metal
oxide, e.g., iron oxide, onto a metal chelating polymer, e.g.,
gelatin, to which at least one active agent is covalently bound,
wherein said polymer is dissolved in an aqueous solution, followed
by stepwise growth of thin layers of the magnetic metal oxide films
onto the polymer/metal oxide nuclei. As shown in these Examples,
the yield of this is almost 100%.
[0045] Examples 1-2 hereinafter describe the preparation of
magnetic nanoparticles of the present invention, consisting of
gelatin as a metal chelating polymer and iron oxide as a magnetic
metal oxide. As shown in these examples, the magnetic nanoparticles
of the present invention can be prepared in a very narrow size
distribution and in sizes ranging from about 10 nm up to about 100
nm. Furthermore, these Examples particularly show the uniformity,
atomic order, magnetic properties and crystalline character of the
nanoparticles. The nanoparticles of the present invention are
superparamagnetic, i.e., they are magnetized in the presence of a
magnetic field, but no remanence is observed in the absence of a
magnetic field.
[0046] Surface analysis of gelatin/iron oxide magnetic composite
nanoparticles prepared as described in Examples 1-2 demonstrated
the presence of gelatin both within and on the surface of the
nanoparticle. As found, the surface gelatin provides additional
stabilization against agglomeration to the nanoparticle, as well as
functional groups such as carboxilate and primary amines through
which appropriate ligands can be covalently bound.
[0047] Preferably, the size of the nanoparticles of the present
invention is less than 300 nm, more preferably less than 100
nm.
[0048] According to the present invention, the metal chelating
polymer used for the preparation of the nanoparticles of the
present invention may be a polymer having functional groups capable
of binding metal ions, particularly iron ions, selected from amino,
hydroxyl, carboxylate, --SH, ether, immine, phosphate or sulfide
groups. In preferred embodiments, the metal chelating polymer is
selected from gelatin, polymethylenimine, chitosan or polylysine,
more preferably gelatin.
[0049] In one embodiment, the magnetic metal oxide coating the
aforesaid metal chelating polymer is an iron oxide or a ferrite
derived from an iron oxide. The iron oxide may be a magnetite,
maghemite, or a mixture thereof, and the ferrite is an oxide of the
formula (Fe,M).sub.3O.sub.4, wherein M represents a transition
metal ion, preferably. selected from Zn.sup.2+, Co.sup.2+,
Mn.sup.2+ or Ni.sup.2+. In a preferred embodiment, the magnetic
metal oxide used for the preparation of the nanoparticles of the
present invention is iron oxide.
[0050] According to the present invention, the at least one active
agent being covalently bound to the metal chelating polymer may be
selected, without being limited to, from a fluorescent dye, a
contrast agent, a peptide, a peptidomimetic, a polypeptide or a
small molecule.
[0051] In one embodiment, the active agent covalently bound to the
metal chelating polymer is a fluorescent dye. Examples of
fluorescent dyes include, without being limited to, rhodamine or
fluorescein.
[0052] In another embodiment, the active agent covalently bound to
the metal chelating polymer is a contrast agent, namely a compound
used to improve the visibility of internal bodily structures in
either an X-ray imaging or magnetic resonance imaging (MRI).
Examples of contrast agents for X-ray imaging include, without
being limited to, barium sulfate-based contrast agents that are
water insoluble, used in the digestive tract only either swallowed
or administered as an enema, and iodine-based water soluble
contrast agents, which can be used almost anywhere in the body, in
particular, intravenously as well as intraarterially, intrathecally
(the spine) and intraabdominally. Commonly used iodinated contrast
agents are diatrizoate (Hypaque 50); metrizoate (Isopaque Coronar
370), ioxaglate (Hexabrix), iopamidol (Isovue 370), iohexol
(Omnipaque 350), ioxilan (Oxilan), iopromide and iodixanol
(Visipaque 320).
[0053] In a further embodiment, the active agent covalently bound
to the metal chelating polymer is a peptide or a
peptidomimetic.
[0054] The arginine-glycine-aspartic acid (Arg-Gly-Asp; RGD) motif
of extracellular matrix components such as fibronectin and
vitronectin binds to integrins, and integrin-mediated adhesion
leads to intracellular signaling events that regulate cell
survival, proliferation and migration. Data obtained by phage
display methods screening for RGD-containing peptides have shown
their selective binding to endothelial lining of tumor blood
vessels. ROD peptides also retard signal transmission, affect cell
migration and induce tumor cell regression or apoptosis. By binding
to integrin of either endothelial or tumor cells, RGD peptides are
capable of modulating in vivo cell traffic by inhibition. of tumor
cell-extracellular matrix and tumor cell-endothelial cell
attachments, which are obligatory for metastatic processes. Several
studies have indicated that RGD-containing compounds can interfere
with tumor cell metastatic processes in vitro and in vivo. Peptides
that are specific for individual integrins are of considerable
interest and of possible medical significance. The
.alpha..sub.v.beta..sub.3 integrin was the first integrin shown to
be associated with tumor angiogenesis, and RGD peptides that
specifically block the .alpha..sub.v.beta..sub.3 integrin show
promise as inhibitors of tumor and retinal angiogenesis, of
osteoporosis and in targeting drugs to tumor vasculature.
Consequently, a great amount of work was invested in designing and
producing integrin-binding peptides and peptidomimetics.
[0055] In one preferred embodiment, the peptide or peptidomimetic
is thus a cyclic ROD (cRGD) peptide or peptidomimetic, or an
acyclic RGD-containing peptide or peptidomimetic. In a more
preferred embodiment, the cRGD peptide is the cRGD peptide of the
sequence cyclo (Arg-Gly-Asp-D-Phe-Lys (SEQ ID NO: 1).
[0056] In yet a further embodiment, the active agent covalently
bound to the metal chelating polymer is a polypeptide.
[0057] Tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL, also referred to as Apo-2 ligand, Apo-2L or TRAIL/Apo2L) is
a member of the tumor necrosis factor (TNF) family of cytokines,
capable of initiating apoptosis through engagement of its death
receptors. Additional members of this family are, e.g., TNF.alpha.
(also referred to as TNF), TNF.beta., TL1A (a TNF-like ligand),
lymphotoxix-3 (LT.beta.), CD30 ligand, CD27 ligand, CD40 ligand,
OX-40 ligand, 4-1BB ligand, Apo-1 ligand (also referred as Fas
ligand or CD95 ligand), Apo-3 ligand (also referred to as RANK
ligand, ODF or TRANCE) and TALL-1 (also referred to as B1yS, BAFF
or THANK) (WO 2004/001009; Wang and El-Deiry, 2003).
[0058] TRAIL is a type II transmembrane protein initially
identified and cloned based on the sequence homology of its
extracellular domain with CD95L (28% identical) and TNF (23%
identical). The native sequence of human TRAIL polypeptide is 281
amino acids long; however, some cells can produce a natural soluble
form of the polypeptide, through enzymatic cleavage of the
polypeptide extracellular region. Like most other TNF family
members, TRAIL forms homotrimers that bind the receptor molecules,
each at the interface between two of its subunits. Indeed, TRAIL
like most of the TNF ligand family occurs in both a membrane-bound
and a soluble form, which can possess different bioactivity. Four
members of the TNF family, i.e., Fas ligand, TNF.alpha., TL1A and
TRAIL, stand out because of their ability to induce apoptosis.
Unlike other TNF family members, soluble TRAIL (sTRAIL) has a
unique structural feature in which the cysteine residues together
coordinate a Zn atom, which is essential for trimer stability and
optimal biological activity. Functional studies showed that TRAIL
has a potent ability to induce apoptosis, in vitro, in a variety of
tumor cell lines including colon, lung, breast, prostate, bladder,
kidney, ovarian and brain tumors, as well as melanoma, leukemia and
multiple myelorha, but not in most normal cells, highlighting its
potential therapeutic application in cancer treatment (WO
2004/001009; Wang and El-Deiry, 2003; Ashkenazi et al., 1999;
Carlo-Stella et al., 2007; Smyth et al., 2003). There are only very
few agents that are truly cancer cell-specific in term of efficacy
or cell death induction as TRAIL. In contrast to other TNF family
members, whose expressions are tightly regulated and are often only
transiently expressed on activated cells, TRAIL mRNA is
constitutively expressed in a wide range of tissues. Although the
main biological function of TRAIL seems to be the induction of
apoptosis, the complete physiological role of this ligand is not
yet fully understood. It appears likely that TRAIL expression on
liver natural killer (NK) cells is regulated by IFN.gamma. secreted
from NK cells in an autocrine manner, since a large portion of NK
cells constitutively produce both TRAIL and IFN.gamma. in wild-type
and T-cell-deficient mice. Mouse gene knockout studies indicated
that TRAIL has an important role in antitumor surveillance by
immune cells, and that it mediates thymocyte apoptosis and it is
important in the induction of autoimmune diseases.
[0059] TRAIL induces apoptosis through interacting with its
receptors. So far, four homologous human receptors for TRAIL have
been identified, including DR4, KILLER/DRS, DcR1 (Trail-R3 TRID)
and DcR2 (TRAIL-R4), as well as a fifth soluble receptor called
osteoprotegerin (OPG), initially identified as a RANKL/OPGL
receptor. Both DR4 and DR5 contain a conserved death domain (DD)
motif and can signal apoptosis. The other three receptors appear to
act as "decoys" for their ability to inhibit TRAIL induced
apoptosis when over expressed. Decoy receptor 1 (DcR1) and DcR2
have close homology to the extra-cellular domains of DR4 and DR5.
DcR2 has a truncated, nonfunctional cytoplasmic DD, while DcR1
lacks a cytosolic region and is anchored to the plasma membrane
though a glycophospholipid moiety. The physiological relevance of
OPG as a receptor for TRAIL is unclear because the affinity for
this ligand at physiological temperatures is very low. On possible
explanation for the selective antitumoral activity of TRAM is that
the decoy receptors are preferentially expressed in normal cells
compared with tumor cells and interfere with TRAIL action (WO
2004/001009; Wang and El-Deiry, 2003; Carlo-Stella, 2007; Shah et
al., 2003). Another possible explanation is that most tumor cell
lines express the agonist TRAIL receptors but no or undetectable
levels of the antagonist receptors, whereas normal cells have been
found to express antagonist TRAIL receptors, and therefore, TRAIL
may allow selective killing of tumor cells only (Wei et al.,
2006).
[0060] Despite early promising results, recent studies have
identified several TRAIL-resistant cancer cells in various tumors.
Resistance of cancer cells to TRAIL appears to occur through the
modulation of various molecular targets, which may include
differential expression of death receptors. Based on molecular
analysis of death-receptor signaling pathways several new
approaches have been developed to increase the efficacy of TRAIL,
including the administration of conventional cancer drugs or
irradiation, in combination with TRAIL (Shankar and Srivastava,
2004; Smyth et al., 2003).
[0061] Thus, in one preferred embodiment, the polypeptide is a
cytokine, for example, tumor necrosis factor (TNF)-.alpha.,
TNF-.beta.; a TNF-related cytokine or an interleukin (IL).
Non-limiting examples of TNF-related cytokines include TNF-related
apoptosis-inducing ligand (TRAIL), TALL-1, the TNF-like ligand
TL1A, lymphotoxin-beta (LT-.beta.), CD30 ligands, CD27 ligands,
CD40 ligands, OX40 ligands, 4-1BB ligands, Apo-1 ligands and Apo-3
ligands, with TRAIL being preferred. In a most preferred
embodiment, the cytokine is TRAIL. Non-limiting examples of
interleukins include any interleukin that has an anti-tumor
activity, with IL-12, IL-23 and IL-27 being preferred.
[0062] In another embodiment, the polypeptide is an enzyme.
[0063] In a further embodiment, the polypeptide is an antibody such
as avastin or remicade. Avastin is a monoclonal antibody against
vascular endothelial grow factor (VEGF), which is used in the
treatment of cancer for inhibiting the growth of tumors by blocking
the formation of new blood vessels. Remicade is a monoclonal
antibody used for treatment of autoimmune disorders by binding to
TNF.alpha., one of the key cytokines that trigers and sustains the
inflammation response, and preventing it from binding to TNF.alpha.
receptors.
[0064] In still a further embodiment, the polypeptide is a hormone,
i.e., a polypeptide hormone such as insulin, obestatin or
ghrelin.
[0065] In another embodiment, the active agent covalently bound to
the metal chelating polymer is a small molecule.
[0066] In one preferred embodiment, the small molecule is an
anthracycline chemotherapeutic agent. The anthracycline
chemotherapeutic agent may be any chemotherapeutic agent of the
anthracycline family including daunorubicin (also known as
adriamycin), doxorubicin, epirubicin, idarubicin and mitoxantrone.
In a more preferred embodiment, the anthracycline chemotherapeutic
agent is doxorubicin, which is a quinine-containing anthracycline
and is the most widely prescribed and effective chemotherapeutic
agent utilized in oncology. Doxorubicin is indicated in a wide
range of human malignancies, including tumors of the bladder,
stomach, ovary, lung and thyroid, and is one of the most active
agents available for treatment of breast cancer and other
indications, including acute lymphoblastic and myelogenous
leukemias, Hodgkin's and non-Hodgkin's lymphomas, Ewing's and
osteogenic bone tumors, soft tissue sarcomas, and pediatric cancers
such as neuroblastoma and Wilms' tumors.
[0067] In another preferred embodiment, the small molecule is an
antifolate drug, i.e., a drug which impairs the function of folic
acid. A well known and a preferred example of an antifolate drug is
methotrexate, which is a folic acid analog that inhibits the enzyme
dihydrofolate reductase, and thus prevents the formation of
tetrahydrofolate that is essential for purine and pyrimidine
synthesis, and consequently leads to inhibited production of DNA,
RNA and proteins. Other examples of antifolate agents include
trimethoprim, pyrimethamine and pemetrexed. As antifolates
interfere with metabolism of nucleotides, their action specifically
targets the fast-dividing cells.
[0068] In a further preferred embodiment, the small molecule is an
antibiotic.
[0069] In still another preferred embodiment, the small molecule is
an amine-derived hormone, i.e., a derivative of the amino acids
tyrosine and tryptophan. Non-limiting examples of amine-derived
hormones include catecholamines, e.g., epinephrine, norepinepluin
and dopamine, and thyroxine.
[0070] In yet another preferred embodiment, the small molecule is a
lipid- or phospholipid-derived hormone, i.e., a hormone derive from
lipids such as linoleic acid and arachidonic acid and
phospholipids. The main classes of lipid- and phospholipid-derived
hormones are the steroid hormones derived from cholesterol, e.g.,
testosterone and cortisol, and the eicosanoids, i.e.,
prostaglandins.
[0071] In still a further preferred embodiment, the small molecule
is an anti-inflammatory agent.
[0072] The anti-inflammatory agent may be selected from a
corticosteroid, the alkaloid colchicine that is the standard
treatment for gout, or non-steroidal anti-inflammatory drugs
(NSAIDs) such as, but not limited to, aspirin, choline and
magnesium salicylates, choline salicylate, celecoxib, diclofenac,
diflunisal, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen,
indomethacin, ketoprofen, magnesium salicylate, meclofenamate
sodium mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin,
piroxicam, rofecoxib, salsalate, sodium salicylate, sulindac,
tolmetin sodium and valdecoxib.
[0073] In view of all the aforesaid, in one preferred embodiment,
the metal chelating polymer is gelatin, the metal oxide is iron
oxide, and the agent covalently bound to the gelatin is selected
from (i) a fluorescent dye, preferably rhodamine or fluorescein;
(ii) a TNF or a TNF-related cytokine, preferably TRAIL; (iii) an
anthracycline chemotherapeutic agent, preferably doxorubicin; (iv)
an antifolate drug, preferably methotrexate; or (v) a combination
thereof. In a more preferred embodiment, said at least one agent
covalently bound to the gelatin is a fuorescent dye or TRAIL.
[0074] According to the present invention, the nanoparticles
defined above may further comprise at least one active agent
physically or covalently bound to the outer surface of the magnetic
metal oxide, wherein said at least one active agent is the same or
different from the at least one active agent cobalently bound to
the polymer.
[0075] In one embodiment, said active agent is covalently bound to
the outer surface of the magnetic metal oxide.
[0076] As demonstrated in Example 4 hereinafter, various techniques
were established to stabilize and covalently bind external
functional groups to these magnetic nanoparticles, by coating them
with a variety of polymers, e.g., polysaccharides, proteins and
polyethyleneglycols. The functional-groups of these coatings were
then used for covalent binding of different reagents, e.g.,
proteins, enzymes and drugs for biomedical applications.
[0077] Thus, in one embodiment, the active agent covalently bound
to the outer surface of the magnetic metal oxide is bound, in fact,
via a molecule containing a functional group attached to the
magnetic metal oxide surface. In certain embodiments, this
molecules comprise a polymer selected from a polysaccharide, more
preferably chitosan, a protein, more preferably gelatin or albumin,
a peptide, or a polyamines.
[0078] In another embodiment, the active agent covalently bound to
the outer surface of the magnetic metal oxide is bound, in fact,
via an activating ligand attached to the magnetic metal oxide outer
surface. In preferred embodiments, the activating ligand is
acryloyl chloride, divinyl sulfone (DVS), dicarbonyl immidazole,
ethylene glycolbis(sulfosuccinimidylsuccinate) or
m-maleimidobenzoic acid N-hydroxysulfosuccinimide ester. In a more
preferred embodiment, the activating ligand is DVS.
[0079] As stated above, in certain cases the activating ligands may
further be attached to the polymer extending outside the metal
oxide coating.
[0080] As shown in Example 5, the active agent may also be
physically bound to the outer surface of the magnetic metal oxide.
This physical binding is based on non-covalent interactions, e.g.,
hydrophobic bonds, ionic interactions and hydrogen bonds, between
the active agent(s). and the outer surface of the magnetic metal
oxide.
[0081] Thus, in another embodiment, the active agent(s) of (b) is
physically bound to the outer surface of the magnetic metal
oxide.
[0082] According to the present invention, the at least one active
agent being bound to the outer surface of the magnetic metal oxide
may be selected, without being limited to, a peptide, a
peptidomimetic, a polypeptide or a small molecule, as defined above
for the active agent covalently bound to the polymer. In one
embodiment, said peptide or peptidomimetic is a cyclic RGD (cRGD)
peptide or peptidomimetic, preferably the cRGD peptide of SEQ ID
NO: 1, or an acyclic RGD-containing peptide or peptidomimetic; said
polypeptide is a cytokine, an enzyme, an antibody, preferably
avastin or remicade, or a hormone, preferably insulin, obestatin or
ghrelin; said cytokine is selected from tumor necrosis factor
(TNF)-.alpha., TNF-.beta., a TNF-related cytokine selected from
TNF-related apoptosis-inducing ligand (TRAIL), TALL-1, the TNF-like
ligand TL1A, lymphotoxin-beta (LT-.beta.), a CD30 ligand, a CD27
ligand, a CD40 ligand, an OX40 ligand, a 4-1BB ligand, an Apo-1
ligand, or an Apo-3 ligand, preferably TRAIL, or an interleukin
(IL), preferably IL having an anti-tumor activity, more preferably
IL-12, IL-23 or IL-27; and said small molecule is selected from an
antifolate drug such as methotrexate, an antibiotic, an
amine-derived hormone, a lipid- or phospholipid-derived hormone, an
anti-inflammatory agent, or an anthracycline chemotherapeutic agent
selected from daunorubicin, doxorubicin, epirubicin, idarubicin or
mitoxantrone, preferably doxorubicin.
[0083] The concentrations of the active agent(s) bound, either
covalently or physically, to the surface of the magnetic metal
oxide may be controlled by changing binding parameters, e.g.,
active agent(s) concentration in the process.
[0084] In one preferred embodiment, the present invention provides
nanoparticles as defined above, wherein said polymer is gelatin,
said magnetic metal oxide is iron oxide, said at least one active
Agent covalently bound to the polymer is selected from (i) a
fluorescent dye, preferably rhodamine or fluorescein; (ii) a TNF or
a TNF-related cytokine, preferably TRAIL; (iii) an anthracycline
chemotherapeutic agent, preferably doxorubicin; (iv) an antifolate
drug, preferably methotrexate; or (v) a combination thereof, and
said at least one active agent bound to the outer surface of the
magnetic metal oxide is selected from: (vi) a TNF or a TNF-related
cytokine, preferably TRAIL; (vii) a cRGD peptide, preferably the
cRGD peptide of SEQ ID NO: 1; (viii) an interleukin having an
anti-tumor activity, preferably IL-12; or (ix) a combination
thereof. In a more preferred embodiment, said at least one active
agent covalently bound to the polymer is a fluorescent dye or
TRAIL, and said at least one active agent bound to the outer
surface of the magnetic metal oxide is TRAIL.
[0085] In another aspect, the present invention provides a
pharmaceutical composition comprising magnetic polymer/metal oxide
composite nanoparticles as defined above and a pharmaceutically
acceptable carrier.
[0086] The pharmaceutical composition of the present invention may
be used for various biological, medical and therapeutical
applications.
[0087] In one embodiment, the pharmaceutical composition of the
present application is used for diagnostics, drug stabilization,
drug delivery and controlled release of drugs.
[0088] In one embodiment, the pharmaceutical composition of the
present invention comprises magnetic polymer/metal oxide composite
nanoparticles as defined above, preferably gelatin/iron oxide
composite nanoparticle, wherein the at least one active agent
covalently bound to the polymer is a fluorescent dye. This
pharmaceutical composition may be used for tumor detection.
[0089] In one embodiment, the pharmaceutical composition of the
present invention comprises magnetic polymer/metal oxide composite
nanoparticles as defined above, preferably gelatin/iron oxide
composite nanoparticles, wherein the at least one active agent
covalently bound to the polymer is a contrast agent. This
pharmaceutical composition may be used for for X-ray imaging or
magnetic resonance imaging (MRI). The present invention thus
relates to a method for X-ray imaging or magnetic resonance imaging
(MRI) comprising administering to an individual in need said
pharmaceutical composition.
[0090] In a preferred embodiment, this composition may further
comprise a molecule capable of binding a tumor specific cellular
marker bound to the outer surface of the magnetic metal oxide. This
composition may be used for tumor detection. The present invention
thus relates to a method for detection of a tumor comprising
administering to an individual in need the pharmaceutical
composition defined above.
[0091] The term "molecule capable of binding a tumor specific
cellular marker" as used herein refers to antibodies or fragments
thereof directed to tumor associated antigens; receptors or
fragments thereof specific for tumor associated ligands; or ligands
of tumor associated receptors.
[0092] In a further embodiment, the pharmaceutical composition of
the present invention comprises magnetic polymer/metal oxide
composite nanoparticles as defined above, preferably gelatin/iron
oxide composite nanoparticles, wherein the at least one active
agent covalently bound to the polymer and said at least one active
agent bound to the outer surface of the magnetic metal oxide, the
same or different, are selected from TNF-.alpha., TNF-.beta., a
TNF-related cytokine selected from TNF-related apoptosis-inducing
ligand (TRAIL), TALL-1, the TNF-like ligand TL1A, lymphotoxin-beta
(LT-.beta.), a CD30 ligand, a CD27 ligand, a CD40 ligand, an OX40
ligand, a 4-1BB ligand, an Apo-1 ligand, or an Apo-3 ligand, an
interleukin having an anti-tumor activity selected from IL-12,
IL-23 or IL-27, a cRGD peptide, preferably the cRGD peptide of SEQ
ID NO:1, a cRGD peptidomimetic, an RGD containing peptide or
peptidomimetic, an antibody, preferably avastin, an anthracycline
chemotherapeutic agent selected from daunorubicin, doxorubicin,
epirubicin, idarubicin or mitoxantrone, an antifolate drug, or a
combination thereof. In a preferred embodiment, said at least one
active agent covalently bound to the polymer is selected from
TRAIL, doxorubicin, methotrexate or a combination thereof, and said
at least one active agent bound to the outer surface of the
magnetic metal oxide is selected from TRAIL, the cRGD peptide of
SEQ ID NO: 1, IL-12, or a combination thereof. This pharmaceutical
composition may be used for reducing or inhibiting the growth of a
tumor, or for reducing or inhibiting the growth of tumor cells
remaining at a site in a patient from which a tumor has been
surgically removed. The present invention thus further relates to a
method for reducing or inhibiting the growth of a tumor or for
reducing or inhibiting the growth of tumor cells left at a site in
a patient from which a tumor has been surgically removed,
comprising administering to said patient said pharmaceutical
composition.
[0093] In still a further embodiment, the pharmaceutical
composition of the present invention comprises magnetic
polymer/metal oxide composite nanoparticles as defined above,
preferably gelatin/iron oxide composite nanoparticles, wherein the
at least one active agent covalently bound to the polymer is a
fluorescent dye or a contrast agent, and said at least one active
agent bound to the outer surface of the magnetic metal oxide is
selected from TNF-.alpha., TNF-.beta., a TNF-related cytokine
selected from TNF-related apoptosis-inducing ligand (TRAIL),
TALL-1, the TNF-like ligand TL1A, lymphotoxin-beta (LT-.beta.), a
CD30 ligand, a CD27 ligand, a CD40 ligand, an OX40 ligand, a 4-1BB
ligand, an Apo-1 ligand, or an Apo-3 ligand, an interleukin having
an anti-tumor activity selected from IL-12, IL-23 or IL-27, a cRGD
peptide, preferably the cRGD peptide of SEQ ID NO: 1, a cRGD
peptidomimetic, an RGD containing peptide or peptidomimetic, an
antibody, preferably avastin, an anthracycline chemotherapeutic
agent selected from daunorubicin, doxorubicin, epirubicin,
idarubicin or mitoxantrone, an antifolate drug, or a combination
thereof. In a preferred embodiment, said at least one active agent
bound to the outer surface of the magnetic metal oxide is selected
from TRAIL, the cRGD peptide of SEQ ID NO: 1, IL-12, or a
combination thereof. This pharmaceutical composition may be used
for reducing or inhibiting the growth of a tumor and monitoring the
size thereof. The present invention thus further relates to a
method for reducing or inhibiting the growth of a tumor and
monitoring the size thereof in a patient comprising administering
to said patient said pharmaceutical composition.
[0094] The term "tumor" as used herein refers to any tumor such as,
without being limited to, brain tumors, preferably glioma, colon
cancer, lung cancer, breast cancer, prostate cancer, bladder
cancer, kidney cancer, ovarioan cancer, melanoma, leukemia or
multiple myeloma, preferably glioma, and metastases thereof. In a
preferred embodiment, the tumor is glioma.
[0095] Brain tumors, in particular, malignant gliomas, belong to
the most aggressive human cancers. Patients with malignant glioma
have a poor prognosis because these brain tumors respond poorly to
radiation or chemotherapy, the conventional treatments of cancer
(Wei et al., 2006). Features responsible for the aggressive
character of glioma include rapid proliferation, diffuse growth and
invasion into distant brain areas in addition to extensive cerebral
edema and high levels of angiogenesis. Patients with malignant
gliomas posses a median survival of less than one year, wherein
there are only occasional long-term survivors. The difficulty in
differentiating tumor and normal brain tissue, and the unusual
ability of gliomas to infiltrate the brain pose a serious challenge
in glioma therapy and diagnosis (Giese et al., 2003), and it is
currently not expected that further advances in neurosurgery,
radiation therapy or chemotherapy will significantly improve the
prognosis of these patients (Desjardins et al., 2005).
[0096] Furthermore, in modern clinical neurooncology,
histopathological diagnosis affects therapeutic decisions and
prognostic estimation more than any other parameter. Unfortunately,
the extensive heterogeneity of astrocytic tumors has made their
pathological classification rather difficult (Sanson et al., 2004).
Currently there are no specific markers for glioblastomas and the
diagnosis of these patients is determined by histological
evaluation of tumor samples. Moreover, no markers are available for
predicting the progression of low-grade astrocytomas to
glioblastomas. Thus, the identification of glioma-specific markers
can assist in the diagnosis of brain tumors and in the prediction
of the prognosis and tumor progression of low-grade
astrocytomas.
[0097] Malignant gliomas, including the anaplastic astrocytoma and
glioblastoma multiform, are the most common primary brain tumors.
Current treatment options include surgery, radiation therapy and
chemotherapy; however, prognosis remains extremely poor and the
development of alternative therapeutic approaches is thus highly
desirable. Gene therapy has been considered as an innovative
therapeutic approach for malignant gliomas, and in the last decade
there has been a great interest in the development of delivery
systems that will allow the expression of exogenous genes in the
central nervous system. For this purpose, plasmid or vector
encoding tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL), incorporated into cationic albumin nanoparticles,
liposomes and replication-deficient herpes simplex virus (HSV),
have been developed (Shah et al., 2003; Van Meir and Bellail, 2004;
Carlo-Stella et al., 2007; Wei Lu et al., 2006; Zhang et al.,
2002). However, no convincing clinical trial has emerged, providing
objective proof of the superiority of gene therapy strategy as
compared to conventional treatment. An alternative approach to gene
therapy includes the direct delivery of TRAIL into the tumors. This
approach, however, presents a challenge due to the limited
stability of the TRAIL as well as its low bioavailability,
inefficiency in crossing cell membranes and poor in vivo metabolic
stability (Denicourt and Dowdy, 2004; Shir and Levitzki, 2001; Shah
et al., 2003; WO 2004/045494).
[0098] Thus, in a preferred embodiment, the tumor is glioma.
[0099] The pharmaceutical composition of the present invention,
when aimed for reducing or inhibiting the growth of a tumor, either
with or without monitoring the size thereof, or for reducing or
inhibiting-the growth of tumor cells left at a site in a patient
from which a tumor has been surgically removed, may be be used in
combination with radiotherapy. In view of that, the various methods
defined above for (i) reducing or inhibiting the growth of a tumor;
(ii) reducing or inhibiting the growth of tumor cells left at a
site in a patient from which a tumor has been surgically removed;
or (iii) reducing or inhibiting the growth of a tumor and
monitoring the size thereof, may be performed in combination with
radiotherapy.
[0100] Since it is known that iron oxide is attracted to sites of
inflammation, in yet a further embodiment, the pharmaceutical
composition of the present invention comprises magnetic
polymer/metal oxide composite nanoparticles as defined above,
wherein said magnetic metal oxide is iron oxide, said at least one
active agent covalently bound to the polymer is a fluorescent dye
or a contrast agent and said at least one active agent bound to the
outer surface of the magnetic metal oxide is an anti-inflammatory
agent. This pharmaceutical composition may be used for detection of
a site of inflammation and treatment of said inflammation in an
individual. The present invention thus relates to a method for
detection of a site of inflammation and treatment of said
inflammation comprising administering to an individual in need said
pharmaceutical composition.
[0101] In still another embodiment, the pharmaceutical composition
of the present invention comprises magnetic polymer/metal oxide
composite nanoparticles as defined above, preferably gelatin/iron
oxide composite nanoparticles, wherein said at least one active
agent covalently bound to the polymer and said at least one active
agent bound to the outer surface of the magnetic metal oxide, each
independently, is insulin. This pharmaceutical composition may be
used for treatment of type 2 diabetes. The present invention thus
relates to a method for treatment of type 2 diabetes comprising
administering to an individual in need said pharmaceutical
composition.
[0102] In yet another embodiment, the pharmaceutical composition of
the present invention comprises magnetic polymer/metal oxide
composite nanoparticles as defined above, preferably gelatin/iron
oxide composite nanoparticles, wherein said at least one active
agent covalently bound to the polymer and said at least one active
agent bound to the outer surface of the magnetic metal oxide, each
independently, is obestatin. This pharmaceutical composition may be
used for treatment of obesity. The present invention thus relates
to a method for treatment of obesity comprising administering to an
individual in need said pharmaceutical composition.
[0103] In a further embodiment, the pharmaceutical composition of
the present invention comprises magnetic polymer/metal
oxide-composite nanoparticles as defined above, preferably
gelatin/iron oxide composite nanoparticles, wherein said at least
one active agent covalently bound to the polymer and said at least
one active agent bound to the outer surface of the magnetic metal
oxide, each independently, is ghrelin. This pharmaceutical
composition may be used for treatment of anorexia. The present
invention thus relates to a method for treatment of anorexia
comprising administering to an individual in need said
pharmaceutical composition.
[0104] In a further aspect, the present invention relates to a
method for evaluating responsiveness of tumor cells to treatment
with a candidate compound, which comprises contacting cells from a
biopsy taken from said tumor with magnetic polymer/metal oxide
composite nanoparticles as defined above, preferably gelatin/iron
oxide composite nanoparticles, and monitoring the viability of the
tumor cells, wherein the active agent bound to the outer surface of
the magnetic metal oxide in the nanoparticles is the candidate
compound to be evaluated and is selected from TNF-.alpha.,
TNF-.beta., a TNF-related cytokine selected from TNF-related
apoptosis-inducing ligand (TRAIL), TALL-1, the TNF-like ligand
TL1A, lymphotoxin-beta (LT-.beta.), a CD30 ligand, a CD27 ligand, a
CD40 ligand, an OX40 ligand, a 4-1BB ligand, an Apo-1 ligand, or an
Apo-3 ligand, an interleukin having an anti-tumor activity selected
from IL-12, IL-23 or IL-27, a cRGD peptide, preferably the cRGD
peptide of SEQ ID NO: 1, a cRGD peptidomimetic, an RGD containing
peptide or peptidomimetic, an antibody, preferably avastin, an
anthracycline chemotherapeutic agent selected from daunorubicin,
doxorubicin, epirubicin, idarubicin or mitoxantrone, an antifolate
drug, or a combination thereof, and the nanoparticles comprise a
fluorescent dye or a contrast agent covalently bound to the
polymer.
[0105] In another aspect, the present invention provides a
nanoparticle consisting of a polymer which is a metal chelating
agent coated with a magnetic metal oxide, wherein at least one
agent having an anti-tumor activity selected from a peptide, A
peptidomimetic, a polypeptide or a small molecule is bound to the
outer surface of the magnetic metal oxide.
[0106] The various definitions with respect to the size of the
nanoparticles having an anti-tumor agent bound exclusively to their
outer surface, as well as to the metal chelating polymer and the
magnetic metal oxide are identical to those defined with respect to
the magnetic nanoparticles defined above, in which at least one
active agent is covalently bound to the polymer. Furthermore, and
as defined with respect to the magnetic nanoparticles, the agent
having an anti-tumor activity may be either covalently or
physically bound to the outer surface of the magnetic metal
oxide.
[0107] As defined above, the agent having anti-tumor activity may
be a peptide, a peptidomimetic, a polypeptide or a small
molecule.
[0108] In one embodiment, the agent having anti-tumor activity is a
peptide or peptidomimetic such as a cRGD peptide or peptidomimetic,
or an acyclic RGD-containing peptide or peptidomimetic. In a
preferred embodiment, the cRGD peptide is the cRGD peptide of SEQ
ID NO: 1.
[0109] In a further embodiment, the agent having anti-tumor
activity is a polypeptide such as a cytokine, for example,
TNF-.alpha., TNF-.beta.; a TNF-related cytokine or an interleukin.
Non-limiting examples of TNF-related cytokines include TNF-related
apoptosis-inducing ligand (TRAIL), TALL-1, the TNF-like ligand
TL1A, lymphotoxin-beta (LT-.beta.), CD30 ligands, CD27 ligands,
CD40 ligands, OX40 ligands, 4-1BB ligands, Apo-1 ligands, and Apo-3
ligands. In preferred embodiment, the cytokine is TRAIL, IL-12,
IL-23 or IL-27, more preferably TRAIL.
[0110] In yet another embodiment, the agent having anti-tumor
activity is a small molecule. Non-limiting examples of small
molecules include anthracycline chemotherapeutic agents and
antifolate drugs, as defined above, preferably doxorubicin and
methotrexate, respectively.
[0111] In one embodiment, the present invention provides
nanoparticles having an anti-tumor agent bound exclusively to their
outer surface, wherein said peptide or peptidomimetic is a cRGD
peptide or peptidomimetic, preferably the cRGD peptide of SEQ ID
NO: 1, or an acyclic RGD-containing peptide or peptidomimetic; said
polypeptide is a cytokine selected from TNF-.alpha., TNF-.beta., a
TNF-related cytokine selected from TNF-related apoptosis-inducing
ligand (TRAIL), TNF-.alpha., TNF-.beta., TALL-1, the TNF-like
ligand TL1A, lymphotoxin-beta (LT-.beta.), a CD30 ligand, a CD27
ligand, a CD40 ligand, an OX40 ligand, a 4-1BB ligand, an Apo-1
ligand, or an Apo-3 ligand, preferably TRAIL, an interleukin (IL),
preferably IL-12, IL-23 or IL-27, or an antibody, preferably
avastin; said small molecule is an antifolate drug selected from
methotrexate or an anthracycline chemotherapeutic' agent selected
from daunorubicin, doxorubicin, epirubicin, idarubicin and
mitoxantrone, preferably doxorubicin; or a combination thereof. In
a preferred embodiment, said polymer is gelatin, said magnetic
metal oxide is iron oxide, and said at least one agent having an
anti-tumor activity is a TNF or a TNF-related cytokine, an
anthracycline chemotherapeutic agent, an antifolate drug or a
combination thereof. In a most preferred embodiment, said at least
one agent having anti-tumor activity is TRAIL.
[0112] In still another aspect, the present invention provides a
pharmaceutical composition comprising nanoparticles having an
anti-tumor agent bound exclusively to their outer surface as
defined above and a pharmaceutically acceptable carrier, for use in
reducing or inhibiting the growth of a tumor.
[0113] In a preferred embodiment, this pharmaceutical composition
comprising nanoparticles having an anti-tumor agent bound
exclusively to their outer surface, wherein the at least one agent
having an anti-tumor activity is TRAIL, the cRGD peptide of SEQ ID
NO: 1, IL-12, doxorubicin, methotrexate or a combination
thereof.
[0114] The nanoparticles having an anti-tumor agent bound
exclusively to their outer surface, as defined above, may be used
for reducing or inhibiting the growth of a tumor or for reducing or
inhibiting the growth of tumor cells left at a site in a patient
from which a tumor has been surgically removed. The present
invention thus further relates to a method for reducing or
inhibiting the growth of a tumor or for reducing or inhibiting the
growth of tumor cells left at a site in a patient from which a
tumor has been surgically removed, comprising administering to said
patient the aforesaid pharmaceutical composition. As explained
above, these nanoparticles may be used in combination with
radiotherapy. Therefore, this method may be performed in
combination with radiotherapy.
[0115] In yet another aspect, the present invention. relates to a
method for evaluating responsiveness of tumor cells to treatment
with a candidate compound, which comprises contacting cells from a
biopsy taken from said tumor with nanoparticles having, an
anti-tumor agent bound exclusively to their outer surface, as
defined above, preferably gelatin/iron oxide composite
nanoparticles having an anti-tumor agent bound exclusively to their
outer surface, and monitoring the viability of the tumor cells,
wherein the antitumor agent bound to the outer surface of the
magnetic metal oxide of the nanoparticles is the candidate compound
to be evaluated.
[0116] The pharmaceutical composition provided by the present
invention may be prepared by conventional techniques, e.g., as
described in Remington: The Science and Practice of Pharmacy, 19th
Ed., 1995. The composition may be in solid, semisolid or liquid
form and may further include pharmaceutically acceptable fillers,
carriers or diluents, and other inert ingredients and excipients.
Furthermore, the pharmaceutical composition can be designed for a
slow release of the nanoparticles. The composition can be
administered by any suitable route, e.g. intravenously, orally,
parenterally, rectally, transdermally or topically. The dosage will
depend on the state of the patient, and will be determined as
deemed appropriate by the practitioner.
[0117] The route of administration may be any route which
effectively transports the active compound to the appropriate or
desired site of action, the intravenous route being preferred. If a
solid carrier is used for oral administration, the preparation may
be tabletted, placed in a hard gelatin capsule in powder or pellet
form or it can be in the form of a lozenge. If a liquid carrier is
used, the preparation may be in the form of a syrup, emulsion or
soft gelatin capsule. Tablets, dragees or capsules having talc
and/or a carbohydrate carrier or binder or the like are
particularly suitable for oral application. Preferable carriers for
tablets, dragees or capsules include lactose, corn starch and/or
potato starch.
[0118] As stated above, malignant gliomas are the most common
primary intracranial tumors in patients. The prognosis of these
tumors is poor because they poorly respond to radiation or
chemotherapy. The difficulty in differentiating tumor and normal
brain tissue, and the unusual ability of gliomas to infiltrate the
brain pose a serious challenge in glioma therapy and diagnosis.
[0119] TRAIL is a transmembrane protein initially expressed at the
cell membrane and subsequently derived by proteolytic processing.
Soluble TRAIL (sTRAIL) can induce apoptosis in tumor cells of
diverse origins while sparing most normal cells. However, while
most previous studies have been performed in cell culture, the
delivery and efficiency of sTRAIL in vivo is significantly less
established and successful. The major hindrance for in vivo sTRAIL
treatment is due to its short half-life due to proteolytic
cleavage, which therefore requires excess amount of sTRAIL. In vivo
sTRAIL treatment is particularly inefficient for glioma tumors due
to difficulties in the administration of TRAIL to the brain and to
the relative insensitivity of gliomas to TRAIL in vivo.
[0120] Examples 6-8 hereinafter describe in detail various in vitro
and in vivo experiments conducted with gelatin/iron oxide magnetic
composite nanoparticles of the present invention, both when at
least one active agent was covalently bound to the polymer inside
the nanoparticles and, optionally, the same or different active
agent was bound to the outer surface of the magnetic metal oxide;
as well as when no active agent was covalently bound to the
polymer.
[0121] As shown in these Examples, the conjugation of TRAIL both to
fluorescent and to non-fluorescent gelatin/iron oxide composite
nanoparticles significantly stabilized the sTRAIL, minimizing
enzymatic degradation of the sTRAIL and thereby decreasing the
amount of sTRAIL essential for apoptosis of tumor cells. This
approach allows the selective delivery of sTRAIL to tumor cells for
efficient apoptosis. Furthermore, the sTRAIL-conjugated
nanoparticles selectively tracked infiltrating tumor cells, exerted
cytotoxic effects in vivo, and significantly increased the survival
of tumor-bearing animals. Tumor cells which were resistant to the
cytotoxic effect of the sTRAIL-conjugated nanoparticles were
sensitized by using a combined treatment of low-level of
.gamma.-irradiation followed by treatment with the
sTRAIL-conjugated nanoparticles. Alternatively, these cells were
efficiently treated with nanoparticles to which more than one
active agent was conjugated. The active agents used for this
purpose were sTRAIL, and other cancer drug(s) such as a cRGD
peptide, IL-12, a cRGD peptide and adriamycin. These agents, in
different combinations, were bound either to the magnetic metal
oxide surface, to the polymer inside the nanoparticle, or to
both.
[0122] In addition to the therapeutic application, these
sTRAIL-conjugated nanoparticles served as a marker for tumor
imaging by MRI and/or fluorescence. By using these imaging
modalities in animal models implanted with human glioma tumors, it
was demonstrated that the sTRAIL-conjugated nanoparticles migrate
to the site of the tumors and accumulate around and within the
tumors, while the nanoparticles without bound sTRAIL migrate slower
and accumulate only at the periphery of the tumor cells. The
nanoparticles to which the sTRAIL was conjugated are used as a
vehicle for stabilizing the sTRAIL, diagnosis of the tumor and
assisting in targeting the tumors for inducing apoptosis. By using
nanoparticles in which a fluorescent dye was bound to the polymer,
it was further shown that the sTRAIL-conjugated nanoparticles
identify and target infiltrating tumor cells. The ability of the
sTRAIL-conjugated nanoparticles to specifically target glioma cells
can be also employed for determining the border of the tumors in
the brain and for distinguishing between recurrent gliomas and
radiation-induced necrosis. In addition, the efficiency of the
sTRAIL-conjugated nanoparticles for targeting and inducing
apoptosis of tumor cells other then gliomas, e.g. carcinoma, breast
cancer and lung cancer, was demonstrated.
[0123] The invention will now be illustrated by the following
non-limiting Examples.
EXAMPLES
Example 1
Synthesis and Characterization of Gelatin/Iron-Oxide Magnetic
Composite Nanoparticles
[0124] Gelatin/iron oxide magnetic composite nanoparticles of sizes
ranging from ca. 5 nm up to 100 nm with narrow size distribution
were prepared by nucleation followed by controlled growth of
magnetic iron oxide thin films onto gelatin/iron oxide nuclei, as
described in detail in WO 99/062079. The nucleation step was based
on complexation of Fe.sup.+2 ions to chelating sites of the
gelatin, followed by partial oxidation (up to approximately 50%) of
the chelated Fe.sup.+2 to Fe.sup.+3, so that the water soluble
gelatin contained both chelated Fe.sup.+2 and Fe.sup.+3 ions.
Gelatin/iron oxide nuclei were than formed by adding NaOH or,
alternatively, ammonia aqueous solution up to ca. pH 9.5. The
growth of magnetic films onto the gelatin nuclei accomplished by
repeating several times the nucleation step.
[0125] Briefly, nanoparticles of 15 nm average dry diameter were
prepared by adding FeCl.sub.2 solution (10 mmol/5 ml H.sub.2O) to
80 ml aqueous solution containing 200 mg gelatin (Sigma), followed
by NaNO.sub.2 solution (7 mmol/5 ml H.sub.2O). After a reaction
time of 10 min, NaOH aqueous solution (1 N) was added up to pH 9.5.
This procedure was repeated four times, or more, if larger
particles were required. The formed magnetic nanoparticles were
then washed from excess reagents using magnetic gradient columns.
Surface analysis demonstrated the present of gelatin both within
and on the surface of the nanoparticle. The surface gelatin
provides additional stabilization against agglomeration to the
nanoparticle and functional groups such as carboxilate and primary
amines through which appropriate ligands can be covalently bonded.
FIGS. 1A-1D demonstrate a transmission electron microscopy (TEM)
picture of magnetic nanopaticles of increased average diameter
formed by repeating the thin magnetic coating process during the
growth step, 4 to 7 times, respectively. FIG. 2 shows that magnetic
nanoparticles of 15 nm dry average diameter, prepared as described
hereinabove and dispersed in water, posses one narrow population
with hydrodynamic average diameter of ca. 100 nm. FIGS. 3A-3B show
high resolution TEM (HTEM) picture (3A), demonstrating crystalline
structure with d-spacing of 0.479 nm, and electron diffraction (ED)
picture (3B), representing sharp rings indicating the crystalline
character of the magnetic nanoparticles. FIG. 4 shows X-ray
diffraction (XRD) pattern of the gelatin/iron oxide magnetic
composite nanoparticles, indicating that the crystalline cores of
these nanoparticles consist nearly completely of maghemite
(.gamma.-Fe.sub.2O.sub.3). From x-ray line broadening, one deduces
a mean diameter of the magnetic cores of 15 nm. FIG. 5 shows
mossbauer spectrum of the gelatin/iron oxide magnetic composite
nanoparticles, further indicating that these nanoparticles consist
of maghemite. It is assumed that magnetite (Fe.sub.3O.sub.4)
nanoparticles were first produced by this nucleation and growth
process, and were then oxidized to the more thermodynamic stable
iron oxide, maghemite. FIG. 6 represents hysteresis loop at room
temperature of the maghemite nanoparticles of 15 nm dry diameter,
indicating that the M(H) curve of these nanoparticles does not
exhibit any coercivity and does not saturate at 10,000 Oe, while
the magnetic moment obtained at 10,000 Oe is ca. 41 emu g.sup.-1.
Both features are typical of superparamagnetic behavior.
[0126] Uniform gelatin/iron oxide magnetic composite nanoparticles
of various sizes and properties were prepared by changing
preparation conditions, e.g., oxidizing reagents, iron salt type,
pH and temperature, as previously disclosed in WO 99/062079.
Uniform nanoparticles of sizes smaller than 15 nm dry diameter down
to 5 nm were prepared by gradual surface dissolution of the 15 nm
nanoparticles with acids, e.g. HCl at pH ca. 1.0, or iron chelating
ligands such as EDTA and oxalic acid. After achieving the desired
diameter, the magnetic nanoparticles were wash into distilled
water.
Example 2
Synthesis of Fluorescent Dye-Labeled Gelatin/Iron Oxide Magnetic
Composite Nanoparticles
[0127] Fluorescent dye-labeled gelatin/iron oxide magnetic
composite nanoparticles in which the fluorescent dye is mainly
entrapped within the magnetic composite nanoparticles were prepared
as described in Example 1, substituting the gelatin for gelatin
covalently bound to a fluorescent dye, as illustrated in FIG. 7.
For example, rhodamine-labeled nanoparticles were prepared by
adding slowly 0.5 ml dimethyl sulfoxide (DMSO) containing 5 mg of
rhodamine isothiocyanate (RITC) to 20 ml aqueous solution
containing 200 mg gelatin. The pH of the aqueous solution was
raised to 9.5 by adding NaOH aqueous solution (1 N), and the
solution was shaken for 1 h at 60.degree. C. This process involves
the covalent binding, via thiourea and/or thiourethane bonds,
between the part of the hydroxyl and amine groups of the gelatin
and the isothiocyanate of the RITC. Excess of RITC was then removed
from the gelatin conjugated rhodamine chains by extensive dialysis
(cut off: 12-14000) of the former aqueous solution at 60.degree. C.
against H.sub.2O, or by washing through a magnetic column. The
volume of the solution was adjusted to 80 ml and the synthesis was
then continued as described in Example 1.
[0128] A similar process was performed with other appropriate dyes,
e.g. fluorescein.
Example 3
Synthesis of Drug(s) Containing Gelatin/Iron Oxide Magnetic
Composite Nanoparticles
[0129] Drug containing gelatin/iron oxide magnetic composite
nanoparticles were prepared as described in Example 1, substituting
the gelatin for gelatin covalently bound to the drug. For example,
adriamycin (Aldrich) was covalently bound to the gelatin via the
carbodiimide activation method, as described by Melamed and Margel
(2001). Briefly, 123 mg NHS (N-hydroxysuccinimide, Sigma) and 82 mg
CDC (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide
metho-p-toluenesulfonate, Sigma) were added to 20 ml MES buffer
(0.01 m at pH 5.0, Sigma) containing 200 mg gelatin and 5 mg
adriamycin, and the solution was then shaken at 60.degree. C. for 2
h. The solution was washed from excess reagent by dialysis (cut
off: 12-14000) against water. The volume of the solution was
adjusted to 80 ml and the synthesis was then continued as described
in Example 1.
[0130] In a similar process, gelatin/iron oxide magnetic composite
nanoparticles which methotrexate or/and sTRAIL, separately or in
combination, was/were covalently bound to the gelatin, were
prepared.
Example 4
Functionalization of Gelatin/Iron Oxide Magnetic Composite
Nanoparticles
[0131] Various ways for generating functional groups on the surface
of the gelatin/iron oxide magnetic composite nanoparticles prepared
as described in Examples 1-3 have been developed, based on
different principles such as (i) the high affinity of a coating
polymer, e.g., dextran, to the nanoparticle surface; (ii) covalent
binding between functional groups on the nanoparticles surface and
a desired functional ligand, e.g., amino ligands. such as proteins
with activated double bonds on the nanoparticle surface via the
Michael addition reaction or to carboxylate groups (belonging to
the gelatin coating) via the carbodiimide activation method, as
described by Melamed and Margel (2001); and (iii) precipitation of
a coating polymer, e.g., gelatin or albumin, on the nanoparticle
surface. The functional groups on the nanoparticle surface were
then used for covalent or physical binding binding of the various
drugs, e.g. TRAIL and cRGD peptide via different activation
methods.
4(i) Functionalization with Activated Double Bonds
[0132] Divinyl sulfone (DVS) was added to the gelatin/iron oxide
magnetic composite nanoparticles prepared as described in Examples
1-3, wherein the initial [DVS]/[nanoparticles] weight ratio was
4/1, and triethylamine was then added to reach a pH of 10.5,
followed by incubation at 60.degree. C. overnight. The formed
functionalized gelatin/iron oxide-DVS or fluorescent dye-labeled
gelatin/iron oxide-DVS nanoparticles were then washed with 0.1 M
bicarbonate buffer (pH=8.3) by magnetic gradient columns, and
stored at 4.degree. C.
4(ii) Functionalization with Crosslinked Dextran Coating Containing
Activated Double Bonds
[0133] 2% (w/v) dextran (MW 48,000, Sigma) was added to an aqueous
dispersion of gelatin/iron oxide magnetic composite nanoparticles
prepared as described in Examples 1-3, and after dissolution of the
dextran, the aqueous dispersion was shaken for 3 h at 85.degree. C.
The dextran coated nanoparticles were washed from excess dextran
with water by magnetic columns. Crosslinking of the dextran coating
was performed with DVS as described in 4(i) hereinabobe, to prevent
leakage of the physical adsorbed dextran into the aqueous
continuous phase and for generation of surface activated double
bonds.
4(iii) Functionalization with Gelatin or Albumin Coating in Absence
or Presence of Activated Double Bonds
[0134] 0.2% (w/v) gelatin was added to an aqueous dispersion of
gelatin/iron oxide magnetic composite nanoparticles prepared as
described in Examples 1-3, and the aqueous dispersion was then
shaken for 3 h at 85.degree. C. The aqueous dispersion was cooled
to room temperature, and the gelatin-coated nanoparticles were then
washed. by magnetic columns. DVS derivatization was performed, if
necessary, as described in 4(i) hereinabobe.
[0135] An albumin coating on the nanoparticles was performed
similarly, substituting the gelatin for bovine or human serum
albumin.
Example 5
Immobilization of Drugs onto the Functional Gelatin/Iron Oxide
Magnetic Composite Nanoparticles
[0136] 5(i) Immobilization of Human TRAIL or IL12 onto Gelatin/Iron
Oxide-DVS Magnetic Composite Nanoparticles 20 .mu.g of human TRAIL
(hTRAIL, CytoLab Ltd., Israel) was added to 1 ml bicarbonate buffer
dispersion (0.1. M, pH=8.3) containing 1 mg gelatin/iron oxide
magnetic composite nanoparticles, prepared as described in Example
4, and the dispersion was then mixed at room temperature for 60
min. This process involves the binding, via Michael addition, of
residual double bonds of the nanoparticles and primary amino groups
of the TRAIL. Blocking of residual activated double bonds was then
performed with glycine, by adding glycine (1% w/v) and continuing
mixing the dispersion for additional 60 min at room temperature.
Excess of unbound hTRAIL and glycine were removed with PBS (pH=7.4)
by magnetic gradient columns. The concentration of bound hTRAIL,
determined by ELISA Development Kit (900-K141 lot no. 11041410,
CytoLab Ltd., Israel), was found to be ca. 2000 ng/mg
nanoparticles.
[0137] IL12 was bound onto the gelatin/iron oxide-DVS magnetic
composite nanoparticles similarly, substituting the hTRAIL for
IL12.
5(ii) Immobilization of cRGD Peptide onto Gelatin/Iron Oxide-DVS
Magnetic Composite Nanoparticles
[0138] The experiment described in 5(i) hereinabove was repeated
substituting the 20 .mu.g of hTRAIL for 3 mg of the cRGD peptide of
the sequence cyclo (Arg-Gly-Asp-D-Phe-Lys (SEQ ID NO: 1) (Peptides
International, Louisville, Ky. 40224 USA). For negative control, a
sithilar process was performed substituting the cRGD peptide for
the cRAD peptide of the sequence cyclo (Arg-Ala-Asp-D-Phe-Lys,
Peptides International).
5(iii) Immobilization of hTRAIL and a cRGD Peptide onto
Gelatin/Iron Oxide DVS Magnetic Composite Nanoparticles
[0139] The experiment described in 5(i) hereinabove was repeated
substituting the glycine for 3 mg cRGD peptide, and as a
consequence, the remaining residual activated double bonds of the
DVS. were conjugated to the cRGD peptide.
5(iv) Immobilization of Bioactive Reagents, e.g., hTRAIL and/or
cRGD Peptide, onto Gelatin/Iron Oxide Composite Nanoparticles
[0140] Human TRAIL and/or cRGD peptide were covalently bound to the
gelatin/iron oxide composite nanoparticles prepared according to
Examples 1-3 and Example 4(iii) via the carbodiimide activation
method, as described in Melamed and Margel (2001). Briefly, 123 mg
NHS and 82 mg CDC were added to 20 ml MES buffer (0.01 M at pH 5.0,
Sigma) containing 200 mg nanoparticles, and the nanoparticles
dispersion was then shaken at room temperature for 3 h. The
activated nanoparticles were washed extensively by magnetic
gradiant columns with PBS, and 5 ml PBS solution containing 8 mg
hTRAIL or 50 mg cRGD peptide were then added to 20 ml of the washed
activated nanoparticles dispersion. The suspension was shaken at
room temperature for about 8 h, and blocking of residual activated
groups was then performed with either glycine (200 mg) or cRGD
peptide (50 mg) as described in 5(i) or 5(iii), respectively.
5(v) Immobilization of Avastin or Remicade onto Gelatin/Iron
Oxide-DVS Magnetic Composite Nanoparticles 20 .mu.g of the
monoclonal antibodies Avastin (Bevacizumab, Genentech) or Remicade
(Infliximab) is added to 1 ml bicarbonate buffer dispersion (0.1 M,
pH=8.3) containing 1 mg gelatin/iron oxide magnetic composite
nanoparticles, prepared as described in Example 4, and the
dispersion is then mixed at room temperature for 60 min. This
process involves the binding, via Michael addition, of residual
double bonds of the nanoparticles and primary amino groups of the
Avastin or Remicade. Blocking of residual activated double bonds is
then performed with glycine, by adding glycine (1% w/v) and
continuing mixing the dispersion for additional 60 min at room
temperature. Excess of unbound Avastin or Remicade and glycine are
removed with PBS (pH=7.4) by magnetic gradient columns. 5(vi)
Physical Immobilization of Bioactive Reagents, e.g., hTRAIL, onto
Gelatin/Iron Oxide Magnetic Composite Nanoparticles
[0141] 20 .mu.g of human TRAIL (hTRAIL, CytoLab Ltd., Israel) was
added to 1 ml bicarbonate or PBS buffer dispersion (0.1 M, pH=8.3)
containing 1 mg non-functionalized gelatin/iron oxide magnetic
composite nanoparticles prepared as described in Example 3, or
albumin coated gelatin/iron oxide magnetic composite nanoparticles
prepared as described in example 4(iii). The dispersion was then
mixed at room temperature for 120 min, and as a consequence, the
hTRAIL was non-covalently bound the non-coated, or albumin coated,
nanoparticles. This physical binding is based on non-covalent
interactions, e.g., hydrophobic bonds, ionic interactions and
hydrogen bonds, between. the TRAIL and the nanoparticles. Excess of
unbound hTRAIL were removed with PBS (pH=7.4) by magnetic gradient
columns.
5(vii) Immobilization of Human TRAIL onto Gelatin/Iron Oxide
Magnetic Composite Nanoparticle
[0142] Human sTRAIL was bound directly to the gelatin/iron oxide
nanoparticles via the carbodiimide activation method, as described
by Melamed and Margel (2001). In a typical experiment, 123 mg NHS
and 82 mg CDC were added to 15 ml MES buffer (0.1M at pH 5.0)
containing 10 mg nanoparticles. The nanoparticles mixture was then
shaken at room temperature for ca. 3 h. The activated nanoparticles
were washed by magnetic gradient columns. PBS solution (2 ml)
containing 0.5 mg sTRAIL was then added to 8 ml of the washed
activated nanoparticles PBS dispersion. The dispersion was then
shaken at room temperature for ca. 2 h. Blocking of residual
activated double bonds was then performed with glycine or primary
amino polyethylene glycol, by adding glycine (1% w/v) (or primary
amino polyethylene glycol, 5 mg) and continuing mixing the
dispersion for additional 60 min at room temperature. Excess of
unbound hTRAIL and glycine (or amino polyethylene glycol) were
removed with PBS (pH=7.4) by magnetic gradient columns.
[0143] The concentrations of the bound bioactive agents described
in 5(i) to 5(vi) were controlled by changing binding parameters,
e.g. hTRAIL concentration.
Example 6
In Vitro Studies: Targeting, Imaging and Apoptosis of Glioma
Cells
Materials and Methods
[0144] Cell lines. All the cells used in the following experiments
were obtained from the American Tissue Culture Collection (ATCC) or
from the Hermelin Brain Tumor Center (Henry Ford Hospital, Detroit,
MI). All cells were cultured in DMEM supplemented with 10% FBS
(Hyclone, Logan, UT), 2 mM L-glutamine and 100 ug/ml
streptomycin-penicillin (Invitrogen) at 37.degree. C. under 5%
CO.sub.2.
[0145] Measurements of cell apoptosis. Cell apoptosis was measured
using propidium iodide (PI) staining and analyzed by flow cytometry
as described by Riccardi and Nicoletti (2006) as well as by ELISA
(Cell Death Detection ELISA Kit) using anti-histone antibodies as
described by Blass et al. (2002). Cells (10.sup.6/ml) were plated
in six-well plates at 37.degree. C. and treated by the indicated
treatments (addition of 10 .mu.l or less phosphate-buffered saline
(PBS) or PBS containing the peptide, e.g. TRAIL, or unbound or
peptide bound nanoparticles) for. 24 h. Detached cells and
trypsinized adherent cells were pooled, fixed in 70% ethanol for 1
h on ice, washed with PBS and treated for 15 min with RNase (50
.mu.M) at room temperature. Cells were then stained with PI (5
.mu.g/ml) and analyzed on a Becton-Dickinson cell sorter.
[0146] Cell viability was further quantitatively assessed by the
measurement of the cytoplasmatic enzyme lactate dehydrogenase (LDH)
released from dead cells to the medium (kit purchased from Sigma).
For this purpose, supernatants were collected from control and
treated cells, and following centrifugation (10 min 1,400.times.g),
supernatants were transferred to 96-well plates and LDH was
measured according to the manufacturer's instructions.
6(i) Stabilization of TRAIL by Conjugation to Gelatin/Iron Oxide
Magnetic Composite Nanoparticles
[0147] In order to examine the stability of the free and conjugated
TRAIL against degradation, 1 ml PBS containing either free TRAIL
(100 ng) or TRAIL-conjugated gelatin/iron oxide magnetic composite
nanoparticles (NP-TRAIL, 100 ng TRAIL) were incubated at 10.degree.
C., and the concentration of TRAIL was measured during 35 days.
FIG. 8 shows that the conjugation of TRAIL to the nanoparticles
stabilized the TRAIL so that the original concentration of the
conjugated TRAIL was maintained up to 35 days in 10.degree. C. In
contrast, the free TRAIL significantly degraded under these
conditions.
6(h) TRAIL-Conjugated Gelatin/Iron Oxide Magnetic Composite
Nanoparticles Induce Apoptosis in Human Glioma Cells and in Glioma
Spheres Established from Primary Tumors
[0148] Human glioma cells A172 were incubated with either TRAIL
(100 ng/ml) or NP-TRAIL (10 ng TRAIL/ml) for 5 hours. Cell
apoptosis was determined by propidium iodide staining and analyzed
by both flow cytometry (FACS) and the morphological appearance of
the bells. FIG. 9A shows that free TRAIL induced apoptosis of about
48% of the cells whereas the NP-TRAIL induced apoptosis of about
57% of the cells and the nanoparticles alone did not induce
significant cell apoptosis. In other words, the apoptosis activity
induced by the conjugated TRAIL was at least 10 times higher than
that induced by the free one.
[0149] The apoptotic effect of free TRAIL (100 ng/ml) and NP-TRAIL
(10 ng TRAIL/ml) was further tested using glioma spheroids derived
from three different tumors, i.e., HF2020, HF1254 and HF1308. These
cultures resemble more the original tumors as they maintain their
three dimensional structure and cell-cell interaction. As found,
glioma spheroids derived from HF2020, shown in FIG. 9B, and HF1308
underwent cells apoptosis in response to TRAIL or NP-TRAIL, whereas
HF 1254 underwent only a low degree of cell apoptosis. In
particular, as presented in FIG. 9B and FIG. 10, both TRAIL and
NP-TRAIL significantly increased the level of LDH in the HF2020 and
the HF 1308 glioma spheroids.
6(iii) The Cytotoxic Effect of Free TRAIL and TRAIL-Conjugated
Gelatin/Iron Oxide Magnetic Composite Nanoparticles in Various
Glioma Cell Lines and Glioma Primary Cultures
[0150] Glioma cells U87, A172 and U251, as well as primary cultures
of glioma cells HF1308, HF1254 and HF 1316, were incubated with
TRAIL (100 ng/ml), NP-TRAIL (10 ng TRAIL/ml) or gelatin/iron oxide
magnetic composite nanoparticles (NP) alone for 24 h. The cells
were then collected, stained with propidium iodide and analyzed for
the sub-G.sub.0 population (apoptotic cells) by FACS analysis. FIG.
11 shows that for all cases, the cytotoxic effect of NP alone did
not differ from the effect caused by PBS, and that NP-TRAIL
exhibited significantly higher cytotoxic activity than that of free
TRAIL.
6(iv) The Cytotoxic Effect of TRAIL-Conjugated Non Fluorescent and
Fluorescent Dye-Labeled Gelatin/Iron Oxide Magnetic Composite
Nanoparticles in Various Glioma Cells
[0151] Human glioma cells A172 cells were incubated for 5 h with
non-fluorescent nanoparticles (NP-Control), NP-TRAIL (10 ng
TRAIL/ml), rhodamine-labeled gelatin/iron oxide magnetic composite
nanoparticles (NPR) and TRAIL-conjugated rhodamine-labeled
gelatin/iron oxide magnetic composite nanoparticles (NPR-TRAIL, 10
ng TRAIL/ml). Apoptosis was determined using propidium iodide
staining and FACS analysis. As shown in FIG. 12, the apoptosis
induced by NPR-TRAIL (51%) was similar to that induced by NP-TRAIL
(45%). Similar results were observed also for other glioma cells,
e.g., U251 and U87. These results illustrate that the fluorescent
TRAIL-conjugated nanoparticles induce apoptosis of glioma cells
similar to their non-fluorescent counterparts and about 10 times
more than that of free TRAIL.
6(v) Specific Internalization of the Fluorescent TRAIL-Conjugated
Gelatin/Iron Oxide Magnetic Composite Nanoparticles into Glioma
Cells
[0152] The specificity of NP-TRAIL for glioma cells as compared to
normal astrocytes was examined with NPR-TRAIL. In particular, human
glioma cells A172 and normal human astrocytes were incubated with
NPR-TRAIL (10 ng TRAIL/ml) for 30 min and the cells were viewed and
photographed using confocal microscopy. As shown in FIG. 13,
NPR-TRAIL entered the A172 cells within 30 min of treatment and
accumulated in the ER/golgi region, whereas very weak fluorescent
was observed in the normal astrocytes. Similarly, NPR that were not
conjugated to TRAIL did not enter the cells in both cell types
(data not shown). These results indicate that NPR-TRAIL probably
entered the glioma cells via internalization following binding to
TRAIL receptors; and they may have important implications for the
ability of TRAIL-conjugated nanoparticles to deliver drugs into the
cells.
6(vi) Synergistic Effect of .gamma.-Irradiation And
TRAIL-Conjugated Gelatin/Iron Oxide Magnetic Composite
Nanoparticles
[0153] Although NP-TRAIL induced apoptosis in many glioma cell
lines, there were some glioma cells that were resistant to this
treatment. Thus, and as .gamma.-radiation has been reported to
increase the sensitivity of tumor cells to TRAIL by increasing the
expression of TRAIL receptors, the effect of combined treatment
with NP-TRAIL and .gamma.-radiation on glioma cell apoptosis was
examined. In particular, A172 and U251 cells that were sensitive to
TRAIL, as well as U87 cells that exhibited low response to TRAIL
and LN-18 cells that were resistant to TRAIL treatment, were
employed, and sub-optimal concentration of NP-TRAIL (5 ng TRAIL/ml)
and .gamma.-radiation (10 Gy) were used. Cells were treated with NP
alone, NP-TRAIL (5 ng. TRAIL/ml), .gamma.-radiation (10 Gy), or a
combination of NP-TRAIL (5 ng TRAIL/ml) and .gamma.-radiation (10
Gy). For the combined treatment, cells were first irradiated and
after 2 h were treated with NP alone or with NP-TRAIL. Cell
apoptosis was determined 24 h later. FIG. 14 shows that the
apoptosis induced by the combined treatment was significantly
increased and that the addition of low dosage of
.gamma.-irradiation overcame the resistance of some glioma cells to
NP-TRAIL.
6(vii) Crgd Peptide-Conjugated Gelatin/Iron Oxide Magnetic
Composite Nanoparticles Induce Autophagy in Glioma Cells
[0154] Another type of cell death induced in glioma cells is type
II cell death or autophagy, as described in Gozuacik and Kimchi
(2004). In order to characterize autophagy, cells were transfected
with LC3-GFP plasmid and the accumulation of autophagic
vacuolization was examined. As found, whereas in the control cells
IC3-GFP appeared throughout the cells, in autophagic cells
punctuate staining was observed. In view of that, the pattern of
LC3-GFP in the control cells treated with NP only and in the cells
treated with cRGD peptide-conjugated gelatin/iron oxide magnetic
composite nanoparticles (NP-cRGD) was examined. For this purpose,
U251 cells were transfected with LC3-GFP for 24 h, the cells were
treated with cRGD peptide (10 .mu.g/ml), NP only or NP-cRGD (ca. 4
.mu.g cRGD/ml) for additional 24 h, and the percentage of cells
with punctuated GFP staining was then calculated. As shown in FIG.
15, about 24 h, about 30% of the cRGD-treated cells and about 18%
of the NP-cRGD-treated cells exhibited an increased punctuated
pattern of LC3-GFP. Contrary to that, only 2-3% of the control or
NP-treated cells exhibited punctuated staining.
6(viii) Synergistic Effect of TRAIL and Crgd Peptide-Conjugated
Gelatin/Iron Oxide Magnetic Composite Nanoparticles
[0155] In this experiment, LN-18 cells that were resistant to TRAIL
treatment were incubated for 24 h with NP-TRAIL, TRAIL and cRGD
peptide-conjugated nanoparticles (prepared as described in Example
5(iii)) or adriamycin containing TRAIL and cRGD peptide-conjugated
nanoparticles (prepared as described in Examples 3 and 5(iii)). The
synergistic effect of the additional drugs (cRGD peptide and
adriamycin) was demonstrated by significant increased in the
apoptosis percent, which was about 8% for the NP-TRAIL, about 25%
for the TRAIL and cRGD peptide-conjugated nanoparticles, and 45%
for the adriamycin containing TRAIL and cRGD peptide-conjugated
nanoparticles.
6(ix) Effect of TRAIL-Conjugated Conjugated Gelatin/Iron Oxide
Magnetic Composite Nanoparticles on Apoptosis of Various Tumor
Cells
[0156] In this experiment, the effect NP-TRAIL on tumor cells other
than glioma was examined, using the cervical carcinoma cell line
HeLa, the breast cancer cell line MCF-7 and the lung cancer cells
A549. FIG. 16 shows the apoptotic effect of TRAIL (100 ng/ml),
NP-TRAIL (50 ng TRAIL/ml), NP alone or PBS (control) after
incubation time of 5 h. As shown, whereas both PBS and NP had
insignificant apoptotic effect, NP-TRAIL induced a significant
apoptosis that was at least twice of that induced by TRAIL,
indicating that conjugation of TRAIL to the nanoparticles
maintained its apoptotic effect and even increased it towards
various cancer cells. Cell apoptosis was measured using propidium
iodide (PI) staining and analyzed by flow cytometry.
6(x) Synergistic Effect of .gamma.-Irradiation and TRAIL-Conjugated
Gelatin/Iron Oxide Magnetic Composite Nanoparticles on Human Glioma
Stem Cell Spheres
[0157] Glioma stem cells were established from the tumor specimens
2355 and 2303, were grown as spheroids and maintained in culture
for two months. The glioma stem cells exhibited self-renewal and
differentiated on poly-L-Lysine to astrocytes, neurons and
oligodendrocytes. In order to examine the effect of NP and of
NP-TRAIL on the cells, sphroids were placed in a 6-well plate and
were treated with TRAIL (100 ng/ml), NP alone or NP-TRAIL (50 ng
TRAIL/ml) for 24 h. The supernatants were then collected and LDH
analysis was performed.
[0158] In order to evaluate the combined effect of
.gamma.-radiation and TRAIL, the cells were first irradiated with 5
Gy radiation and after 4 hours were treated with either TRAIL or
NP-TRAIL for additional 24 h. Cell death was determined using LDH
levels in culture supernatants. The apoptotic effect induced by the
different treatments was similar in both glioma stem cell lines and
illustrated in FIGS. 17A-17B. Insignificant apoptotic effect was
observed for NP only, .gamma.-irradiation alone or free TRAIL.
NP-TRAIL induced moderate apoptotic effect; however, both NP-TRAIL
and free TRAIL, together with .gamma.-irradiation, induced
significant apoptosis.
6(xi) IL-12-Conjugated Gelatin/Iron Oxide Magnetic Composite
Nanoparticles Induce Apoptosis in Human Ovarian Cancer Cells
[0159] Ovarian cancer cells were treated with medium atone, IL-12
(50 ng/ml), NP (50 .mu.g/ml) or IL-12-conjugated gelatin/iron oxide
magnetic composite nanoparticles (NP-IL-12, 50 ng IL-12/ml),
incubated for 24 h and were then analyzed for cell death using the
LDH assay. As shown in FIG. 18, both IL-12 and NP-IL-12 induced
cell death in the cultured cells, as further reflected in the cell
morphology.
Example 7
In Vivo Studies: Targeting, Imaging and Apoptosis of Glioma
Cells
Materials and Methods
[0160] Cell lines and animals. U251 cells were obtained from ATCC
(Manassas, Va.) or from the Hermelin Brain Tumor Center (Henry Ford
Hospital, Detroit, Mich.). All cells were cultured in DMEM
supplemented with 10% FBS (Hyclone, Logan, UT), 2 mM L-glutamine,
and 100 ug/ml streptomycin-penicillin (Invitrogen) at 37.degree. C.
under 5% CO.sub.2. Female Nu/Nu rats were obtained from NCI
Fredericks (Fort Detricks, Md.). All rats were 6-8 weeks old at the
time of tumor implantation.
[0161] Tumor implantation and nanoparticles administration. Rats
were allowed to acclimate for one week after arrival prior to use.
Prior to tumor cell implantation, animals were anesthetized and
prepared for sterile surgery. A 1 cm long incision was made through
the scalp and a 26-gauge needle was used to gently puncture, by
twisting, a hole through the skull 2 mm to the right and 2 mm to
the dorsal of the midline. The animal was then placed into a
stereotaxic device (Kopf, Tujunga, Calif.), equipped with a
micro-manipulator and a syringe holder. The needle was lowered 3 mm
into the pre-made hole, raised 0.5 mm, and the tumor volume (5
.mu.l) was slowly injected over a total of 2.5 min. The needle was
left in place for 1 min and was then slowly raised over 1 min. The
animal was removed from the stereotaxic device, the hole sealed
with bone wax, the scalp was sutured and the animal was monitored.
for recovery. On day 4, 7 or 10 after tumor implantation, PBS (10
.mu.l) in absence or presence of control nanoparticles (0.05 mg),
TRAIL-conjugated nanoparticles (100 ng TRAIL conjugated to 0.05 mg
nanoparticles) or free TRAIL (100, 200 or 800 ng) were implanted in
the same manner in the ipsi-lateral or the contra-lateral
hemisphere. At either select time points, or at signs of morbidity,
standard perfusion with saline by 10% formalin was preformed. The
brains were removed and placed in 10% neutral buffered formalin
overnight for further processing. For visualization of
fluorescently labeled tumors and nanoparticles, at select time
points, standard perfusion with saline was preformed. Brains were
removed and snap-frozen for sectioning using a cryostat for further
visualization.
[0162] Histochemistry, immunohistochemistry, nanoparticle
visualization and apoptosis detection. Formalin-fixed tissues were
embedded in paraffin, sectioned (5 .mu.m), and hematoxylin and
eosin (H&E) stained for histomorphological assessment.
Hematoxylin stains with blue color the nucleus, the acidic regions
of the cytoplasm and the cartilage matrix. Eosin stains with pink
color the basic regions of the cytoplasm and the collagen fibers
(Gartner and Hiatt, 2001). The 5 .mu.m sections were dried in a
60.degree. C. oven for 1 h and routinely deparafinized to
ddH.sub.2O. For detection of human cells, sections were incubated
with a 1:250 dilution of human mitochondria antibody (Chemicon,
cat#H10060). Immunohistochemistry was performed using the Biocare
Medical Nemesis 7200 (Concord, Calif.) stainer and reagents. The
sections were blocked ith Biocare Sniper block for 7 minutes and
incubated with primary antibody in Biocare diluent for 60 min.
After buffer rinses, the sections were avidin biotin blocked for 15
min. Following rinses, antigens were detected using universal link,
followed by HP (Biocare) and Betazoid DAB. Buffer rinses are then
followed by dH2O and 10 second counterstain with CAT Hematoxylin.
Control sections were processed omitting the primary antibody.
[0163] Gomori's Iron reaction staining (Sheehan and Hrapchak, 1980)
for nanoparticle visualization (blue color) was done utilizing 5
.mu.m sections from formalin-fixed paraffin embedded tissue. The 5
.mu.m sections were dried in a 60.degree. C. oven for 1 h and
routinely deparanfinized to ddH.sub.2O. For all remaining steps,
acid cleaned glassware was used. Slides were immersed in equal
parts of 20% HC1 and 10% aqueous potassium ferrocyanide for 10-20
minutes. Slides were then rinsed well in ddH.sub.2O and
counterstained with Nuclear Fast Red for 2 minutes, followed by
dehydration, cleared and then cover slipped. Sections of spleen
were used as positive controls for each staining.
[0164] Staining of apoptotic cells was performed using TUNEL
staining (brown staining) that specifically detects apoptotic cells
(Shah et al., 2003). Briefly, staining of formalin-fixed paraffin
embedded tissue was performed utilizing the Apoptag Peroxidase In
Situ Apoptosis Detection Kit (Chemicon, Temecula, Calif.) as per
manufacturer's instructions. Briefly, 5 .mu.m sections from
formalin-fixed paraffin embedded tissue were dried in a 60.degree.
C. oven for 1 h and routinely deparafinized. Tissue was then
pretreated with proteinase K (20 .mu.g/ml) for 15 mins at room
temp. Slides were washed two times in ddH.sub.2O and endogenous
peroxidase was quenched in 3.0% hydrogen peroxide in PBS for 5 mins
at room temperature. Slides were wash twice in PBS and 75 .mu.l/5
cm.sup.2 of equilibration buffer was added to each slide for at
least 10 seconds. Excess liquid was tapped off and 55 .mu.l/5
cm.sup.2 of TdT enzyme was added. Slides were incubated for 1 hour
and were then washed in Stop/wash buffer for 15 seconds with
agitation, then 10 mins at room temp. Next, the slides were washed
in 3 changes of PBS for 1 min each, followed by incubation with 65
.mu.l/5 cm.sup.2 anti-digoxignenin conjugate for 30 min. Following
4 changes of PBS for 2 mins each, slides were incubated for 3-6
mins at room temp with 75 .mu.l/5 cm.sup.2 peroxidase substrate.
Slides were washed for a final 3 changes of ddH.sub.2O for 1 min
each, incubated in ddH.sub.2O for 5 min, and then counterstained
with methyl green for 10 mins at room temperature, washed with
ddH.sub.2O, cleared with 100% n-butanol, dehydrated through xylene
and cover slips mounted with permount.
[0165] Imaging. Immunohistological and histochemical images were
taken at room temperature using a Nikon Eclipse E800M microscope
with X10, X20 and X40 objectives connected to a Nikon DXM1200C
digital camera, and digitized using ACT-1C software on Dell
Optiplex GX620 computers. Tiff images were imported into Adobe
Photoshop for composite production. Insets are magnifications
performed using Photoshop.
[0166] Fluorescent images were taken at room temperature using a
Nikon C 1 confocal microscope with X4, X10 and X20 objectives
connected to a digital camera. Bitmap images were imported into
Adobe Photoshop for composite production.
[0167] In vivo MR imaging studies: An appropriate state of
anesthesia was obtained with halothane (3% for induction, 0.7% to
1.5% for maintenance in a 2:1 mixture of N.sub.2:O.sub.2). The
anesthetized rats were placed in a 7 Tesla, 20 cm bore
superconducting magnet (Magnex Scientific, Abingdon, England)
interfaced to a BRUKER console (Bellerica, Mass.). A 12 cm
self-shielded gradient set with maximum gradients of 45 gauss/cm
was used. The radio frequency (RF) pulses were applied by a 7.5 cm
diameter saddle coil actively decoupled by TTL control from the 3.2
cm diameter surface receive coil which was positioned over the
center line of the animal skull. Stereotaxic ear bars was used to
minimize movement during the imaging procedure. Rectal temperature
was maintained at 37.+-.0.5.degree. C. using a feedback controlled
water bath. A modified fast low angle shot (FLASH) imaging sequence
was employed for reproducible positioning of the animal in the
magnet at each MRI session. MR studies were performed using T1-,
T2- and T2*-weighted MRI scans. For detection of iron oxide labeled
cells, scans typically employed are T2*W gradient echoes. Average
examination times for the T1-, T2- and T2*-weighted MRI scans were
approximately 9, 13 and 13 minutes, respectively, for in vivo
studies of brain tumors. The following parameters were used to
acquire MRI: T1-weighted multislice sequence (TR/TE=500/10 ms,
128.times.128 matrix, 13-15 slices, 1 mm thick, 32 mm field of view
(FOV), number of excitation (NEX)=4). T2 weighted images were
obtained using standard two-dimensional Fourier transformation
(2DTF) multislice (13-15) multiecho (6 echoes) MRI. A series of six
sets of images (13-15 slices for each set) were obtained using TEs
of 10, 20, 30, 40, 50 and 60 msec and a TR of 3000 msec. The images
were produced using 32 nun FOV, 1 mm slice thickness, 128.times.128
matrix, and NEX=2. T2* weighted images were obtained using standard
multislice (13-15 slices) multi gradient echo (6 echoes) MRI. A
series of six sets of images (13-15 slices for each set) were
obtained using TEs of 5, 10, 15, 20, 25 and 30 msec and a TR of
3000 msec. The images were produced using 32 nun FOV, 1 mm slice
thickness; 128.times.128 matrix and NEX=2. Both the T2 and T2*
images were used to measure the T2 and T2* maps. Three dimensional
(3D) gradient echo MR images were obtained with TR=100 msec, TE=6
msec, 10.degree. of flip angle (FA), 32.times.32.times.16 mm.sup.3
FOV, 256.times.192.times.64 matrix, and NEX=1. The total time for
entire sequence was approximately 20 minutes. To maintain the body
temperature, heating pad was used. Rectal probe were used to
monitor the body temperature. To compare the image quality between
the 3 tesla and 7 tesla MR systems, randomly selected animals
underwent MRI using 3 tesla. For imaging with 3 tesla MRI,
appropriate anesthesia was obtained by injecting ketamine chloride
and xylazine. Rats were properly rapped with heated drapes to
maintain body temperature.
7(i) Establishment of Glioma Animal Models
[0168] In order to examine the in vivo effect of TRAIL-conjugated
gelatin/iron oxide magnetic composite nanoparticles (NP-TRAIL), two
glioma animal models were established. In the first model, human
U87 or U251 cells were intracranially implanted into nude mice. In
the second model, we employed human glioma cells from fresh
operative tumor samples were implanted in nude rats, generating
tumors that maintain the original properties of the original
tumors, thus providing a system that can predict the response of
these tumors to different anti-cancer treatments.
7(ii) TRAIL-Conjugated Gelatin/Iron Oxide Magnetic Composite
Nanoparticles Induce Cell Apoptosis in Glioma
[0169] In order to examine the effect of NP-TRAIL in vivo, human
U251 glioma cells were employed as xenografts. Tumors were allowed
to develop for 7 days at which time NP-TRAIL, gelatin/iron oxide
magnetic composite nanoparticles (NP) alone or PBS were
intracranially injected at the site of the tumor. The degree of
cell apoptosis was examined after 7 days of treatment. Cell
apoptosis was determined using TUNEL staining (brown staining) that
specifically detects apoptotic cells. FIG. 19 shows tumor sections
from rats treated with either NP alone or NP-TRAIL. As shown, NP
alone did not induce a significant degree of cell apoptosis;
however, NP-TRAIL induced a large degree of cell apoptosis as
determined by TUNEL (brown staining)-positive cells. In the
PBS-treated rats, no significant cell apoptosis was indicated as
well (data not shown).
7(Iii) TRAIL-Conjugated Gelatin/Iron Oxide Magnetic Composite
Nanoparticles Prolong Survival in Glioma
[0170] In order to determine whether NP-TRAIL (100 ng TRAIL) could
provide a survival benefit, PBS, NP alone or NP-TRAIL were
delivered intratumoraly directly into xenogenic human U251 tumors
in the brains of nude rats. Animals were then monitored for signs
of morbidity and euthanized at the appearance of any signs or at
day 85 post tumor implant. As shown in FIG. 20, treatment with
NP-TRAIL significantly prolonged survival over that of NP alone
(p=0.0248) or PBS (p=0.0288). Photomicrographs of representative
tumors at time of morbidity are shown in FIGS. 21A-21C. Large
tumors are evident in animals euthanized due to tumor morbidity,
but no tumors are evident in censored animals. Some scaring is
evident in these animals however (arrows). Each series is taken
from the same animal and from sections 2 mm apart. A similar
experiment was also accomplished with sTRAIL substituting the
injected 10 .mu.l PBS containing NP-TRAIL (100 ng TRAIL) for 100,
200 and 800 ng sTRAIL. These experiments indicated similar survival
results to that observed with PBS. However, the experiment with the
higher concentration of sTRAIL indicated some damage to
oligodendrocytes.
7(iv) TRAIL-Conjugated Gelatin/Iron Oxide Magnetic Composite
Nanoparticles Decrease Tumor Volume in Glioma
[0171] In order to determine the effect of NP-TRAIL on tumor
burden, PBS, NP alone or NP-TRAIL were delivered intratumoraly into
xenogenic human U251 tumors in the brains of nude rats. Animals
were euthanized on day 21 post tumor implant and brains were
harvested for sectioning and H&E staining. Brains were cut into
2 mm blocks, processed, and blocks with evident tumor were cut in 5
um sections, with every 15.sup.th section kept and stained with
H&E, and tumor volume on this slide determined. Max width and
height of tumor on each 15.sup.th slide was measured, and total
tumor burden determined by multiplying max W.times.HX(5.times.15)
for each slide with evident tumor and these numbers added.
Statistical significance was determined with Statview using
Fischer's PLSD (significance level 5%). As shown in FIG. 22,
NP-TRAIL administration significantly decreased tumor burden as
compared with NP alone or PBS. No significant difference was seen
between NP alone and PBS administration.
7(v) Tumor Tracking Ability
[0172] The ability of NP to travel from the site of implantation to
the site of tumor growth is a useful therapeutic potential of NP,
particularly for brain tumor therapy. Thus, in order to determine
that ability, U251 tumor cells were implanted into the left
hemisphere of nude rat brains on day 0 and rhodamine-labeled
gelatin/iron oxide magnetic composite nanoparticles (NPR) were
administered parallel to the tumor implantation but in the
contralateral hemisphere 7 days later. On day 11 (4 days post NPR
administration), animals were euthanized and perfused with saline,
and brains were removed and snap frozen for cryosectioning and
analysis with confocoal microscopy. As shown in FIG. 23, NPR are
easily seen tracking along the corpus collosum. Although some NPR
are indeed migrating away from the tumor mass, the majority of NPR
were seen to be migrating towards the tumor mass in the left
hemisphere. Similar behavior was observed for rhodamine-labeled
TRAIL-conjugated gelatin/iron oxide magnetic composite
nanoparticles (NPR-TRAIL).
7(vi) Tumor Eestruction
[0173] In this experiment, the ability of NP-TRAIL to destruct the
tumor was determined. In particular, nude rats were implanted with
U25 1n tumors on day 0. NP-TRAIL, NP alone or PBS were
intraneoplastically administered on day 7, and animals were
euthanized and tissue harvested on day 14. As shown in FIG. 24,
NP-TRAIL administration led to the development of large areas of
tumor destruction in the lower part of the tumor mass, which were
not seen in NP alone- or PBS-administered animals. Many necrotic
and apoptotic cells were seen within this area (right panels)
following NP-TRAIL administration; however were not seen following
NP or PBS administration. FIG. 25 demonstrates high magnification
of the areas of tumor destruction following therapy.
7(vii) TRAIL-Conjugated Gelatin/Iron Oxide Magnetic Composite
Nanoparticles Arrive at the Borders and Inside Human Glioma
Xenograft Implanted within Nude Rats as Shown by MRI
[0174] U251n human glioma cells were implanted in nude rats and 11
days later, NP or NP-TRAIL were intracranially injected in the
contra lateral side of the tumor. MR images were obtained 8 days
later. As shown in FIGS. 26A-26B, NP-TRAIL (26B) induced lower
signal intensity both at the margin and inside the tumor, compared
to NP only (26A). The lower intensity implies the presence of
nanoparticles.
7(viii) In Vivo Uptake of TRAIL-Conjugated Rhodamine-Labeled
Gelatin/Iron Oxide Magnetic Composite Nanoparticles by Tumor
Cells
[0175] NPR alone or NPR-TRAIL were implanted directly within the
tumor mass 7 days after GFP-U251 tumor cells implantation. Four
days later, animals were euthanized, and brains were harvested and
snap frozen for sectioning and imaging (red color and green color
indicate the presence of NPR and GFP-U251 tumor cells,
respectively). As shown in FIG. 27, whereas NPR alone were found in
areas around tumor cells but were not colocalized with tumor cells
(panels D, E, F), NPR-TRAIL were found in areas of tumor
destruction colocalized with tumor cells (panels G, H, I). It is
further shown that the tumor visible in the NPR-TRAIL-treated
animal is degraded compared to the tumor visible in both the PBS-
or NPR-treated-animals, with only a few viable tumor cells clearly
visible in this section. This experiment indicates that TRAIL led
to tumor cell uptake of NP, as shown in vitro in Example 6(v).
7(ix) Migration of cRGD Peptide-Conjugated Rhodamine-Labeled
Gelatin/Iron Oxide Magnetic Composite Nanoparticles to Gliomas
[0176] cRGD peptide-conjugated rhodamine-labeled gelatin/iron oxide
magnetic composite nanoparticles (NPR-cRGD, 10 .mu.l containing
0.05 mg nanoparticles conjugated to about 2 .mu.g cRGD peptide)
were injected to the contra-lateral side of the brain, 7 days after
GFP-U251 tumor cells implantation. Four days later, animals were
euthanized, and brains were harvested and snap frozen for
sectioning and imaging (red color and green color indicate the
presence of NPR and GFP-U251 tumor cells, respectively). FIG. 28
shows that NPR-cRGD migrated to the tumor site
7(x) Migration of cRGD Peptide- and TRAIL-Conjugated
Rhodamine-Labeled Gelatin/Iron Oxide Magnetic Composite
Nanoparticles Toward Injury Site
[0177] Injury was induced by needle injection of PBS at the left
side of the brain. Following 4 days, 5 .mu.l (25 .mu.g) of NPR
alone, NPR-TRAIL (50 ng TRAIL) and NPR-cRGD were injected to the
contra-lateral side of the brain. After 4 days, the animals were
sacrificed and the fluorescence of the NPR was visualized under
fluorescent microscope. As shown in FIG. 29, few NPR were present
at the other side of the brain but their distribution was abundant.
NPR-TRAIL were localized mainly along the site of injury. In
contrast, NPR-cRGD were distributed all over the side of the
injured brain and also along the corpus callosum. These results
suggest that the nanoparticles have some ability to track to site
of injury and that NPR-TRAIL show selective accumulation in the
site of injury. NPR-cRGD have tracking ability but their
distribution is not limited to the site of injury and they may bind
to inflammatory cells that accumulate there as well. These results
have important implications for the use of the NP system to deliver
drugs in brain injury, stroke and inflammatory diseases in the
brain.
Example 8
The Cytotoxic Effects of TRAIL-Conjugated Gelatin/Iron Oxide
Magnetic Composite Nanoparticles on Bladder Carcinoma Cells, Breast
Cancer Cells and Normal Breast Cells
[0178] In these experiments, the cytotoxic effects of
TRAIL-conjugated gelatin/iron oxide magnetic composite
nanoparticles (NP-TRAIL) on various cancer cells other than glioma
cells were determined. The cancer cell lines particularly used were
the bladder carcinoma cells TSU-PR1 and the breast cancer cells
MDA-MB, and the normal breast cells MCF10A were used as controls.
In addition, the effect on these cells of NP-TRAIL in combination
with proteasome inhibitor (PS), a multicatalytic proteinase complex
responsible for the majority of intracellular protein degradation,
was studied. Pharmacologic inhibitors of the proteasome possess in
vitro and in vivo antitumor activity. Preclinical studies
demonstrate that proteasome inhibition potentiates the activity of
other cancer therapeutics such as TRAIL in part by down regulating
chemoresistance pathways.
[0179] Cells (1.times.10.sup.5/well) were treated with different
concentrations of TRAIL (10-100 ng/ml), NP or NP-TRAIL (10-40 ng
TRAM/ml), in the absence or presence of PS (5 mM). Cell death was
determined after 24 h using LDH assay. 100% cell death was
determined in Triton X-100-treated cells and data normalized.
[0180] As shown in FIG. 30A-30B, normal breast cells MCF10A did not
respond to all of the treatments described above, indicating that
these treatments are non-toxic to normal cells. As further shown,
the activity of NP-TRAIL was potentiated after treatment with PS.
This fording may be of high significance, in particular when
planning effective treatment strategies.
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Sequence CWU 1
1
115PRTArtificialThis is a cyclic peptide 1Arg Gly Asp Phe Lys1
5
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