U.S. patent application number 12/989402 was filed with the patent office on 2011-07-14 for biocompatible polymer and magnetic nanoparticle with biocompatibility.
This patent application is currently assigned to Industrial Technology Research Institute. Invention is credited to Wen-Hsiang Chang, Wen-Uan Hsieh, Shiu-Hua Huang, Chin-1 Lin, Kelly Teng, Shian-Jy Jassy Wang.
Application Number | 20110171715 12/989402 |
Document ID | / |
Family ID | 41216385 |
Filed Date | 2011-07-14 |
United States Patent
Application |
20110171715 |
Kind Code |
A1 |
Chang; Wen-Hsiang ; et
al. |
July 14, 2011 |
BIOCOMPATIBLE POLYMER AND MAGNETIC NANOPARTICLE WITH
BIOCOMPATIBILITY
Abstract
The invention discloses a biocompatible polymer for covalently
modifying magnetic nanoparticles. The biocompatible polymer may be
coupled to a targeting agent and/or a fluorescent dye. The
invention also discloses a magnetic nanoparticle with
biocompatibilities comprising the biocompatible polymer.
Inventors: |
Chang; Wen-Hsiang; (Taipei,
TW) ; Hsieh; Wen-Uan; (Hsinchu County, TW) ;
Huang; Shiu-Hua; (Taipei County, TW) ; Lin;
Chin-1; (Tainan County, TW) ; Wang; Shian-Jy
Jassy; (Hsinchu County, TW) ; Teng; Kelly;
(Taipei, TW) |
Assignee: |
Industrial Technology Research
Institute
Hsinchu County
TW
|
Family ID: |
41216385 |
Appl. No.: |
12/989402 |
Filed: |
April 22, 2008 |
PCT Filed: |
April 22, 2008 |
PCT NO: |
PCT/CN2008/000823 |
371 Date: |
March 16, 2011 |
Current U.S.
Class: |
435/188 ;
428/402; 530/300; 530/391.3; 530/395; 530/400; 536/121; 536/22.1;
556/419; 977/773; 977/810; 977/811; 977/838; 977/930 |
Current CPC
Class: |
A61K 49/1848 20130101;
C08G 65/3328 20130101; C08G 65/323 20130101; Y10T 428/2982
20150115; A61K 49/12 20130101; A61K 49/186 20130101; C07F 7/1804
20130101; C08G 65/33306 20130101; A61K 49/0093 20130101; A61K
49/085 20130101; C08G 65/326 20130101; C08G 65/336 20130101; B82Y
5/00 20130101 |
Class at
Publication: |
435/188 ;
556/419; 530/391.3; 530/300; 530/400; 536/121; 536/22.1; 530/395;
428/402; 977/838; 977/773; 977/810; 977/811; 977/930 |
International
Class: |
C12N 9/96 20060101
C12N009/96; C07F 7/18 20060101 C07F007/18; C07K 16/00 20060101
C07K016/00; C07K 2/00 20060101 C07K002/00; C07H 23/00 20060101
C07H023/00; B32B 5/16 20060101 B32B005/16 |
Claims
1. A biocompatible polymer of formula (I), ##STR00005## wherein
R.sub.1 is alkyl, aryl, carboxyl, or amino; R.sub.2 is alkyl or
aryl; n is an integer from 5 to 1000; and m is an integer from 1 to
10.
2. The biocompatible polymer as claimed in claim 1, wherein each of
R.sub.1 and R.sub.2 independently is a C.sub.1-C.sub.20 straight
chain or branched alkyl.
3. The biocompatible polymer as claimed in claim 1, wherein each of
R.sub.1 and R.sub.2 independently is a C.sub.6-C.sub.12 substituted
or unsubstituted aryl.
4. The biocompatible polymer as claimed in claim 1, wherein R.sub.1
is methyl.
5. The biocompatible polymer as claimed in claim 1, wherein R.sub.2
is ethyl.
6. The biocompatible polymer as claimed in claim 1, wherein n is
15.
7. The biocompatible polymer as claimed in claim 1, wherein m is
3.
8. A magnetic nanoparticle with bio compatibility, comprising: a
magnetic nanoparticle; a biocompatible polymer of formula (II)
covalently coupled to the magnetic nanoparticle, ##STR00006##
wherein R.sub.1 is alkyl, aryl, carboxyl, or amino; n is an integer
from 5 to 1000; and m is an integer from 1 to 10.
9. The magnetic nanoparticle with biocompatibility as claimed in
claim 8, wherein R.sub.1 is carboxyl or amino.
10. The magnetic nanoparticle with biocompatibility as claimed in
claim 8, wherein the magnetic nanoparticle is a superparamagnetic
nanoparticle.
11. The magnetic nanoparticle with biocompatibility as claimed in
claim 8, wherein the magnetic nanoparticle comprises at least one
of Fe, Co, Ni, and oxides thereof.
12. The magnetic nanoparticle with biocompatibility as claimed in
claim 9, wherein R.sub.1 is coupled to a specific targeting agent
comprising an antibody, a protein, a peptide, an enzyme, a
carbohydrate, a glycoprotein, a nucleotide or a lipid.
13. The magnetic nanoparticle with biocompatibility as claimed in
claim 12, wherein the magnetic nanoparticle has a diameter of about
3-500 nm.
14. The magnetic nanoparticle with biocompatibility as claimed in
claim 12, wherein the biocompatible polymer is coupled to a
fluorescent dye.
15. The magnetic nanoparticle with biocompatibility as claimed in
claim 14, wherein the magnetic nanoparticle containing the
fluorescent dye and the specific targeting agent has a diameter of
about 15-200 nm.
16. The magnetic nanoparticle with biocompatibility as claimed in
claim 14, wherein the fluorescent dye exhibits at least one of
ultraviolet (UV), near-infrared (NIR), and visible (VIS) light
excitation or emission wavelength.
17. The magnetic nanoparticle with biocompatibility as claimed in
claim 14, wherein the fluorescent dye comprises an organic dye, an
inorganic dye, or an organometallic complex.
18. The magnetic nanoparticle with biocompatibility as claimed in
claim 14, wherein the fluorescent dye and the specific targeting
agent are coupled to the biocompatible polymer by a covalent
bond.
19. The magnetic nanoparticle with biocompatibility as claimed in
claim 14, wherein the specific targeting agent and the
biocompatible polymer are coupled by a --CONH-- linkage.
20. The magnetic nanoparticle with biocompatibility as claimed in
claim 8, wherein the biocompatible polymer is coated on the
magnetic nanoparticle to form a core/shell structure.
21. The magnetic nanoparticle with biocompatibility as claimed in
claim 20, wherein the biocompatible polymer forms a monolayer
coating on the magnetic nanoparticle.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to a biocompatible polymer and in
particular to a biocompatible polymer for covalently modifying
magnetic nanoparticles.
[0003] 2. Description of the Related Art
[0004] Magnetic resonance imaging (MRI) is an appealing noninvasive
approach for early cancer diagnostics and therapeutics. MRI
utilizes radio frequency pulses and magnetic field gradients
applied to a subject in a strong field to produce images. MRI is
capable of showing several different characteristics of tissues.
The level of tissue magnetization at specific signal recording
periods during the MR imaging cycle generally determines the
brightness of a particular tissue in the MRI images. Contrast is
produced when tissues do not have the same level of
magnetization.
[0005] While the imaging capabilities of MRIs have revolutionized
imaging technology, the resolution is limited to the elucidations
of lesions within the body on the order of 1 mm. This limitation
has led to the development of contrast enhancement agents. Because
of the superparamagnetic property, iron oxide nanoparticles have
been found effective as contrast enhancement agents for MRIs. The
magnetic nanoparticle can be modified with a biocompatible polymer
to prolong the particle circulation time in blood and reduce
immunogenicity. Furthermore, the magnetic nanoparticle can be
modified with a fluorescent dye and a specific targeting agent to
provide fluorescent properties and specific targeting
functions.
[0006] U.S. Patent Publication No. 20070148095 discloses a
multi-modality contrast agent with specificity for both magnetic
and optical imaging. The multi-modality contrast agent includes a
magnetic nanoparticle, a biocompatible polymer chemically modifying
the magnetic nanoparticle, a fluorescent dye coupled to the
biocompatible polymer, and a specific targeting agent coupled to
the biocompatible polymer. The biocompatible polymers include
polyethylene glycol (PEG), polylactic acid (PLA), PLA-PEG,
poly(glycolic acid) (PGA), poly(.epsilon.-caprolactone) (PCL),
poly(methyl methacrylate) (PMMA), and the like.
[0007] U.S. Patent Publication No. 20070148095 discloses a silane
compound for modifying magnetic nanoparticle and a method for using
the nanoparticle to detect and treat tissues of interest.
[0008] Commercially available MRI contrast enhancement agents
include Feridex.RTM. (dextran-coated iron oxide) and Resovist.RTM.
(carboxydextran-coated iron oxide).
BRIEF SUMMARY OF THE INVENTION
[0009] In one aspect, the invention provides a biocompatible
polymer of formula (I),
##STR00001##
[0010] wherein R.sub.1 is alkyl, aryl, carboxyl, or amino, R.sub.2
is alkyl or aryl, n is an integer from 5 to 1000, and m is an
integer from 1 to 10.
[0011] In another aspect, the invention provides a magnetic
nanoparticle with biocompatibility, comprising a magnetic
nanoparticle and a biocompatible polymer of formula (II) covalently
coupled to the magnetic nanoparticle,
##STR00002##
[0012] wherein R.sub.1 is alkyl, aryl, carboxyl, or amino, n is an
integer from 5 to 1000, and m is an integer from 1 to 10.
[0013] A detailed description is given in the following embodiments
with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The invention can be more fully understood by reading the
subsequent detailed description and examples with references made
to the accompanying drawings, wherein:
[0015] FIG. 1 is a schematic drawing showing the synthesis of the
biocompatible polymer of the invention; and
[0016] FIG. 2 is a schematic drawing showing a magnetic
nanoparticle modified with the biocompatible polymer of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The following description is of the best-contemplated mode
of carrying out the invention. This description is made for the
purpose of illustrating the general principles of the invention and
should not be taken in a limiting sense. The scope of the invention
is best determined by reference to the appended claims.
[0018] The biocompatible polymer of the invention is represented by
general formula (I),
##STR00003##
[0019] wherein R.sub.1 is alkyl, aryl, carboxyl, or amino, R.sub.2
is alkyl or aryl, n is an integer from 5 to 1000, and m is an
integer from 1 to 10. FIG. 1 is a schematic drawing showing the
synthesis of the biocompatible polymer of the invention, wherein
R1, R2, n, and m have the same meaning as described above. As shown
in FIG. 1, the synthetic scheme involves converting the hydroxyl
end group of polyethylene glycol (PEG) to a carboxyl group by using
a succinic anhydride compound, and coupling a silane group to the
PEG. Suitable alkyl groups for R.sub.1 and R.sub.2 include
C.sub.1-C.sub.20 straight chain or branched alkyl groups. In one
embodiment, each of R.sub.1 and R.sub.2 independently, is a
C.sub.1-C.sub.6 straight chain or branched alkyl such as methyl,
ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl,
isopentyl, tert-pentyl, n-hexyl, and isohexyl. Suitable aryl groups
for R.sub.1 and R.sub.2 include C.sub.6-C.sub.12 substituted or
unsubstituted aryl groups such as phenyl, biphenyl, and naphthyl,
and examples of substituents thereof include hydroxyl, haloalkyl,
alkoxyl, cyano, nitro, amino, or alkylamino. The number of
methylene units m is preferably an integer from 1 to 10. The number
of oxyethylene units n is preferably an integer from 5 to 1000,
equivalent to a molecular weight of about 200-50000 g/mole of the
PEG. In one embodiment, m is about 3, and n is about 15.
[0020] The biocompatible polymer synthesized in FIG. 1 is useful in
that it can chemically modify the surface of the iron oxide
nanoparticle to increase biocompatibility. In addition, the
biocompatible polymer is useful in that it can label particles
(e.g., nanoparticles, magnetic particles, magnetic nanoparticles,
superparamagnetic particles), to render the particles to be further
reactive toward one or more targeting, fluorescent, therapeutic, or
diagnostic agents.
[0021] The invention also provides a magnetic nanoparticle with
biocompatibility, comprising a magnetic nanoparticle; a
biocompatible polymer of formula (II) covalently coupled to the
magnetic nanoparticle,
##STR00004##
[0022] wherein R.sub.1 is alkyl, aryl, carboxyl, or amino, n is an
integer from 5 to 1000, and m is an integer from 1 to 10. FIG. 2 is
a schematic drawing showing a magnetic nanoparticle modified with
the biocompatible polymer of the invention. The magnetic
nanoparticle is preferably made of at least one of Fe, Co, Ni, and
oxides thereof. It will be appreciated that the nanoparticle can be
made of any single or composite magnetic material, although
superparamagnetic materials are particularly preferred. After the
biocompatible polymer is chemically bonded to the magnetic
nanoparticle, the terminal groups R.sub.1 are transformed into
active functional groups such as carboxyl or amino groups to allow
coupling with fluorescent dye and/or specific targeting agents.
However, R.sub.1 is not alkyl or aryl, since alkyl or aryl are not
capable of coupling with targeting agents or fluorescent dye. In
one embodiment, R.sub.1 is a carboxyl group. The number of
methylene units m is preferably an integer from 1 to 10. The number
of oxyethylene units n is preferably an integer from 5 to 1000. In
one embodiment, m is about 3, and n is about 15. The biocompatible
polymer is preferably coated on the entire surface of the magnetic
nanoparticle to form a core-shell structure. More preferably, the
biocompatible polymer forms a monolayer coating on the magnetic
nanoparticle.
[0023] Experimental results indicate that the biocompatible polymer
of the invention may increase the r2 value of the magnetic
nanoparticle to about 2 times that of commercial contrast agents,
Feridex.RTM. and Resovist.RTM.. Accordingly, the magnetic
nanoparticle may provide greater contrast enhancement when being
used as an MRI contrast agent.
[0024] The targeting agent is preferably coupled to the
biocompatible polymer via covalent bonds. Commonly used targeting
agents include an antibody, a protein, a peptide, an enzyme, a
carbohydrate, a glycoprotein, a nucleotide, and a lipid. The
magnetic nanoparticle may have a diameter of about 3-500 nm after
coupling with the targeting agent. Those skilled in the art can
attach any suitable targeting agents on the nanoparticle to give
specificity thereto. For example, folic acid can be used to specify
breast cancer cells with a folate receptor. The structure of the
folic acid allows coupling with an amine-terminated or
carboxy-terminated biocompatible polymer. For example, the folic
acid allows coupling with the amine-terminated biocompatible
polymer by forming a --CONH-- linkage.
[0025] A fluorescent dye may be further coupled to the magnetic
nanoparticle to provide an optical signal for optical imaging
techniques such as NIR imaging, thus allowing real-time monitoring
of foci by different imaging techniques. Preferably, the
fluorescent dye is coupled to the biocompatible polymer via
covalent bonds. Suitable fluorescent dyes include organic or
inorganic dyes and organometallic complexes. The excitation and
emission wavelengths of the fluorescent dye may be ultraviolet
(UV), near-infrared (NIR), or visible (VIS) light. The magnetic
nanoparticle coupled with the targeting agent and fluorescent dye
preferably has a diameter of about 15-200 nm.
[0026] Without intending to limit the present invention in any
manner, the present invention will be further illustrated by the
following examples.
Example 1
Nanoparticle Preparation
[0027] 11.6 g (0.058 mole) of FeCl.sub.2. 4H.sub.2O, 11.6 g (0.096
mole) of FeCl.sub.3.6H.sub.2O and 400 ml of deionized water were
stirred in a three-necked flask at 300 rpm at 25.degree. C. 170 ml
of a 2.5N NaOH solution was added to the flask at a rate of 47
.mu.l/sec. When a pH value of 11-12 was measured after the addition
of the 2.5N NaOH solution, 20 ml of oleic acid was added and
stirred for 30 minutes. Thereafter, a 6N HCl solution was slowly
added to adjust the pH value to about 1, thus precipitating oleic
acid encapsulated-iron oxide particles. The precipitates were
collected, washed with deionized water for 4-5 times to remove
excess oleic acid, and dried.
Example 2
Synthesis of Biocompatible Polymer mPEG-silane
[0028] 300 g (0.4 mole) of methoxy-PEG (mPEG, molecular weight:
750) and 600 ml of N-methyl-2-pyrrolidone were placed in a 1000 ml
round bottom flask under vacuum (20 Ton) for more than 2 hours. 48
g (0.48 mole) of succinic anhydride and 19.5 g (0.159 mole) of
4-dimethylamino-pyridine (DMAP) were added for reaction at
30.degree. C. for two days.
[0029] 36 ml (0.48 mole) of thionyl chloride was added at a rate of
1 ml/min and the mixture was stirred for 2-3 hours. Thereafter,
133.8 ml (0.96 mole) of triethylamine was added at a rate of 1
ml/min. After cooled to room temperature, the mixture was filtered
to remove precipitates. 94.5 ml (0.4 mole) of 3-aminopropyl
triethoxysilane was added for reaction for at least 8 hours.
[0030] The reaction mixture was added to 9 L of isopropyl ether for
re-precipitation, and the precipitates were collected, re-dissolved
in 500 ml of toluene, and centrifuged at 5000 rpm for 5 minutes to
collect a supernatant. The supernatant was again, added to 9 L of
isopropyl ether for re-precipitation. Brown oily liquid was
collected and dried under vacuum to obtain the biocompatible
polymer, mPEG-silane.
Example 3
Synthesis of Biocompatible Polymer COOH-PEG-silane
[0031] 300 g (0.4 mole) of PEG (molecular weight: 750) and 600 ml
of N-methyl-2-pyrrolidone were placed in a 1000 ml round bottom
flask under vacuum (20 Ton) for more than 2 hours. 96 g (0.96 mole)
of succinic anhydride and 39 g (0.318 mole) of
4-dimethylamino-pyridine (DMAP) were added for reaction at
30.degree. C. for two days, thus obtaining dicarboxy-terminated PEG
(COOH-PEG).
[0032] 36 ml (0.48 mole) of thionyl chloride was added at a rate of
1 ml/min and stirred for 2-3 hours. Thereafter, 133.8 ml (0.96
mole) of triethylamine was added at a rate of 1 ml/min. After
cooled to room temperature, the mixture was filtered to remove
precipitates. Then, 94.5 ml (0.4 mole) of 3-aminopropyl
triethoxysilane was added for reaction for at least 8 hours.
[0033] The reaction mixture was added to 9 L of isopropyl ether for
re-precipitation, and the precipitates were collected, re-dissolved
in 500 ml of toluene, and centrifuged at 5000 rpm for 5 minutes to
collect a supernatant. The supernatant was again, added to 9 L of
isopropyl ether for re-precipitation. Brown oily liquid was
collected and dried under vacuum, thus obtaining the biocompatible
polymer, COOH-PEG-silane.
Example 4
Coupling with Biocompatible Polymer
[0034] 250 g of mPEG-silane or COOH-PEG-silane was added to 1-1.2 L
of a toluene solution containing 10 g of iron oxide of Example 1
and the mixture was sonicated for 2-3 hours. After addition of 1.5
L of deionized water, the mixture was purified by an
ultra-filtration device and concentrated to 100 ml to obtain iron
oxide nanoparticles modified by a biocompatible polymer.
Example 5
Coupling with Targeting Agent
[0035] 226 .mu.l of folate solution (folate/dimethyl sulfoxide: 10
mg/ml) was placed in a 50 ml brownish round bottom flask. 5 ml of
dimethyl sulfoxide (DMSO) and 176.5 .mu.l of dicyclohexyl
carbodiimide solution (dicyclohexyl carbodiimide/DMSO: 5 mg/ml) was
added to the solution and stirred for 1 hour. Thereafter, 98.5
.mu.l of NHS solution (N-hydroxysuccinimide/DMSO: 5 mg/ml) was
added and stirred for 1 hour. Then, 289 .mu.l of ethylenediamine
was added to give a solution A.
[0036] 1 ml of the COOH-PEG-silane modified iron oxide nanoparticle
of Example 4 (4.48 mg/ml) and 10 ml of DMSO were placed in a 50 ml
round bottom flask under vacuum for 1 hour. 176.5 .mu.l of
dicyclohexyl carbodiimide solution (dicyclohexyl carbodiimide/DMSO:
5 mg/ml) was added to the solution and stirred for 1 hour.
Thereafter, 98.5 .mu.l of NHS solution (N-hydroxysuccinimide/DMSO:
5 mg/ml) was added and stirred for 1 hour to give a solution B.
[0037] 2895 .mu.l (half-volume) of solution A was added to solution
B and stirred for 8 hours. The resulting solution was added into a
dialysis membrane (Mw: 3000) and water was used for dialysis. Then,
the solution was concentrated to 2 ml by an ultra-filtration device
to obtain iron oxide nanoparticles coupled with a targeting
agent.
Example 6
Coupling with Fluorescent Dye
[0038] 1 ml of CypHer5E (NIR dye from Amersham Bioscience Co.,
10.sup.-6 mole/ml) was mixed with 10.sup.-6 mole of ethylenediamine
and stirred for 1 hour, thus giving a solution C.
[0039] The iron oxide nanoparticles coupled with folate (2 mg/ml)
of Example 5 were dissolved in 10 ml of deionized water, followed
by addition of 10.sup.-6 mole of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC). After the mixture was stirred for one hour,
10.sup.-6 mole of N-hydroxysuccinimide (NHS) was added and stirred
for another hour, thus giving a solution D.
[0040] Solution C was added to solution D and stirred for 8 hours.
The resulting solution was added into a dialysis membrane (Mw:
3000) and water was used for dialysis. Then, the solution was
concentrated to 2 ml by an ultra-filtration device to obtain iron
oxide nanoparticles coupled with a targeting agent and a
fluorescent dye.
Example 7
Relaxivity Test
[0041] The modified iron oxide nanoparticles of Example 5 were
compared for the r1 and r2 relaxivity with the product of U.S.
Patent Publication No. 2006/0216239 and commercial contrast agents,
i.e., Feridex.RTM. and Resovist.RTM..
[0042] Iron oxide solutions of various concentrations (0.1, 0.2,
0.3, 0.4, 0.5 mM) were prepared and measured for the T1 or T2
relaxation time by a Minispec mq 20 from the Bruker Corporation. A
linear relationship was established between the reciprocal of
relaxation time as the ordinate axis and the concentration of the
solution as the abscissa axis. The slope of the linear relationship
was the r1 and r2 relaxivity.
[0043] As shown in Table 1, the r2 relaxivity of the modified iron
oxide nanoparticles of the invention was about 2 times that of
Feridex.RTM. and Resovist.RTM., and about 1.4 times that of the
prior art product of U.S. Patent Publication No. 2006/0216239.
Accordingly, the contrast enhancement was improved due to the
higher r2 relaxivity.
TABLE-US-00001 TABLE 1 The US invention 2006/0216239 Resovist .RTM.
Feridex .RTM. Diameter* 8-12 nm 8-12 nm 4.2 nm 4.8-5.6 nm r2 (mM
321.8 .+-. 2.3 229 164 160 s).sup.-1 r1 (mM 33.4 .+-. 0.3 23.6 25.4
40 s).sup.-1 *The diameter was determined by TEM
[0044] While the invention has been described by way of example and
in terms of preferred embodiment, it is to be understood that the
invention is not limited thereto. To the contrary, it is intended
to cover various modifications and similar arrangements (as would
be apparent to those skilled in the art). Therefore, the scope of
the appended claims should be accorded the broadest interpretation
so as to encompass all such modifications and similar
arrangements.
* * * * *