Patent Application
20110171715
1. Field of the Invention
The invention relates to a biocompatible polymer and in particular to a biocompatible polymer for covalently modifying magnetic nanoparticles.
2. Description of the Related Art
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.
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.
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(ε-caprolactone) (PCL), poly(methyl methacrylate) (PMMA), and the like.
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.
Commercially available MRI contrast enhancement agents include Feridex® (dextran-coated iron oxide) and Resovist® (carboxydextran-coated iron oxide).
In one aspect, the invention provides a biocompatible polymer of formula (I),
wherein R1 is alkyl, aryl, carboxyl, or amino, R2 is alkyl or aryl, n is an integer from 5 to 1000, and m is an integer from 1 to 10.
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,
wherein R1 is alkyl, aryl, carboxyl, or amino, n is an integer from 5 to 1000, and m is an integer from 1 to 10.
A detailed description is given in the following embodiments with reference to the accompanying drawings.
The invention can be more fully understood by reading the subsequent detailed description and examples with references made to the accompanying drawings, wherein:
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.
The biocompatible polymer of the invention is represented by general formula (I),
wherein R1 is alkyl, aryl, carboxyl, or amino, R2 is alkyl or aryl, n is an integer from 5 to 1000, and m is an integer from 1 to 10.
The biocompatible polymer synthesized in
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,
wherein R1 is alkyl, aryl, carboxyl, or amino, n is an integer from 5 to 1000, and m is an integer from 1 to 10.
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® and Resovist®. Accordingly, the magnetic nanoparticle may provide greater contrast enhancement when being used as an MRI contrast agent.
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.
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.
Without intending to limit the present invention in any manner, the present invention will be further illustrated by the following examples.
11.6 g (0.058 mole) of FeCl2. 4H2O, 11.6 g (0.096 mole) of FeCl3.6H2O and 400 ml of deionized water were stirred in a three-necked flask at 300 rpm at 25° C. 170 ml of a 2.5N NaOH solution was added to the flask at a rate of 47 μl/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.
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° C. for two days.
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.
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.
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° C. for two days, thus obtaining dicarboxy-terminated PEG (COOH-PEG).
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.
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.
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.
226 μl 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 μl of dicyclohexyl carbodiimide solution (dicyclohexyl carbodiimide/DMSO: 5 mg/ml) was added to the solution and stirred for 1 hour. Thereafter, 98.5 μl of NHS solution (N-hydroxysuccinimide/DMSO: 5 mg/ml) was added and stirred for 1 hour. Then, 289 μl of ethylenediamine was added to give a solution A.
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 μl of dicyclohexyl carbodiimide solution (dicyclohexyl carbodiimide/DMSO: 5 mg/ml) was added to the solution and stirred for 1 hour. Thereafter, 98.5 μl of NHS solution (N-hydroxysuccinimide/DMSO: 5 mg/ml) was added and stirred for 1 hour to give a solution B.
2895 μl (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.
1 ml of CypHer5E (NIR dye from Amersham Bioscience Co., 10−6 mole/ml) was mixed with 10−6 mole of ethylenediamine and stirred for 1 hour, thus giving a solution C.
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 10−6 mole of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). After the mixture was stirred for one hour, 10−6 mole of N-hydroxysuccinimide (NHS) was added and stirred for another hour, thus giving a solution D.
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.
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® and Resovist®.
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.
As shown in Table 1, the r2 relaxivity of the modified iron oxide nanoparticles of the invention was about 2 times that of Feridex® and Resovist®, 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.
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.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN2008/000823 | 4/22/2008 | WO | 00 | 3/16/2011 |
