Abstract:
The present invention relates to a process for preparing water-soluble and dispersed iron oxide (Fe 3 O 4 ) nanoparticles and application thereof, characterized in which two-stage additions of protective agent and chemical co-precipitation are employed in the process. In the first stage, Fe 3 O 4  nanoparticles are obtained using absorbent-reactant coexistence technology. In the second stage, proper amount of adherent is added to cover the nanoparticle surface entirely. The resulting water-soluble and dispersed Fe 3 O 4  nanoparticles can easily bind with thiols or biomolecules, such as nucleic acid and peptide. The Fe 3 O 4  nanoparticles of the present invention may be used as magnetic resonance imaging contrast agent and used in magnetic guiding related biomolecular technologies for clinical testing, diagnosis and treatment.

Description:
CROSS REFERENCES TO THE RELATED APPLICATIONS 
       [0001]    This application is a Continuation-in-part of pending U.S. application Ser. No. 10/882,210, filed Jul. 2, 2004. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention provides a method for preparing water-soluble and dispersed iron oxide nanoparticles and its applications in magnetic resonance imaging as contrast agent, and in magnetic guiding related biomolecular technologies and clinical testing, diagnosis and treatment. 
         [0004]    2. Description of the Related Art 
         [0005]    Nanoparticles are very small particles with general size ranging from 1 nm to 100 nm. Given their tiny dimensions, nanoparticles exhibit many special properties related to their surface and volume, for example, very high surface area and surface energy, discrete electronic energy level, special light absorption, and single magnetic domain. Therefore nanoparticles provide great potential in the development of new materials. Every magnetic nanoparticle has specific magnetic orientation. But when the particle is very small, its magnetic field becomes unstable. Such magnetic nanoparticles may be used to carry drug into the body of patients, in which the drug is delivered to different parts of the body through magnetic force. Magnetic nanoparticles can also improve the magnetic resonance imaging (MRI) technology by enhancing the imaging contrast to help doctors identify tumor cells, arterial plaques and central nervous system diseases. 
         [0006]    Magnetic Fe 3 O 4  nanoparticles are usually prepared by the standard aqueous precipitation technique of Fe 2+  and Fe 3+  ions from a basic solution. But those nanoparticles would aggregate in the solution if without any stabilizer. A coating of polymer or surfactant on the surface of nanoparticles helps the nanoparticles to become better dispersed in solution, either in aqueous phase (water-soluble) or in oil phase (oil-soluble). Markovich et al. (Adv. Mater, 2001, 13, 1158-1161) discloses oil-soluble Fe 3 O 4  nanoparticles in hexane or heptane by the coating of oleic acid. But the application of oil-soluble Fe 3 O 4  nanoparticles is limited. Most iron oxide nanoparticles used in biomedicine are required to be water-soluble, and coated with a layer of substance, such as protein, hydrophilic polymers, starch and glucan, so as to increase their water solubility and dispersibility. However, the above-mentioned substance have very high molecular weight, which increases the volume of the nanoparticles. When used in intravenous injection, iron oxide nanoparticles coated with those substance are 30 to 150 nm in size and mainly in aggregate form. 
         [0007]    For the preparation of water-soluble nanoparticles, Huang et al. (US2004/0115345A1) discloses water-soluble gold nanoparticles protected by tiopronin or coenzyme A monolayers. After the formation of gold nanoparticles, a thiol group containing organic compound is used to protect the nanoparticles by the strong binding affinity between gold and thiol group. However, the unique and well-known binding affinity only presents between gold and thiol group. Iron oxide nanoparticle has no such property. One cannot use the same mechanism to stabilize water-soluble iron oxide nanoparticles. 
         [0008]    Additionally, contrast agents for magnetic resonance imaging currently available on the market are mainly Gd 3+  based. Gadolinium (Gd) is a heavy metal with cytotoxicity. Using improper dosage or formulation of Gd 3+  contrast agent might produce adverse health effect. Sometimes Gd 3+  contrast agent produces “false positive signal”, or “false negative signal” when its concentration is diluted by body fluids. 
         [0009]    To overcome the above mentioned problems, it is desirable to develop novel super-paramagnetic iron oxide nanoparticles as a contrast agent, which have better stability and biocompatible property. 
       SUMMARY OF THE INVENTION 
       [0010]    To address the drawbacks of prior arts for making water-soluble and dispersed iron oxide nanoparticles and the limitation of magnetic resonance imaging contrast agents currently on the market, the present invention discloses a technology for preparing highly water-soluble iron oxide in aqueous phase process that displays super-paramagnetic behavior and may be used as MRI contrast agent. The iron oxide also easily binds biomolecules and drugs due to its simple coating interface and aqueous phase process. Thus the technology disclosed in the present invention may be further developed into a platform technology for functional imaging and target treatment. 
         [0011]    An object of the present invention is to provide a method for preparing water-soluble and dispersed Fe 3 O 4  nanoparticles, comprising the steps of: (a) mixing solutions containing Fe 2+  and Fe 3+  at the concentration of 1:2 to 1:4; (b) adding an organic acid as adherent, said organic acid is selected from the group consisting of acetic acid, cysteine, alanine, and glycine; (c) adjusting pH value of the foregoing solution to over 10 to produce a precipitate; (d) collecting and washing said precipitate; (e) adding in relation to step (b), an amount of an organic acid to provide a molar equivalent ratio of organic acid/Fe 3+  of greater than 112 to achieve an entire coverage of the surface of the nanoparticles, said organic acid is selected from the group consisting of acetic acid, cysteine, alanine, and glycine; (f) adding organic solvent and water to remove the excess amount of organic acid in step (e); and (g) collecting purified Fe 3 O 4  nanoparticles. 
         [0012]    The pre-determined mixing ratio of Fe 2+  and Fe 3+  solutions in step (a) is preferably 1:2. 
         [0013]    The organic acid in steps (b) and (e) is preferably glycine. The amount of the organic acid in step (b) provides a molar equivalent ratio of organic acid/Fe 3+  of 6 to 7, preferably. The organic acids used in steps (b) and (e) may be the same or different, preferably the same. Said organic acid is used as an adherent. In step (b), Fe 3 O 4  nanoparticles are obtained using the adherent-reactant coexistence technology; in step (e), the adherent is added to achieve complete coating of the nanoparticle surface and result in water-soluble and dispersed Fe 3 O 4  nanoparticles. 
         [0014]    In step (c) a base, e.g. NaOH, NH 4 OH or other similar substances, is added to adjust the pH. 
         [0015]    The organic solvent in step (f) is selected from a group consisting of acetone, methanol, ethanol, and n-hexane, preferably acetone. 
         [0016]    The aforesaid method is preferably carried out under 20˜40° C., preferably 25° C. 
         [0017]    Another object of the present invention is to provide Fe 3 O 4  nanoparticles, characterized in which said nanoparticles are water-soluble and well dispersed averaging 6.2 nm±2.2 nm in size; wherein said Fe 3 O 4  nanoparticles are coated with organic acid as adherents; said organic acid is selected from the group consisting of acetic acid, cysteine, alanine, and glycine. 
         [0018]    The Fe 3 O 4  nanoparticle of the present invention has —NH 2  group on its surface, and molecules (such as protein, enzyme or drugs) could directly attach on the nanoparticles through —NH 2  group. The —NH 2  group is provided by small molecule weight organic acids (acetic acid, cysteine, alanine, or glycine), hence the nanoparticle have reduced volume. 
         [0019]    A further object of the present invention is to provide a contrast agent for magnetic resonance imaging containing primarily water-soluble and dispersed Fe 3 O 4  nanoparticles prepared according to the method described above and water. 
         [0020]    In summary, the present invention uses small molecule weight organic acids (glycine, acetic acid, cysteine and alanine) to prepare uniformly distributed and water-soluble Fe 3 O 4  nanoparticles without adding any polymer or surfactant. Such Fe 3 O 4  nanoparticle is advantageous of its dispersibility and biocompatibility, and therefore may be use as MRI contrast agent and widely applied in biomedical testing and treatment in the future. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0021]      FIG. 1  shows the flow chart for preparing Fe 3 O 4  nanoparticles according to the invention. 
           [0022]      FIG. 2  shows a TEM image of Fe 3 O 4  nanoparticles of the invention dissolved in water. 
           [0023]      FIG. 3A  shows a liver MRI scan prior to using Fe 3 O 4  nanoparticle contrast agent. 
           [0024]      FIG. 3B  shows a liver MRI scan after using Fe 3 O 4  nanoparticle contrast agent. 
           [0025]      FIG. 4A  is a kidney MRI scan prior to using Fe 3 O 4  nanoparticle contrast agent. 
           [0026]      FIG. 4B  is a kidney MRI scan after using Fe 3 O 4  nanoparticle contrast agent. 
           [0027]      FIG. 5  shows the survival rate of rats after being injected with Fe 3 O 4  nanoparticle contrast agent. 
           [0028]      FIG. 6  shows the thermogravimetric analysis data of Fe 3 O 4  nanoparticles prepared in example 1. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0029]    The method for preparing water-soluble and dispersed Fe 3 O 4  nanoparticles according to the present invention as shown in  FIG. 1  comprises the steps of: (a) mixing solutions containing Fe 2+  and Fe 3+  at the concentration of 1:2 to 1:4; (b) adding an organic acid as adherent, said organic acid is selected from the group consisting of acetic acid, cysteine, alanine, and glycine; (c) adjusting pH value of the foregoing solution to over 10 to produce a precipitate; (d) collecting and washing the precipitate of Fe 3 O 4  nanoparticles; (e) adding, in relation to step (b), and amount of an organic acid to provide a molar equivalent ratio of organic acid/Fe 3+  of greater than 112 to achieve an entire coverage of the surface of the nanoparticles, said organic acid is selected from the group consisting of acetic acid, cysteine, alanine, and glycine; (f) adding organic solvent and water to remove the excess amount of organic acid in step (e); and (g) collecting purified Fe 3 O 4  nanoparticles. 
         [0030]    Examples are illustrated below to depict the preparation of water-soluble and dispersed Fe 3 O 4  nanoparticles and its application as MRI contrast agent. 
       EXAMPLE 1 
     Preparation of Water-Soluble Fe 3 O 4  Nanoparticles 
       [0031]    For the preparation of water-soluble Fe 3 O 4  nanoparticles of the present invention, the amount of adherents required to achieve an entire coverage of the surface of the nanoparticles is calculated as follows: 
         [0032]    Each Fe 3 O 4  molecule contains one Fe 2+  ion and two Fe 3+  ions, so the molar ratio of Fe 3+ :Fe 3 O 4 =2:1. As taking the amount of Fe 3+  as reference, one mole Fe 3+  ion and 0.5 mole Fe 2+  could obtain 0.5 mole Fe 3 O 4  molecules, theoretically. According to the size of nanoparticles (6.22 nm±2.2 nm obtained by TEM) and the volume of Fe 3 O 4  crystal lattice, each Fe 3 O 4  nanoparticle contains 1785 Fe 3 O 4  molecules, which means 0.5 mole Fe 3 O 4  molecules would obtain 0.5÷1785=0.00028 mole Fe 3 O 4  nanoparticles. 
         [0033]    According to the TGA (Thermogravimetric Analysis) data of Fe 3 O 4  nanoparticle coated with adherents as shown in  FIG. 6 , each Fe 3 O 4  nanoparticle has totally 250˜400 adherent molecules attached on its surface. By using the maximum value, 400, 0.00028 mole Fe 3 O 4  nanoparticles will need 0.00028×400=0.112 mole adherent molecules. But this is the minimum amount just enough for covering whole surface. Since the covering efficiency is positively related to the amount of adherents added into the solution. 1000 fold relative to the minimum amount will be used to make sure of 100% covering efficiency. Therefore, the amount of adherent molecule added is at least 1000×0.112=112 mole, when using 1 mole of Fe 3+  and 0.5 mole Fe 2+  as raw material. Accordingly, a molar ratio of greater than 112 for adherent molecule to Fe 3+  would be used in the following preparation of Fe 3 O 4  nanoparticles. 
         [0034]    First mix 1 ml of 0.2M FeCl 2  and 4 ml of 0.1M FeCl 3  in 2M HCl solution, then add 1 g of glycine (preferably 0.5˜1.5 g) slowly drip 5M NaOH solution into the mixture to adjust its pH to over 10 to provide an alkaline environment for Fe 3 O 4  in the solution to precipitate; next agitate for 10 minutes, then wash with D.I. water several times to collect the black precipitate (Fe 3 O 4 ); next add 3 g of glycine as adherent (the total molar ratio of glycine to Fe 3+  is about 117.5); agitate 10˜15 minutes and then vibrate for 30 minutes to let the adherent cover the surface of Fe 3 O 4  nanoparticles entirely; subsequently add obtained Fe 3 O 4  nanoparticles to acetone and water mixture to remove excess organic acid adherent; centrifuge at 8000 rpm for 20 minutes to precipitate the Fe 3 O 4  nanoparticles to obtain water-soluble and dispersed Fe 3 O 4  nanoparticles disclosed in the invention.  FIG. 2  shows the electron microscope image of resulting Fe 3 O 4  nanoparticles dissolved in D. I. water with particle size of 6.2 nm±2.2 nm and exhibiting good, stable and long-lasting water solubility and dispersibility. 
       EXAMPLE 2 
     Using Fe 3 O 4  Nanoparticles as MRI Contrast Agent-Injected in Liver 
       [0035]    In this example, Fe 3 O 4  nanoparticles prepared in Example 1 were used as MRI contrast agent. The contrast agent was prepared by dissolving the Fe 3 O 4  nanoparticles in D. I. water, and if necessary, adding to it proper amount of serum or similar body fluid. 
         [0036]      FIG. 3A  shows the MRI scan before Fe 3 O 4  nanoparticles were injected into the liver;  FIG. 3B  shows the MRI scan after the liver was injected with 0.86 μM Fe 3 O 4  nanoparticles. By comparing where the arrows are pointed at in  FIGS. 3A and 3B , it is clearly shown that Fe 3 O 4  nanoparticles indeed entered the liver to provide the contrast enhancement effect. 
       EXAMPLE 3 
     Using Fe 3 O 4  Nanoparticles as MRI Contrast Agent-Injected in Kidney 
       [0037]    In this example, Fe 3 O 4  nanoparticles as described in Example 2 were used as MRI contrast agent and injected in kidney to observe its enhancement effect. 
         [0038]      FIG. 4A  shows the MRI scan before Fe 3 O 4  nanoparticles were injected into the kidney;  FIG. 4B  shows the MRI scan after the kidney was injected with 0.86 μM Fe 3 O 4  nanoparticles. By comparing where the arrows are pointed at in  FIGS. 4A and 4B , it is clearly shown that Fe 3 O 4  nanoparticles indeed entered the kidney to provide the contrast enhancement effect. 
       EXAMPLE 4 
     The Safety of Using Fe 3 O 4  Nanoparticles as MRI Contrast Agent 
       [0039]    In this test, rats were injected with 5 mg/kg of Fe 3 O 4  nanoparticles and observed for survival at week 0, 2, 4, and 6. The finding as shown in  FIG. 5  indicates that none of the rates died; the survival rate was 100%. Thus Fe 3 O 4  nanoparticles were considered safe as a contrast agent. 
         [0040]    To sum up, in comparison with prior art, the technology disclosed herein have the following advantages: 
         [0041]    1. The technology disclosed in the invention can produce highly water-soluble and uniformly dispersed Fe 3 O 4  nanoparticles without using hydrophilic polymer, surfactant, protein, starch or glucan as protective agent, and offers greater room for subsequent design of surface modification and binding. 
         [0042]    2. The Fe 3 O 4  nanoparticles of the present invention can bind with nucleic acids, proteins and other biomolecules by forming covalent bond or non-covalent bond for applications in biomedical field. 
         [0043]    3. In comparison with contrast agents currently available on the market, the Fe 3 O 4  nanoparticle contrast agent herein have very small particle size (6.2 nm±2.2 nm). And because the particle is of nano size and exhibits super-paramagnetic characteristics, its relaxation rate T 1  is far lower than the SPIO system on the market (also Fe 3 O 4  nanoparticle contrast agent). Table 1 compares the relaxation rate T 1  and T 2  of Fe 3 O 4  nanoparticles herein, SPIO contrast agent, and Gd 3+  contrast agent. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Fe 3 O 4  nanoparticle 
                   
                   
               
               
                   
                 contrast agent of the 
               
               
                   
                 invention 
                 SPIO contrast agent 
                 Gd 3+  contrast agent 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 T1 
                 34 ms 
                  176 ms 
                 74.4 ms 
               
               
                 T2 
                 23 ms 
                 0.77 ms 
               
               
                   
               
               
                 * All contrast agents have the same concentration of 4.61 mM (concentration of metal ion). 
               
             
          
         
       
     
         [0044]    4. As shown in Table 1, T 1  of the Fe 3 O 4  nanoparticles of the invention is much lower than that of SPIO and Gd 3+  contrast agent. In the aspect of contrast enhancement effect, Gd 3+  is superior to iron oxide (under ionic concentration of 1E-1˜1E-2M). But the Fe 3 O 4  nanoparticle contrast agent of the invention exhibits better contrast enhancement effect than SPIO with serum or water as solvent. 
         [0045]    5. The T 2  of Fe 3 O 4  nanoparticle contrast agent of the invention is not lower than SPIO. But the T 2  effect of Fe 3 O 4  nanoparticle contrast agent of the invention under ionic concentration of 1E-1 ˜1E-2M is comparable to that of SPIO. 
         [0046]    6. In comparison with SPIO system available on the market (also iron oxide nanoparticle contrast agent), the Fe 3 O 4  nanoparticle contrast agent of the invention is water soluble and dispersed without the protection of starch or glucan. Its T 1  effect is better than that of SPIO and its T 2  effect is comparable to that of SPIO. 
         [0047]    7. In comparison with Gd 3+  contrast agent, the Fe 3 O 4  nanoparticle contrast agent of the invention is non-toxic, has low immunostimulation and does not precipitate in the body. It also costs less to make than the Gd 3+  process and does not require the protection of chelating agent. 
         [0048]    The preferred embodiments of the present invention have been disclosed in the examples. However the examples should not be construed as a limitation on the actual applicable scope of the invention, and as such, all modifications and alterations without departing from the spirits of the invention and appended claims, including the other embodiments shall remain within the protected scope and claims of the invention.