Iron oxide nanocapsules, method of manufacturing the same, and MRI contrast agent using the same

Abstract

Provided are iron oxide nanocapsules for an MRI contrast agent having high contrast, in which a plurality of iron oxide nanoparticles having a hydrophobic ligand attached thereto are encapsulated in an encapsulation material including a biodegradable polymer and a surfactant, and which satisfy Relations 1, 2, 3, 4 and 5 below. Also a method of manufacturing the iron oxide nanocapsules is provided.
5≦100*D.sub.μ(IO)/C.sub.ω(IO)  [Relation 1]
2.5≦100*D.sub.μ(Cap)/C.sub.ω(Cap)  [Relation 2]
0.5 wt %≦F(IO)≦50 wt %  [Relation 3]
1 nm≦D.sub.μ(IO)≦25 nm  [Relation 4]
50 nm≦D.sub.μ(Cap)≦200 nm  [Relation 5]

Claims

1. Iron oxide nanocapsules, in which a plurality of iron oxide nanoparticles having an oleate ligand attached thereto are encapsulated in an encapsulation material including a biodegradable polymer and a surfactant, and which satisfy Relations 1, 2, 3, 4 and 5 below:
10≦100*D.sub.m(IO)/C.sub.v(IO)  Relation 1
2.5≦100*D.sub.m(Cap)/C.sub.v(Cap)  Relation 2
7 wt %≦F(IO)≦35 wt %  Relation 3
1 nm≦D.sub.m(IO)≦25 nm  Relation 4
50 nm≦D.sub.m(Cap)≦200 nm  Relation 5 in which: in Relation 1, D.sub.m(IO) is an average size of iron oxide nanoparticles, and C.sub.v(IO) is a standard deviation in size distribution of iron oxide nanoparticles, and in Relation 2, D.sub.m(Cap) is an average size of iron oxide nanocapsules, and C.sub.v(Cap) is a standard deviation in size distribution of iron oxide nanocapsules, and in Relation 3, F(IO) is encapsulation efficiency which refers wt % of iron oxide nanoparticles encapsulated in the iron oxide nanocapsules, and in Relation 4, D.sub.m(IO) is defined as in Relation 1, and in Relation 5, D.sub.m(Cap) is defined as in Relation 2.

2. The iron oxide nanocapsules of claim 1, which further satisfy Relation 7 below:
5≦100*D.sub.m(Cap)/C.sub.v(Cap)  Relation 7 in which, D.sub.m(Cap) and C.sub.v(Cap) are defined as in Relation 2.

3. The iron oxide nanocapsules of claim 1, which are manufactured by mixing a dispersion solution of iron oxide nanoparticles comprising 0.1˜20 wt % of iron oxide nanoparticles dispersed in an organic solvent and 0.1˜20 wt % of a biodegradable polymer dissolved therein with an aqueous surfactant solution and performing emulsification, thus preparing an emulsion, and adding water to the emulsion.

4. The iron oxide nanocapsules of claim 1, wherein the biodegradable polymer is one or more biodegradable polymers selected from polylactide, polyglycolide, and poly(lactide-co-glycolide).

5. The iron oxide nanocapsules of claim 4, wherein the surfactant is one or more surfactants selected from sodium lauryl sulfate, polyvinylalcohol, poloxamer, polysorbate, alkyldiphenyloxide disulfonate.

6. The iron oxide nanocapsules of claim 1, wherein the biodegradable polymer has a molecular weight of 1,000˜250,000 g/mol.

7. An MRI (Magnetic Resonance Imaging) T2 contrast agent, comprising the iron oxide nanocapsules of claim 1.

8. An MRI T2 liver-specific contrast agent, comprising the iron oxide nanocapsules of claim 1.

9. The iron oxide nanocapsules of claim 1, wherein the surfactant is one or more surfactants selected from sodium lauryl sulfate, polyvinylalcohol, poloxamer, polysorbate, alkyldiphenyloxide disulfonate.

Description

DESCRIPTION OF DRAWINGS

(1) The above and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments given in conjunction with the accompanying drawings, in which:

(2) FIG. 1 is a transmission electron microscope (TEM) image showing iron oxide nanoparticles having a hydrophobic ligand attached thereto, obtained in Preparative Example 1;

(3) FIG. 2 is a TEM image showing iron oxide nanoparticles having a hydrophobic ligand attached thereto, obtained in Preparative Example 2;

(4) FIG. 3 is of optical photographs showing PLGA-iron oxide nanocapsules of Example 1 dispersed in an aqueous phase, before and after encapsulation;

(5) FIG. 4 is a TEM image showing the PLGA-iron oxide nanocapsules of Example 1;

(6) FIG. 5 is of a graph and a magnetic resonance image showing the contrast enhancement in vitro of the PLGA-iron oxide nanocapsules of Example 1, compared to the results of using a commercially available contrast agent Feridex;

(7) FIG. 6 is of magnetic resonance images showing the contrast enhancement in vivo of the PLGA-iron oxide nanocapsules of Example 1, in which pre, 1 HR and 2 HR indicate measurements before injection, 1 hour after injection, and 2 hours after injection;

(8) FIG. 7 is of graphs showing the dose of the PLGA-iron oxide nanocapsules of Example 1 versus the contrast enhancement in vivo, compared to the results of using a commercially available contrast agent Feridex;

(9) FIG. 8 is a graph showing the encapsulation efficiency of the PLGA-iron oxide nanocapsules of Example 1 versus the contrast enhancement in vivo, compared to the results of using a commercially available contrast agent Resovist;

(10) FIG. 9 is a graph showing the size of the PLGA-iron oxide nanocapsules of Example 1 versus the contrast enhancement in vivo, compared to the results of using a commercially available contrast agent Resovist;

(11) FIG. 10 is of graphs showing the cell viability depending on the concentration and incubation time of the PLGA-iron oxide nanocapsules of Example 1, compared to the results of using a commercially available contrast agent Feridex;

(12) FIG. 11 is a TEM image showing PLGA-iron oxide nanocapsules of Comparative Example 1 having a uniformity of iron oxide nanoparticles of 2.8, obtained in Comparative Example 1;

(13) FIG. 12 is a TEM image showing PLGA-iron oxide nanocapsules in which the encapsulation efficiency of iron oxide nanoparticles is 0.5 wt % or less, obtained in Comparative Example 2; and

(14) FIG. 13 is of images and a graph showing the contrast enhancement in vivo of PLGA-iron oxide nanocapsules having a size of 200 nm or more, obtained in Comparative Example 3.

BEST MODE

(15) Hereinafter, the present invention will be described in detail based on the following examples and comparative examples, but is not limited to such examples.

(16) Furthermore, the fundamental concepts and embodiments of the present invention will be able to be easily modified or changed by those skilled in the art.

Preparative Example 1

(17) Mass Production of Monodispersed 10 nm Iron Oxide Nanoparticles Having Oleate Attached Thereto

(18) 10.8 g of iron oxide and 36.5 g of sodium oleate were dissolved in a solvent mixture comprising 80 ml of ethanol, 60 ml of distilled water, and 140 ml of hexane, and the obtained solution was heated to 57° C. and maintained at the same temperature for 1 hour. In this procedure, initial orange color of water phase became clear, and an initial transparent organic phase turned to a red color, after which the upper organic layer containing iron oleate complex was separated, and hexane was then evaporated, thus obtaining a viscous liquid. 36 g of the iron oleate complex (obtained a viscous liquid) was added to a mixture comprising 200 g of octadecene and 5.7 g of oleic acid.

(19) The mixture thus obtained was heated to 70° C. from room temperature at a heating rate of 2.5° C./min in a vacuum, and maintained at the same temperature for 1 hour so that the remaining solvent and moisture were removed leaving behind the reaction material. Thereafter, the reaction material was heated to 320° C. at a rate of 2.5° C./min in a nitrogen atmosphere, and maintained at the same temperature for 1 hour and aged. In this procedure, vigorous reaction occurred, and the initial red solution turned to a black brown color, meaning that the iron oleate complex was completely decomposed and iron oxide nanoparticles were produced. After the reaction completed, when the temperature arrived at an automatic ignition temperature or less (150° C.) via natural cooling, air was fed so that oxidation was carried out.

(20) The solution containing nanoparticles thus formed was cooled to room temperature, and a mixture solution comprising hexane and acetone at a volume ratio of was added in an amount corresponding to three times the volume of a stock solution, thus forming black precipitates, which were then separated using centrifugation (rpm=2,000).

(21) The supernatant was decanted. This washing process was repeated at least two times, and the hexane and acetone contained in the remainder were removed using drying, and the product thus obtained was iron oxide nanoparticles to be easily re-dispersed in hexane. FIG. 1 is a TEM image showing the finally produced iron oxide nanoparticles, having an average size of 10 nm and a size uniformity of 10.1.

Preparative Example 2

(22) Mass Production of Monodispersed 4 nm Iron Oxide Nanoparticles Having Oleate Attached Thereto

(23) Iron oxide nanoparticles were mass synthesized in the same manner as in Preparative Example 1, with the exception that 100 g of hexadecane was used as the solvent, and the final heating temperature was 280° C.

(24) The nanoparticles thus obtained were easily re-dispersed in a non-polar organic solvent such as hexane or toluene. FIG. 2 is a TEM image showing the final iron oxide nanoparticles, having an average size of 4 nm and a size uniformity of 6.15.

Example 1

(25) Manufacture of PLGA-Iron Oxide Nanocapsules Using Changes in Solubility of Biodegradable Polymer Solution

(26) 200 mg of PLGA having a carboxyl terminal group and a molecular weight (Mw) of 5,000 was added to 10 ml of ethylacetate and stirred for 10 minutes to completely dissolve it. 200 mg of the monodispersed iron oxide nanoparticles of Preparative Example 1 was added to the above solution and ultra-sonicated at 45° C. for 60 minutes, thus preparing a dispersion solution of iron oxide nanoparticles. When the iron oxide nanoparticles were not completely dispersed in the solvent, an opaque brown color was evident, and most of them precipitated within 5 minutes. On the other hand, in the case where the iron oxide nanoparticles were completely dispersed using ultrasonication, there was a deep transparent black color and precipitation did not occur for ones of days or longer.

(27) 10 ml of the dispersion solution of iron oxide nanoparticles thus prepared was mixed with 20 ml of a 5 wt % aqueous solution of Pluronic F-127 (P2443, Sigma, Cas No. 10 9003-11-6) and emulsified for 7 minutes at 20,000 rpm using a homogenizer. While the emulsified solution was immediately placed in a 200 ml beaker and stirred at 500 rpm, 100 ml of distilled water was added thereto at one time and stirring was performed for 20 minutes. The prepared solution was then placed in a dialysis membrane and stirred for two days, so that the reaction remainder was removed, after which the resultant product was frozen at −20° C., and lyophilized, thus obtaining nanocapsule powder. FIG. 3 shows images of the above PLGA-iron oxide nanocapsules dispersed in water phase. Before encapsulation the iron oxide nanoparticles having oleate attached thereto were distributed in only the hexane layer, whereas after encapsulation the nanocapsules stabilized with PLGA and Pluronic F-127 were stably distributed in the water layer and observed to be stable without precipitation for ones of weeks.

(28) FIG. 4 is a TEM image showing that the formation of the above PLGA-iron oxide nanocapsules was very uniform, having an average size of 111.6 nm and a size uniformity of 12.5, in which the iron oxide nanoparticles were observed to be uniformly distributed in an encapsulation efficiency of 10.3 wt % in the capsules.

Example 2

(29) Manufacture of PLGA-Iron Oxide Nanocapsules Using Non-Polar Organic Solvent and Distillation

(30) 200 mg of PLGA having a carboxyl terminal group and a molecular weight of 20,000 was added to 150 ml of ethylacetate and stirred for 10 minutes to completely dissolve it, thus preparing a first solution. 200 mg of the monodispersed iron oxide nanoparticles of Preparative Example 1 were added to 100 ml of hexane (a non-polar organic solvent) and ultra-sonicated for 1 hour to completely disperse them, thus preparing a second solution. The first solution and the second solution were mixed together and stirred for 30 minutes, and two solutions were observed to be completely mixed without phase separation.

(31) The mixture solution comprising the first solution and the second solution was heated to 72° C. higher than the boiling point of hexane and lower than the boiling point of ethylacetate and distilled until the amount of the remaining solution was about 10 ml, thus preparing a dispersion solution of iron oxide nanoparticles. Although hexane having an azeotropic point with ethylacetate was distilled together, its evaporation rate was faster, and thus, in the case where 10 ml of the final solution remained, the remaining hexane was 1 vol % or less, and the iron oxide nanoparticles were seen to be dispersed in the final ethylacetate left behind.

(32) 10 ml of the prepared dispersion solution of iron oxide nanoparticles was mixed with 20 ml of 5 wt % aqueous solution of Pluronic F-127 and emulsified for 7 minutes at 20,000 rpm using a homogenizer. While the emulsified solution was immediately placed in a 200 ml beaker and stirred at 500 rpm, 100 ml of distilled water was added thereto at one time and stirring was performed for 20 minutes. The prepared solution was then placed in a dialysis membrane and stirred for two days, so that the reaction remainder was removed, after which the resultant product was frozen at −20° C., and lyophilized, thus obtaining nanocapsule powder.

(33) The PLGA-iron oxide nanocapsules thus manufactured contained iron oxide nanoparticles encapsulated in a very high encapsulation efficiency of 10.3 wt % and had an average size of 178.4 nm and a size uniformity of 7.2, and were regarded as very uniform.

Example 3

(34) Manufacture of PLGA-Iron Oxide Nanocapsules Having High Encapsulation Efficiency

(35) Nanocapsules were manufactured in the same manner as in Example 1, with the exception that 40 mg of PLGA, 3 ml of ethylacetate, 200 mg of iron oxide nanoparticles, and 6 ml of 5 wt % aqueous solution of Pluronic F-127 were used, and a stirring rate upon emulsification was 26,000 rpm.

(36) The PLGA-iron oxide nanocapsules thus manufactured had an average size of 168.9 nm and a size uniformity of 6.4, and were regarded as uniform. In particular, the encapsulation efficiency of iron oxide nanoparticles was 27.4 wt % which was much higher than 7 wt % that was considered to be a threshold due to aggregation of iron oxide nanoparticles in a conventional emulsificaion-diffusion method.

Example 4

(37) Manufacture of PLGA-Iron Oxide Nanocapsules Having Small Average Size

(38) Nanocapsules were manufactured in the same manner as in Example 1, with the exception that 40 mg of PLGA, 3 ml of ethylacetate, 40 mg of iron oxide nanoparticles, and 6 ml of 5 wt % aqueous solution of Pluronic F-127 were used, and a stirring rate upon emulsification was 26,000 rpm.

(39) The PLGA-iron oxide nanocapsules thus manufactured contained the iron oxide nanoparticles encapsulated in a very high encapsulation efficiency of 8.8 wt % and had an average size of 90.9 nm and a size uniformity of 7.2, and were regarded as very small and uniform.

Example 5

(40) Measurement of Magnetic Resonance Relaxivity In Vitro of Nanocapsules

(41) In order to evaluate the usability of the PLGA-iron oxide nanocapsules of Example 1 as an MRI T2 liver-specific contrast agent, T2 relaxivity in vitro was measured using a BGA12 gradient coil in a 4.7 T magnetic resonance imaging system (Biospec 47/40, Bruker Biospin MRI GmbH). The iron concentration of iron oxide-PLGA nanocapsule powder was analyzed via ICP-AES, and dispersed in a concentration of 1˜4 μg Fe/ml in a solution of 0.01M PBS (Phosphate Buffered Saline, pH 7.4). The first solution was diluted by half to prepare a total of five kinds of samples, which were then placed in 250 μl tubes to measure T2 relaxation time at one time. The measurement of T2 relaxation time was performed using MSME (Multi Slice Multi Echo sequence) pulse sequence. Specific parameters are as follows.

(42) TR(repetition time)=10,000 ms, TE(echo time)=8-2048 ms (256 times at intervals of 8 ms), FOV=60×40 mm, Resolution=0.234×0.156 mm/pixel, slice thickness=1 mm, number of acquisition=1, Matrix size=128×128

(43) FIG. 5 is of a graph and a magnetic resonance image showing the contrast enhancement in vitro of the PLGA-iron oxide nanocapsules of Example 1, from which the contrast enhancement of the capsules manufactured using the method according to the present invention can be seen to be much higher than that of the commercially available contrast agent.

Example 6

(44) Measurement of Magnetic Resonance Relaxivity In Vivo of Nanocapsules

(45) In order to evaluate the usability of the PLGA-iron oxide nanocapsules of Example 1 as an MRI T2 liver-specific contrast agent, T2 relaxivity in vivo was measured using BGA12 gradient coil in a 4.7 T magnetic resonance imaging system (Biospec 47/40, Bruker Biospin MRI GmbH).

(46) The MR test in vivo was carried out using a five-week-old male Balb/c mouse weighing about 20˜25 g. The mouse was anesthetized and then placed horizontally in an MRI apparatus to observe the coronal plane, and in order to observe the liver tissue of the same plane, the mouse was subjected to inhalation anesthesia for the total test time so that the mouse barely moved. The iron concentration of iron oxide-PLGA nanocapsule powder was analyzed using ICP-AES, and dispersed in 0.01M PBS solution, after which 200 μl of the PLGA-iron oxide nanocapsule solution was injected at one time through the tail vein of the mouse, and the final dosage of the solution was set to 1 mg Fe/kg in consideration of the body weight of the mouse. The T2 relaxation time was measured using RARE (Rapid Acquisition with Refocused Echoes) pulse sequence, and specific parameters thereof are as follows.

(47) TR(repetition time)=3,500 ms, TE(echo time)=36 ms, FOV=60×40 mm, Resolution=0.234×0.156 mm/pixel, slice thickness=1 mm, number of acquisition=4, Matrix size=256×256

(48) In order to quantitatively measure T2 decay effects of PLGA-iron oxide nanocapsules, a cross-section of the liver tissue was selected and the entire liver portion was adopted as a ROI (Region of Interests) and signal intensity (SI) thereof was analyzed. In order to maximize the reliability of the obtained SI, a 1 wt % agarose solution was placed in a 200 μl tube, cooled, solidified, fixed around the abdominal cavity of the mouse, and used as the control. The T2 decay effect of PLGA-iron oxide nanocapsules was calculated from Equation 1 below and graphed.
T2 decay effect (ΔR2)=100*[1−(SNR).sub.τ/(SNR).sub.0](SNR: Signal to Noise Ratio)  (Equation 1)
(SNR).sub.τ=(SI of ROI).sub.τ/(SI of Agarose).sub.τ
(SNR).sub.0=(SI of ROI).sub.0/(SI of Agarose).sub.0

(49) In Equation 1, SI of ROI means the signal intensity in the liver corresponding to the region of interest, and SI of agarose means the signal intensity of agarose used as a control for the liver tissue, t means the signal intensity at t time after adding the contrast agent, and 0 means the signal intensity just before adding the contrast agent.

(50) The contrast enhancement results of the PLGA-iron oxide nanocapsules of Example 1 and the commercially available contrast agent are compared in FIG. 4. The maximum T2 decay effect (ΔR2.sub.μαξ) of commercially available contrast agent was about 58%, whereas the PLGA-iron oxide nanocapsules according to the present invention exhibited a maximum T2 decay effect of about 73%, which was much higher than that of the commercially available contrast agent. Even when the inventive nanocapsules were used in a small amount, the diseased portion of the liver could be accurately diagnosed.

(51) FIG. 6 is of magnetic resonance images showing the contrast enhancement in vivo of the PLGA-iron oxide nanocapsules of Example 1, in which the contrast enhancement in vivo of the inventive capsules can be seen to be much higher than that of the commercially available contrast agent.

(52) FIG. 7 is of graphs showing the dose of the PLGA-iron oxide nanocapsules of Example 1 versus the contrast enhancement in vivo, in which the contrast enhancement in vivo of the inventive capsules is adjusted depending on the dose and is higher over the entire dose range compared to when using the commercially available contrast agent. In particular, the contrast enhancement thereof can be observed to be much higher compared to using 0.42 mg Fe/kg corresponding to the human dose of the commercially available contrast agent.

(53) FIG. 8 is a graph showing the encapsulation efficiency of the PLGA-iron oxide nanocapsules of Example 1 versus the contrast enhancement in vivo, in which the contrast enhancement in vivo of the inventive capsules can be adjusted depending on the encapsulation efficiency. In particular, when the encapsulation efficiency of iron oxide nanoparticles is 19˜27 wt %, the contrast enhancement can be observed to be higher than when using capsules having an encapsulation efficiency of 11 wt % or less.

(54) FIG. 9 is a graph showing the size of the PLGA-iron oxide nanocapsules of Example 1 versus the contrast enhancement in vivo, in which the contrast enhancement in vivo of the inventive capsules can be adjusted by the size of the capsules. In the case where the capsules have a small size of 100 nm or less, contrast enhancement is observed to increase, and thus the size of the capsules and the size uniformity are regarded as very important.

Example 7

(55) Measurement of Cytotoxicity of Nanocapsules

(56) In order to evaluate the cytotoxicity of the PLGA-iron oxide nanocapsules of Example 1, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)] assay was performed as a cell cytotoxicity test. The test was performed using HEK293 cell as the human kidney cell line and HepG2 cell as the liver cell line, and both of the two cell lines were adherent cells propagating in a state of attached to the surface of test vessel, and were inoculated in a density of 1×10.sup.5 in a 96-well plate.

(57) The cells were incubated for 24 hours after which the culture medium was removed, and the PLGA-iron oxide nanocapsules having a maximum concentration of 9762 ppm were diluted by half up to 19 ppm to prepare a total of nine kinds of solutions, 12.5 μl of each of which was added along with 87.5 μl of the culture medium to each well. Respective wells were incubated for 24 hours, and the culture medium was removed, 20 μl of MTT solution was added thereto, and incubation was carried out for 4 hours. Finally, 100 μl of a solubilization solution was added thereto and incubated, after which absorbance at 550 nm was measured, thus determining the cell viability.

(58) As shown in FIG. 10, regardless of the concentration of PLGA-iron oxide nanocapsules of Example 1 and the incubation time, the viability was high for the entire cell line, and no cell toxicity was observed even at a concentration at least 300 times the use concentration of contrast agent comprising the PLGA-iron oxide nanocapsules according to the present invention.

Comparative Example 1

(59) PLGA-Iron Oxide Nanocapsules Having Uniformity of Iron Oxide Nanoparticles of 10 or Less

(60) Using 10 ml of a solution of iron oxide nanoparticles prepared in the same manner as in Example 1 with the exception that 100 mg of 4 nm iron oxide nanoparticles and 100 mg of 10 nm iron oxide nanoparticles were mixed, the same procedures as in Example 3 were performed, thus manufacturing nanocapsules having a size uniformity of iron oxide nanoparticles of 2.8.

(61) FIG. 11 is a TEM image showing the PLGA-iron oxide nanocapsules having a uniformity of iron oxide nanoparticles of 2.8. The average size of the capsules comprising 25 wt % of iron oxide nanoparticles having an average size of 4 nm and 75 wt % of iron oxide nanoparticles having an average size of 10 nm encapsulated therein was 146 nm, and the size uniformity of the capsules was approximately uniform to the level of about 5.12. However, because of low uniformity of iron oxide nanoparticles, T2 relaxivity was measured to be 202.8 mM.sup.−1 s.sup.−1, which was much lower than 345.7 mM.sup.−1 s.sup.−1 when using only the 10 nm iron oxide nanoparticles.

Comparative Example 2

(62) PLGA-Iron Oxide Nanocapsules Having Encapsulation Efficiency of 0.5 Wt % or Less

(63) Using 10 ml of a solution of iron oxide nanoparticles prepared in the same manner as in Example 1 with the exception that 4 mg of iron oxide nanoparticles was used, the same procedures as in Example 3 were performed, thus manufacturing nanocapsules.

(64) FIG. 12 is a TEM image showing the PLGA-iron oxide nanocapsules having an encapsulation efficiency of iron oxide nanoparticles of 0.5 wt % or less. As results of encapsulating the iron oxide nanoparticles having an average size of 10 nm and a size uniformity of 10 or more, very uniform PLGA-iron oxide nanocapsules having an average size of 147.6 nm and a size uniformity of 20 were manufactured. However, the use thereof as a contrast agent is limited because of the proportion of iron oxide being too low.

Comparative Example 3

(65) PLGA-Iron Oxide Nanocapsules Having Average Size of 200 nm or More

(66) Nanocapsules were manufactured in the same manner as in Example 3 with the exception that 10 ml of the solution of iron oxide nanoparticles prepared in Example 1 was used, the stirring rate upon emulsification was 7,000 rpm. FIG. 13 is of an image and a graph showing the contrast enhancement in vivo of the PLGA-iron oxide nanocapsules having a size of 200 nm or more. As results of encapsulating iron oxide nanoparticles having an average size of 10 nm and a size uniformity of 10 or more, PLGA-iron oxide nanocapsules having an average size of 520.9 nm and a size uniformity of 2.2 were manufactured. The capsules were in the size ranging from 100 nm to ones of μm. In particular, PLGA-iron oxide nanocapsules having a size of 200 nm or more exhibited low T2 relaxivity and low distribution in the liver and thus the maximum T2 decay effect (A R2.sub.μαξ) was only about 34%.

(67) Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions, and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.