Contrast agent for nuclear magnetic resonance imaging comprising melanin nanoparticles stably dispersed in water

Abstract

The present invention relates to a contrast agent for nuclear magnetic resonance imaging, and more particularly, to a contrast agent for nuclear magnetic resonance imaging containing melanin nanoparticles having a uniform shape and size, thereby providing good dispersibility in water, no cell toxicity, and a long retention time in vivo.

Claims

1. A contrast agent for nuclear magnetic resonance imaging, comprising: melanin nanoparticles having stable dispersibility in water; and paramagnetic metal ions which are coordinated on the surface of the melanin nanoparticles.

2. The contrast agent according to claim 1, wherein the melanin is obtained from the ink of cuttlefish and has a diameter of 30 nm to 600 nm.

3. The contrast agent according to claim 1, wherein the melanin nanoparticle is synthesized from a melanin precursor of dopamine, DOPA or cysteine, and has a diameter of 30 nm to 600 nm.

4. The contrast agent according to claim 1, wherein the paramagnetic metal ion is one or more metal ions selected from the group consisting of gadolinium (Gd), iron (Fe), manganese (Mn), nickel (Ni), copper (Cu), erbium (Er), europium (Eu), holmium (Ho) and chromium (Cr).

5. The contrast agent according to claim 1, wherein the surface of the melanin nanoparticles is modified with 3-mercaptopropionic acid.

6. The contrast agent according to claim 1, wherein an antibody is bound to the surface of the melanin nanoparticles.

7. The contrast agent according to claim 6, wherein the antibody is Cetuximab or Trastuzumab.

8. The contrast agent of claim 1, further comprising polyethyleneglycol (PEG) attached to the surface of the melanin nanoparticles for surface modification.

9. The contrast agent according to claim 8, wherein the surface of the melanin nanoparticles is modified with amine- or thiol- functionalized PEG.

10. The contrast agent according to claim 8, wherein the PEG has a molecular weight of 1 KDa to 40 KDa.

11. The contrast agent according to claim 8, wherein the surface of the melanin nanoparticles is modified with 3-mercaptopropionic acid.

12. The contrast agent according to claim 8, wherein an antibody is bound to the surface of the melanin nanoparticles.

13. A method for preparing a contrast agent for nuclear magnetic resonance imaging comprising melanin nanoparticles having stable dispersibility in water and paramagnetic metal ions which are coordinated on the surface of the melanin nanoparticles, the method comprising: adding a solution containing paramagnetic metal ions to a solution containing melanin nanoparticles to form coordinate bonds between the paramagnetic metal ions and melanin of the melanin nanoparticles, and forming melanin nanoparticles having stable dispersibility in water.

14. The method according to claim 13, further comprising adding 3-mercaptopropionic acid.

15. The method according to claim 14, further comprising binding the prepared melanin nanoparticles with an antibody.

16. The method according to claim 15, wherein the antibody is Cetuximab or Trastuzumab.

17. The method according to claim 13, wherein the melanin is obtained from the ink of cuttlefish and has a diameter of 30 nm to 600 nm.

18. The method according to claim 13, wherein the melanin nanoparticles are synthesized from a melanin precursor of dopamine, DOPA or cysteine, and have a diameter of 30 nm to 600 nm.

19. The method according to claim 13, wherein the paramagnetic metal ion is one or more metal ions selected from the group consisting of gadolinium (Gd), iron(Fe), manganese (Mn), nickel (Ni), copper (Cu), erbium (Er), europium (Eu), holmium (Ho) and chromium (Cr).

20. The method of claim 13, further comprising adding polyethyleneglycol (PEG) to the solution containing paramagnetic metal ions and melanin nanoparticles.

21. The method according to claim 20, wherein the PEG has a molecular weight of 1 KDa to 40 KDa.

22. The method according to claim 20, wherein the melanin nanoparticles are synthesized from a melanin precursor of dopamine, DOPA or cysteine, and have a diameter of 30 nm to 600 nm.

23. The method according to claim 20, wherein the paramagnetic metal ion is one or more metal ions selected from the group consisting of gadolinium (Gd), iron(Fe), manganese (Mn), nickel (Ni), copper (Cu), erbium (Er), europium (Eu), holmium (Ho) and chromium (Cr).

24. A contrast agent produced by the method of claim 13.

25. A contrast agent produced by the method of claim 20.

26. The contrast agent according to claim 25, wherein the melanin is obtained from the ink of cuttlefish and has a diameter of 30 nm to 600 nm.

27. The contrast agent according to claim 25, wherein the melanin nanoparticles are synthesized from a melanin precursor of dopamine, DOPA or cysteine, and have a diameter of 30 nm to 600 nm.

28. The contrast agent according to claim 25, wherein the paramagnetic metal ion is one or more metal ions selected from the group consisting of gadolinium (Gd), iron (Fe), manganese (Mn), nickel (Ni), copper (Cu), erbium (Er), europium (Eu), holmium (Ho) and chromium (Cr).

29. The contrast agent according to claim 25, wherein the surface of the melanin nanoparticles is modified with amine-or thiol-functionalized PEG.

30. The contrast agent according to claim 25, wherein the PEG has a molecular weight of 1 KDa to 40 KDa.

31. The contrast agent according to claim 25, wherein the surface of the melanin nanoparticles is modified with 3-mercaptopropionic acid.

32. The contrast agent according to claim 25, wherein an antibody is bound to the surface of the melanin nanoparticles.

33. The contrast agent according to claim 32, wherein the antibody is Cetuximab or Trastuzumab.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic illustration showing formation of coordinate bonds between paramagnetic metal ions and melanins of melanin nanoparticles;

(2) FIG. 2 is a TEM image of melanin nanoparticles (MelNPs) according to one embodiment of the present invention;

(3) FIG. 3 is a TEM image of melanin nanoparticles (sepia melanin) according to one embodiment of the present invention;

(4) FIG. 4 shows IR spectra (FIG. 4A) and UV/Vis spectra (FIG. 4B) of melanin nanoparticles (MelNPs and sepia melanin) according to one embodiment of the present invention;

(5) FIG. 5 is a TEM image of melanin nanoparticles (MelNPs) according to one embodiment of the present invention;

(6) FIG. 6 shows the result of measuring Fe.sup.3+ concentrations of Fe.sup.3+-coordinated melanin nanoparticles (Fe.sup.3+-MelNPs and Fe.sup.3+-sepia melanin) according to one embodiment of the present invention;

(7) FIG. 7 shows ESR signals of Fe.sup.3+-coordinated melanin nanoparticles (Fe.sup.3+-MelNPs and Fe.sup.3+-sepia melanin) according to one embodiment of the present invention;

(8) FIG. 8 shows the result of measuring size-dependent Fe.sup.3+ concentrations of Fe.sup.3+-coordinated melanin nanoparticles (Fe.sup.3+-MelNPs and Fe.sup.3+-sepia melanin) according to one embodiment of the present invention;

(9) FIG. 9 shows a TEM image (FIG. 9A) FT-IR spectra (FIG. 9B), dispersibility (FIG. 9C), and T1 excitation time graph (FIG. 9D) of PEGylated Fe.sup.3+-coordinated melanin nanoparticles (PEGylated Fe.sup.3+-MelNPs) according to one embodiment of the present invention;

(10) FIG. 10 shows viability of HeLa cells by treatment of PEGylated Fe.sup.3+-coordinated melanin nanoparticles (PEGylated Fe.sup.3+-MelNPs) according to one embodiment of the present invention, in which FIG. 10A shows cell viability according to concentrations of melanin nanoparticles, FIG. 10B snows a non-treated control group, and FIG. 10C shows the result of PEGylated Fe.sup.3+-MelNPs treatment;

(11) FIG. 11 shows MRI properties of Fe.sup.3+- coordinated melanin nanoparticles (Fe.sup.3+-MelNPs and Fe.sup.3+-sepia melanin) according to one embodiment of the present invention, in which each concentration of the melanin nanoparticles was 4 mg/mL; (a) T.sub.1 and T.sub.2 weighted MR images obtained from Sepia before and after chelation with Fe.sup.3+ion (b) T.sub.1 and T.sub.2 weighted MR images of MelNP before and after chelation with Fe.sup.3+ion (c) plot of 1/T.sub.1 and 1/T.sub.2 against Fe concentration of Fe.sup.3+-Sepia and (d) Fe.sup.3+-MelNP

(12) FIG. 12 shows T1-weighted MRI image of PEGylated Fe.sup.3+-coordinated melanin nanoparticles (PEGylated Fe.sup.3+-MelNPs) according to one embodiment of the present invention, in which Sp indicates spleen, Ki indicates kidney, Li indicates liver, and St indicates stomach; and

(13) FIG. 13 shows T1-weighted MRI image of PEGylated Fe.sup.3+-coordinated melanin nanoparticles (PEGylated Fe.sup.3+-MelNPs) according to one embodiment of the present invention, in which FIG. 13A shows the result of PEGylated Fe.sup.3+-MelNPs, FIG. 13B shows the result of IgE-bound PEGylated Fe.sup.3+-MelNPs, and FIG. 13C shows the result of Cetuximab-bound PEGylated Fe.sup.3+-MelNPs.

MODE FOR DISCLOSURE

(14) Hereinafter, the present invention will be described in more detail with reference to Examples. however, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.

EXAMPLE 1

Preparation of Melanin Nanoparticles

(15) 1) Example 1-1 (MelNPs)

(16) 180 mg of dopamine hydrochloride (Aldrich Chemical) was dissolved in 90 mL of deionized water. 760 μl of 1 N NaOH solution was added to the dopamine hydrochloride solution at 50° C. with vigorous stirring. When NaOH was added, the solution immediately changed to light yellow, and gradually to deep brown. After reaction for 5 hours, melanin nanoparticles were recovered by centrifugation (18,000 rpm), and washed with deionized water several times. Precipitates were removed after low speed centrifugation (4000 rpm), and then melanin nanoparticles were stored as a dispersibility solution.

(17) 2) Example 1-2 (MelNPs)

(18) Melanin nanoparticles were prepared in the same manner as in Example 1-1, except that the addition amount, of NaOH was from 400 to 950 μ, the amount of deionized water was from 45 to 180 mL, and the reaction temperature was from 20 to 70° C.

(19) In detail, 180 mg of dopamine hydrochloride was dissolved in 90 mL of tri-distilled water, and then 760 μl of 1 N NaOH was added thereto with vigorous stirring at room temperature. After 5 hours, melanin nanoparticles having a size of 316 nm were obtained by several centrifugations. Further, melanin nanoparticles having a size of 577 nm were obtained in the same manner, except that the amount of 1 N NaOH was reduced to 450 μl.

(20) 3) Example 1-3 (Sepia Melanin)

(21) Ink sacs were obtained from dissection of Korean cuttlefish, and sepia melanin was extracted therefrom by syringe. Sepia melanin was centrifuged (18,000 rpm) and washed five times, and then re-dispersed in water and stored.

(22) TEM images of the melanin nanoparticles prepared in Examples 1-1 and 1-3 were obtained on Hitachi-7600 electron microscope, and the results are shown in FIG. 2 (Example 1-1) and FIG. 3 (Example 1-3).

(23) As shown in FIG. 2, the melanin nanoparticles prepared in Example 1-1 had an average diameter of approximately 95 nm, and the shape and size of the prepared nanoparticles were uniform. In contrast, as shown in FIG. 3, the sepia melanin prepared in Example 1-3 had a diameter of 30 to 200 nm, and the size of the particles was irregular.

(24) Further, the infrared spectra of the melanin nanoparticles prepared in Examples 1-1 and 1-3 were obtained with a JASCO FT-IR-600 Plus, and UV/vis spectra were obtained on a SINCO S-3100. The results are shown in FIG. 4. As shown in FIG. 4, the infrared spectra and Uv/vis spectra of the melanin nanoparticles prepared in Examples 1-1 and 1-3 were similar to each other.

(25) Further, TEM images of the melanin nanoparticles prepared in Example 1-2 were also obtained on Hitachi-7600 electron microscope, and the results are shown in FIG. 5.

EXAMPLE 2

Preparation of Paramagnetic Metal Ion-coordinated Melanin Nanoparticles

(26) 1) Example 2-1 (Fe.sup.3+-MelNPs)

(27) 100 μl of Fe.sup.3+ solution (1 mg/mL) was added to 10 ml of melanin nanoparticles solution (1 mg/mL) prepared in Example 1-1 with vigorous stirring. After 3 hours, Fe.sup.3+-coordinated melanin nanoparticles were recovered by centrifugation (19,000 rpm), and the supernatant was fettered using a membrane filter (0.45 μm pore size) and Fe.sup.3+ concentration was measured by ICP-AES to calculate the amount of Fe.sup.3+ bound to the melanin nanoparticles.

(28) The Fe.sup.3+-coordinated melanin nanoparticles thus recovered were washed with deionized water several times, and diluted and stored.

(29) 2) Example 2-2 (Fe.sup.3+-MelNPs)

(30) Fe.sup.3+-coordinated melanin nanoparticles were prepared in the same manner as in Example 2-1, except that the melanin nanoparticles prepared in Example 1-2 were used instead of the melanin, nanoparticles prepared in Example 1-1.

(31) 2) Example 2-3 (Fe.sup.3+-sepia Melanin)

(32) Fe.sup.3+-coordinated melanin nanoparticles were prepared in the same manner as in Example 2-1, except that the melanin nanoparticles prepared in Example 1-3 were used instead of the melanin nanoparticles prepared in Example 1-1.

(33) The concentrations of Fe.sup.3+ coordinated, in Examples 2-1 and 2-3 were measured and the results are shown in FIG. 6.

(34) Further, the ESR spectra of Fe.sup.3+-coordinated melanin nanoparticles prepared in Examples 2-1 and 2-3 and non-Fe.sup.3+-coordinated melanin nanoparticles prepared in Examples 1-1 and 1-3 were recorded on a JEOL JES-FA200, and the results are shown in FIG. 7. As shown in FIG. 7, ESR signal intensities of Examples 2-1 and 2-3 were reduced compared, to Examples 1-1 and 1-3, respectively. The reduction in ESR signal intensities indicates formation of coordinate bonds between the paramagnetic metal ions, Fe.sup.3+ ions and the dihydroxyl groups of melanin nanoparticles.

(35) Further, the concentration of the coordinated Fe.sup.3+ of the Fe.sup.3+ coordinated melanin nanoparticles prepared, in Example 2-2 was measured and the results are shown in FIG. 8. As shown in FIG. 8, as the size of melanin nanoparticles increases, the amount of the coordinated Fe.sup.3+ decreases.

EXAMPLE 3

Preparation of PEGylated Paramagnetic Metal Ion-coordinated Melanin Nanoparticles (PEGylated Fe3+-MelNPs)

(36) 150 mg of methoxy-poly(ethylene glycol)thiol (mPEG-SH; 2 kDa; SunBio (Korea)) was added, to 10 mL of Fe.sup.3+-coordinated melanin nanoparticles solution (1 mg/mL) prepared in Example 2-1, and NH.sub.4OH solution (28 wt %) was added to adjust the pH of the solution to approximately 10.3. After stirring for 1 hour. surface-modified melanin nanoparticles were recovered by centrifugation (18,000 rpm), and washed with deionized water several times using redispersibility/centrifugation processes so as to prepare PEGylated Fe.sup.3+-coordinated melanin nanoparticles.

(37) The TEM images and FT-IR spectra of the prepared melanin nanoparticles were measured and shown in FIGS. 9A and 9B, respectively. Further, the dispersibility of the prepared melanin nanoparticles was observed with the naked eye (FIG. 9C), and plots of T1 excitation time vs Fe.sup.3+ concentration were obtained (FIG. 9D).

EXAMPLE 4

Preparation of MPA/PEG-bound Paramagnetic Metal Ion-coordinated Melanin Nanoparticles

(38) 160 mg of methozy-poly(ethylene glycol)thiol (mPEG-SH; 2 kDa; SunBio (Korea)) and 10 mL of MPA (3-mercaptopropionic acid) were added to 10 mL of Fe.sup.3+-coordinated melanin nanoparticles solution (1 mg/mL) prepared in Example 2-1, and NH.sub.4OH solution (28 wt %) was added to adjust the pH of the solution to approximately 10.3. After stirring for 1 hour, surface-modified melanin nanoparticles were recovered by centrifugation (18,000 rpm), and washed with deionized water several times using redispersibility/centrifugation processes so as to prepare PEG bound Fe.sup.3+-coordinated melanin nanoparticles.

EXAMPLE 5

Preparation of Antibody-bound Melanin Nanoparticles

(39) Example 5-1) Preparation of Cetuximab-bound Melanin Nanoparticles

(40) 0.2 μmol of EDC hydrochloride (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC) hydrochloric) was added to 1 mL of melanin nanoparticles solution (1 mg/mL) prepared in Example 4. After stirring for 1 hour, 4 μl of Cetuximab solution (1 mg/mL) was added and stirred for 4 hours and recovered Cetuximab-bound melanin nanoparticles by a purification process of centrifugation/redispersibility.

EXAMPLE 5-2

Preparation of IgG-bound Melanin Nanoparticles

(41) IgE-bound melanin nanoparticles were prepared in the same manner as in Example 5-1, except that IgG antibody was used instead of Cetuximab.

EXPERIMENTAL EXAMPLE 1

Cytotoxicity Assay

(42) Cell viability was tested by the WST-1 assay. Cells (HeLa cell) were cultured on 96-well plates at a density of 3/10.sup.3 cells/well for 24 hours, followed by treatment with mPEG-SH-surface treated melanin nanoparticles (PEGylated Fe.sup.3+-MelNPs) prepared in Example 3. After 24 hours of culture with increasing concentrations, 10 μl of WST-1 solution (2-(4-nitrophenyl)-5-(2-sulfophenyl)-3-[4-(4-sulfophenylazo)-2-sulfophenyl]-2H-tetrazolium disodium salt, Daeli Science, Korea) was added to each well, and the plates were cultured for an additional 1 hour at 37° C. The absorbance of each well at 455 nm was measured with a reference at 630 nm by using a Bio-Tek model ELx800™ microplate reader (Bio-Tek Instruments, Winooski, Vt.), and the absorbance from the melanin nanoparticles themselves was compensated. The percentage of cell viability was calculated using the following formula:

(43) % cell viability=(mean absorbance in test wells)/(mean absorbance in control well)×100.

(44) Each experiment was performed in triplicate, and the results are shown in FIG. 10. As shown in FIG. 10, when HeLa cells were treated with 200 g/mL, their viability was 100%, indicating that mPEG-SH-surface treated melanin nanoparticles (PEGylated Fe.sup.3+-MelNPs) did not show any cytotoxicity, and thus they leave high biocompatibility.

EXPERIMENTAL EXAMPLE 3

MRI Relaxation Properties

(45) The Fe.sup.3+-coordinated melanin nanoparticles (Examples 2-1 (Fe.sup.3+-MelNPs) and 2-3 (Fe.sup.3+-sepia melanin)) were prepared in Eppendorf tubes at varying concentrations. Their T1 and T2 relaxation times were measured on a 3.0 T clinical MRI scanner (Philips, Achieve ver. 1.2, Philips Medical Systems, Best, The Netherlands, 80 mT/m gradient amplitude, 200 ms/m slew rate). A Loot-Locker sequence (TR/TE=10/1 ms; flip angle=5°) was used to acquire 17 gradient echo images at different inversion delay times (minimum inversion time: 87 ms, phase interval: 264 ms, in-plane image resolution: 625×625 mm.sup.2, slice thickness: 500 mm). The images were fitted into 3-parameter function to calculate T1 values by using a Matlab program. T2 measurements were performed using 10 different times in a multislice turbo spin echo sequence (TR/TE=5000/20, 40, 60, 80, 100, 120, 140, 160, 180, 200 ms, in-plane resolution; 200×200 mm.sup.2, slice thickness: 500 mm). The images were processed using the Levenberg-Marquardt method to calculate T2 values using a Matlab program. r.sub.1 and r.sub.2 were calculated from the plots of T1-1 and T2-1 versus concentration of the contrast agent. The signal intensities for each of the ROIs on the T1 map (60-80 pixels) and the T2 map (200-300 pixels) were measured for each concentration, which were then used for r.sub.1 and r.sub.2 calculations, respectively. Relaxivities were derived based on the molar concentration of iron atoms measured using ICP-AES. The results are shown in FIG. 11.

(46) Further, Gd-DTPA, Fe.sub.2O.sub.3, MnO, and Hollow Mn.sub.3O.sub.4 were measured as control groups in the same manner. The results are shown in the following Table 1.

(47) TABLE-US-00001 TABLE 1 Contrast Diameter r.sub.1 r.sub.2 agent (nm) (mM.sup.−1 S.sup.−1) (mM.sup.−1 S.sup.−1) r.sub.2/r.sub.1 B.sub.0 (T) Gd-DTPA molecule 4.5 5 1.1 3 Fe.sub.2O.sub.3 2.2 4.7 17.5 3.6 3 MnO 7 0.3 1.7 4.7 3 Hollow 20 1.4 7.7 5.5 3 Mn.sub.3O.sub.4 Example 95 17 18 1.1 3 2-1 Example 30-200 10 16 1.6 3 2-3

EXPERIMENTAL EXAMPLE 4

In vivo MRI Experiment

(48) In vivo MRI was carried on a 7T/20 micro-MRI System (Bruker-Biospin, Fallanden, Switzerland) equipped with a 20 cm gradient set capable of supplying up to 400 mT/m in a 100 μs rise time. A birdcage coil (72 mm i.d.; Bruker-Biospin, Fallanden, Switzerland) was used for excitation, and an actively decoupled phased array coil was used to receive the signal.

(49) During MRI, the animals were anesthetized with inhalation of 2% isoflurane. The rectal temperature was carefully monitored and maintained at 36±1° C. The melanin nanoparticles prepared in Examples 3, 5-1 and 5-2 were intravenously administered through a tail vein of a mouse in an amount of 20 mg per 1 kg of body weight, of the mouse. The amount of Fe injected was 144 μg per 1 kg of body weight of the mouse, when measured, by ICP-AES. To investigate the time course distributions of the injected melanin nanoparticles in the mouse body, MRI was performed before and 1, 3, 6, 24, 48 hrs after the administrations.

(50) High-resolution melanin nanoparticle contrast-enhanced MR images were obtained from each mouse abdomen by using a FSE (fast spin-echo) T.sub.1-weighted MRI sequence and a FSE (fast spin-echo) T.sub.2-weighted MRI sequence. All images were analyzed using Paravasion software (Bruker-Biospin, Fallanden, Switzerland). The sequence parameters are the same as follows. FSE (fast spin-echo) T.sub.1-weighted MRI sequence

(51) Repetition time (TR)/echo time (TE)=300/7.9 ms, number of experiment (NEX)=4, echo train length=2, 100×100 μm.sup.2 in plane resolution, a slice thickness: 800 μm, 10 slices) FSE (fast spin-echo) T.sub.2-weighted MRI sequence

(52) Repetition time (TR)/echo time (TE)=3000/60 ms, number of experiment (NEX)=4, echo train length=4, 100/100 μm.sup.2 in plane resolution, a slice thickness: 800 μm, 10 slices)

(53) The results are shown in FIG. 12. After injection of PEGylated Fe.sup.3+-MelNPs, T1-weighted MRI images in the spleen and the liver were observed within 1 hour, respectively, because of the selective accumulation of PEGylated Fe.sup.3+-MelNPs in the cells of RES (reticuloendotherial system).

(54) After 6 hrs, the liver seemed to return to a similar contrast to that before administration, out apparently high T1-weighted MRI images in the spleen persisted. After 24 hours, all organs seemed to return to normal contrast, indicating the degradation and/or clearance of PEGylated Fe.sup.3+-MelNPs, and also indicating that melanins snow biocompatibility similar to other biomaterials, unlike other inorganic nanoparticles.

(55) Experiments were performed in the same manner as above, except that liver cancer-transplanted mice were used, and the results are shown in FIG. 13A.

(56) 1 hour after injection of PEGylated Fe.sup.3+-MelNPs, T.sub.1-weighted MPI images were obtained from the normal liver tissue, but not from the cancer tissue (FIG. 13A). Such selective accumulation in the normal liver is attributed to pseudonegative contrast of tumor, which can be also explained by the difference in the activity or amount of RES cells between cancer cells and normal liver cells.

(57) Experiments were also performed in the same manner as above, except that liver cancer-transplanted mice were used, and antibody-bound PEGylated Fe.sup.3+-MelNPs were used, and the results are shown in FIGS. 13B and 13C.

(58) Up to 6 hours after injection of Cetuximab-bound PEGylated Fe.sup.3+-MelNPs for selective targeting liver cancer, the same images as in the normal liver were observed. After 24 hours, the normal liver returned, to the contrast similar to that before injection but the tumor was clearly visualized (FIG. 13C).

(59) These tissue-specific targeting results can be compared with the results (FIG. 13B) of pseudonegative contrast of tumor by IgG-bound PEGylated Fe.sup.3 +-MelNPs.