METHOD FOR INCREASING DISPERSION STABILITY OF NANOPARTICLES AS T1 MRI CONTRRAST AGENT AND T1 MRI CONTRAST NANOPARTICLES

Abstract

The present invention improves an existing contrast agent, especially, a T1 contrast agent, and adopts a strategy in which the T1 contrast material is partially coated on a support surface to which a hydrophilic functional group is exposed. The partial coating strategy adopted in the present invention improves both the stability and contrast performance of T1 contrast agent nanoparticles, and such a strategy leads to very interesting technical development.

Claims

1. A method for increasing dispersion stability of nanoparticles as T1 contrast agents, the method comprising: (a) selecting a support material capable of, when particlized, exposing hydrophilic chemical functional groups on a surface; (b) using the support material to manufacture support particles, wherein hydrophilic chemical function groups are exposed on a surface of each of the support particles and the hydrodynamic size of the support particles is 1-20 nm; and (c) coating a T1 contrast material on the support particles to manufacture nanoparticles as constant agents, wherein the coating of the T1 contrast material is achieved by bonding between the contrast material and the hydrophilic functional groups on the surface of the support particle; the coating of the T1 contrast material is a partial coating on the surface of the support; some of the hydrophilic functional groups on the support particle are still exposed on the surface of the support; and the hydrodynamic size of the nanoparticles is 2-30 nm.

2. The method of claim 1, wherein the support material is an organic polymer, silica, or gold (Au) each comprising hydrophilic chemical functional groups.

3. The method of claim 2, wherein the organic polymer is a polysaccharide.

4. The method of claim 3, wherein the polysaccharide is dextran.

5. The method of claim 1, wherein the coating of the T1 coating material on the support is achieved through chemical bonding.

6. The method of claim 1, wherein the hydrodynamic size of the support particles is 1-15 nm, 1-10 nm, 1-8 nm, 1-5 nm, 1-4 nm, 2-15 nm, 2-10 nm, 2-8 nm, 2-5 nm, 2-4 nm, 3-15 nm, 3-10 nm, 3-8 nm, 3-5 nm, 3-4 nm, 4-15 nm, 4-10 nm, 4-8 nm, 4-7 nm, or 4-5 nm.

7. The method of claim 1, wherein the hydrodynamic size of the nanoparticles as contrast agents is 2-25 nm, 2-20 nm, 2-15 nm, 2-10 nm, 2-8 nm, 2-6 nm, 2-5 nm, 3-25 nm, 3-20 nm, 3-15 nm, 3-10 nm, 3-8 nm, 3-6 nm, 3-5 nm, 4-25 nm, 4-20 nm, 4-15 nm, 4-10 nm, 4-8 nm, 4-7 nm, or 4-6 nm.

8. The method of claim 2, wherein step (b) is carried out by reacting the organic polymer and a cross-linker having hydrophilic chemical functional groups to crosslink the organic polymer.

9. The method of claim 1, wherein the hydrophilic chemical functional groups exposed on the surface of the support particle in step (b) are originated from the support material per se.

10. The method of claim 8, wherein the hydrophilic chemical functional groups exposed on the surface of the support particle in step (b) are originated from the cross-linker.

11. The method of claim 8, wherein the cross-linker having hydrophilic chemical functional groups is a cross-linker having amine groups.

12. The method of claim 11, wherein the amine groups are exposed on the surface of the support particle and step (b) comprises a step for substituting the amine groups with carboxyl groups.

13. The method of claim 1, wherein the T1 contrast material is an iron oxide.

14. The method of claim 1, wherein in step (c), the amount of the T1 contrast material used relative to 100 weight of the support particles is 0.5-10 wt %, 0.8-7.0 wt % 0.5-5.0 wt %, or 0.9-5.0 wt %.

15. The method of claim 1, wherein the T1 contrast material is bound to 14-70%, 14-60%, 14-50%, 14-45%, 15-70%, 15-60%, 15-50%, 15-45%, 16-70%, 16-60%, 16-50%, or 16-45% of the hydrophilic functional groups on the surface of the support particle.

16. The method of claim 1, wherein the nanoparticles as T1 contrast agents showed dispersion stability in which the hydrodynamic size change is 10% or less for a NaCl concentration change between 125 mM to 500 mM in an aqueous solution, 10% or less for a change between pH 6 and pH 8, and 10% or less for a temperature range between 4 C. and 37 C.

17. The method of claim 1, wherein the nanoparticles as T1 contrast agents show a T1 relaxivity of 2.7-5.0 s.sup.1 for 1 mM concentration (metal basis).

18. T1 contrast agent nanoparticles comprising: (a) support particles; and (b) a T1 contrast material coated on the support particles, wherein hydrophilic functional groups are exposed on a surface of each of the support particles; the hydrodynamic size of the support particles is 1-20 nm; the T1 contrast material is bound to the hydrophilic functional groups on the surface of the support particle while the T1 contrast material being bound to some of the hydrophilic functional groups on the surface of the support; some of the hydrophilic functional groups on the support particle are still exposed on the surface of the support; and the hydrodynamic size of the nanoparticles is 2-30 nm.

19. The T1 contrast agent nanoparticles of claim 18, wherein the support material is an organic polymer, silica, or gold (Au) each comprising hydrophilic chemical functional groups.

20. The T1 contrast agent nanoparticles of claim 19, wherein the organic polymer is a polysaccharide.

21-33. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIG. 1 shows, relative to the overall hydrophilic functional groups (amine groups) on a surface of a nano-support manufactured by the present invention, the proportion of contrast material-bound functional groups (upper graph) and the proportion of still exposed contrast material-bound functional groups (lower graph).

[0096] FIG. 2 shows the results of analyzing stability of nano-contrast agents of the present invention.

[0097] FIG. 3 shows the results of analyzing T1 relaxivity rate of the present invention.

[0098] FIG. 4 shows the results of analyzing animal toxicity of nano-contrast agents of the present invention.

[0099] FIG. 5 shows the results of analyzing the stability of nano-support particles of the present invention manufactured by using a protein or gold as a support.

DETAILED DESCRIPTION

[0100] Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLES

Example 1: Synthesis of Dextran Nano-Supports and Partial Coating of T.SUB.1 .Contrast Material

[0101] Dextran nano-supports were synthesized through cross-linking of dextran (molecular weight: 10 kDa, Pharmacosmos, Denmark). Specifically, 1.8 g of dextran was dissolved in a basic aqueous solution, and then epichlorohydrin (6 mL, Sigma, USA) and ethylenediamine (26 mL, Sigma, USA) as cross-linkers were added thereto, followed by 24 hr-reaction in a thermostatic bath at room temperature. The reaction product was purified using a hollow fiber membrane filter (MWCO 10,000, GE Healthcare, Netherlands). The synthesized dextran nano-supports showed a dynamic diameter of about 4.1 nm. FeCl.sub.2, FeCl.sub.3, and NaOH were respectively added at a molar ratio of 1:2:8 to the synthesized dextran nano-supports, followed by strong magnetic stirring for 30 minutes at room temperature, thereby introducing Fe.sub.3O.sub.4 as a T.sub.1 contrast material. The synthesized nanoparticles were purified using a hollow fiber membrane filter (MWCO 10,000, GE Healthcare, Netherlands).

Example 2: Control of Proportion of T.SUB.1 .Contrast Material Bound to Dextran Nano-Support

[0102] In the T.sub.1 contrast material introduction step in example 1 above, the proportion of the T.sub.1 contrast material bound to the dextran nano-support was controlled by adjusting the amount (Fe metal basis) of T.sub.1 contrast material (FeCl.sub.2 or FeCl.sub.3) to 0.1% (wt), 1% (wt), 2.5% (wt), 5.0% (wt), 10.0% (wt), 25% (wt), 50% (wt), and 100% (wt) when the weight of the overall dextran nano-supports is 100. The hydrodynamic size of the synthesized nanoparticle contrast agents was measured as about 4.7 nm, 4.8 nm, 5.8 nm, 6.5 nm, 7.2 nm, 9.0 nm, 10.0 nm, and 25 nm, respectively, as the amount of the T.sub.1 contrast material increased. The quantification of T.sub.1 contrast material bound to the dextran nano-supports conducted using inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer, USA). Specifically, a piranha solution (mixture solution of 1:3 H.sub.2O.sub.2 and H.sub.2SO.sub.4) was added to each material, and the mixture was heated at 70 C. for 24 hours and then diluted with distilled water to reach a volume of 10 mL. As a result of analysis, the amounts (Fe metal basis) of the contrast material bound relative to 100 weight of the support particles showed 0.1% (wt), 0.99% (wt), 2.44% (wt), 4.76% (wt), 9.09% (wt), 20.0% (wt), 33.3% (wt), and 50.0% (wt), respectively.

Example 3: Quantification of Hydrophilic Functional Groups on Surface of Nano-Support

[0103] Since amine (NH.sub.2) functional groups were introduced to a surface of each of the nano-supports manufactured in example 1 above, the quantification of hydrophilic functional groups on the surface of each of the nano-supports was conducted using TNBSA assay, which is a well-known amine quantification method. Specifically, an aqueous solution of 0.01% (w/v) 2,4,6-trinitrobenzene sulfonic acid (TNBSA, Thermo, USA) was added to 0.5 mL of 10 mg/mL nano-supports, followed by reaction at 37 C. for 2 hours, and then 0.25 mL of an aqueous solution of 10% sodium dodecyl sulfate (SDS, Sigma, USA) and 0.125 mL of 1 M HCl (Sigma, USA) were added thereto, and the absorbance was measured at a wavelength of 339 nm. Here, to quantify the amount of amine groups from the absorbance, a total of five different concentrations of lysine (Sigma, USA) solutions were subjected to TNBSA assay and the absorbance was measured to plot a calibration curve.

Example 4: Quantification of Proportion of Contrast Material-Bound Functional Groups Relative to Overall Functional Groups on Surface of Nano-Support

[0104] The quantification of the proportion was conducted by measuring the amount of amine groups of contrast agent-introduced nano-supports and the amount of amine groups of pure nano-supports and then comparing the amounts. Here, the amount of nano-supports was the same, and was calculated as a percentage by the following equation.

[0105] {(amine groups of pure nanosupports amine groups of constant material-introduced nanosupports)/(amine groups of pure nanosupports)}100

[0106] As the amount of T.sub.1 contrast material bound relative to 100 wt of the dextran supports increased to 0.1% (wt), 0.99% (wt), 2.44% (wt), 4.76% (wt), 9.09% (wt), and 20.0% (wt), the proportion of contrast material-bound functional groups in the overall functional groups on the surface of the nano-support increased to 13.81%, 16.57%, 19.34%, 44.20%, 82.87%, and 85.64%. That is, after the contrast material was bound, the proportion of functional groups exposed on the surface of the nano-support was 86.19%, 83.43%, 80.66%, 55.80%, 17.13%, and 14.36% for 0.1% (wt), 0.99% (wt), 2.44% (wt), 4.76% (wt), 9.09% (wt), and 20.0% (wt) in the amount of T.sub.1 contrast material bound, respectively. No exposed functional groups were detected when the amount of T.sub.1 contrast material bound was 33.3% (wt) and 50.0% (wt) (FIG. 1).

Example 5: Test on Stability of Nano-Contrast Agents

[0107] The dextran nano-contrast agents were tested for stability in various salt conditions, pH conditions, and temperature conditions including physiological environments. An aqueous solution of 2 M NaCl (Sigma, USA) was properly added to the same amount of nano-contrast agents to prepare salt conditions of 500 mM, 250 mM, and 125 mM, respectively. For pH conditions, the same amount of nano-contrast agents were incubated in buffer solutions of 0.1 M NaCl with pH 6, 7, and 8. For temperature conditions, the same amount of nano-contrast agents were incubated in PBS solutions with pH 7.4 at 37 C. and 4 C.

Example 6: Measurement of Stability of Nano-Contrast Agents

[0108] The stability of the dextran nano-contrast agents were measured through a change rate in hydrodynamic size of the nano-contrast agents. The change rate of the hydrodynamic size at each condition, implemented in example 7, relative to the hydrodynamic size in the pH 7.4 PBS solution at room temperature was calculated as a percentage. All the hydrodynamic sizes were measured using a zeta potential system (NanoZS Zetasizer, Malvern, UK).

[0109] The stability of the support varied according to the amount of T1 contrast material bound (i.e., the proportion of exposed functional groups) relative to 100 wt of the dextran supports. Specifically, when the proportion of contrast agent-bound functional groups relative to the overall functional groups is 16.57% (exposure proportion: 83.43%), 19.34% (exposure proportion: 80.66%), and 44.20% (exposure proportion: 55.80%), the change rate in hydrodynamic size was less than 10% in the salt conditions and pH conditions. Whereas, for 13.81% (exposure proportion: 86.19%), 82.87% (exposure proportion: 17.13%), and 85.64% (exposure proportion: 14.36%), significantly high change rates of hydrodynamic size was shown, indicating instability. As a result of observation for 168 hours at temperature conditions of 37 C. and 4 C. when the proportion of functional groups was 19.34% (exposure proportion: 80.66%), the change rate of hydrodynamic size was less than 10%, and thus high stability was maintained (FIG. 2).

Example 7: Measurement of MRI Contrast Effect of Nano-Contrast Agent

[0110] T.sub.1-MRI contrast effect (T.sub.1 relaxivity) of the dextran nano-contrast agents was measured using the 3 Tesla MRI equipment (Philips Achieva). A specific experiment method was as follows. Each sample was dispersed in water at a concentration of 1 mM (iron basis), placed in PCR tube, and fixed on a supporting member. The supporting member was placed in the center of an MRI animal coil (Custume made, China), and T.sub.1 relaxation time was measured using the following inverse recovery MRI sequence. [TI=100, 500, 1000, 2000, and 3000 ms, time of echo (TE)=7.4 ms, FOV=100 mm, matrix=256256, slice thickness=2 mm, and acquisition number=1].

[0111] As a result of testing, when the proportion of contrast agent-bound functional groups relative to the overall functional groups was 16.57%, 19.34%, 44.20%, and 82.87%, T.sub.1 relaxivity (the inverse of T.sub.1 relaxation time, R.sub.1), which is a barometer of T.sub.1 contrast effect, was 4.77 s.sup.1, 4.17 s.sup.1, 3.69 s.sup.1, and 2.83 s.sup.1, respectively, which were at least 2 times to at most 9 times higher compared with 13.81% (R.sub.1=0.53 s.sup.1) and 85.64% (R.sub.1=1.43 s.sup.1).

Example 8: Substitution of Functional Group with Carboxyl Group on Surface of Nano-Contrast Agent

[0112] The dextran nano-contrast agent containing amine groups (nano-contrast agent having 2.44 wt % of T.sub.1 contrast material), manufactured in the example above, was reacted with a succinic anhydride (100 molar excess compared with amine groups existing on a surface of a nano-support, Acros, USA) in dimethyl sulfoxide (DMSO, Daejung Chemical, Korea) at room temperature for 12 hours under magnetic stirring. Thereafter, the reaction produce was diluted with distilled water such that the proportion of DMSO in the entire solution was less than 5%, and purified using a centrifugal membrane filter (MWCO 10,000, UltraCone, Millipore, USA).

Example 9: Test of Animal Toxicity of Nano-Contrast Agent

[0113] ICR mice (6 weeks old, 8 female/male each in a total of 16 animals) were intravenously administered with dextran nano-contrast agents (a nano-contrast agent containing amine groups and a nano-contrast agent having substituted carboxyl groups, both having 2.44 wt % of a T1 contrast material).

[0114] The mice were divided into four groups, each of which had 2 female/male each (a non-injection group, a saline injection group, a high-dose group, and a medium-dose group). The total dose was 25 mL/kg, and was divided into two times each 12.5 mL/kg. The dose of each individual was calculated based on the weight of the day of administration. Tail veins of test systems were intravenously administered at a rate of about 2 mL/min at intervals of 12 hours using a disposable syringe (1 mL, 26 G). For toxicity evaluation, general conditions (type of toxic symptom, timing of onset, recovery period, etc.) and death or not were observed and weight measurement was conducted after the administration. First, as a result of injection of the dextran nano-contrast agent containing amine groups on the surface thereof, all mice were dead regardless of the dose of injection. When the nano-contrast agent with substituted carboxyl groups was injected, no death cases were observed in not only the medium-dose group but also the high-dose group, and the mouse weight change over time was increased equally in the dose groups and the control groups, indicating no toxicity (FIG. 4).

Example 10: Synthesis of Protein Nano-Supports and Partial Coating of T.SUB.1 .Contrast Material

[0115] Dextran nano-supports were synthesized through cross-linking of bovine serum albumin (BSA, Sigma, USA). Specifically, 12.6 mg of BSA was dissolved in a solution of 50 mM NaCl (Sigma, USA), and then ethylene diamine (26 L, Sigma, USA) as a cross-linker,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 9.58 mg, Thermofisher, USA), and sulfo-NHS (1.08 mg, Thermofisher, USA) were added, followed by reaction at room temperature for 2 hours. The reaction product was purified using a centrifugation membrane filter (MWCO 10,000, UltraCone, Millipore, USA). The synthesized protein nano-supports showed a dynamic diameter of about 6.1 nm. FeCl.sub.2, FeCl.sub.3, and NaOH were respectively added at a molar ratio of 1:2:8 to the synthesized protein nano-supports, followed by strong magnetic stirring for 30 minutes at room temperature, thereby introducing Fe.sub.3O.sub.4 as a T.sub.1 contrast material. The synthesized nanoparticles were purified using a centrifugation membrane filter (MWCO 10,000, UltraCone, Millipore, USA). The finally obtained protein nano-contrast agents showed a hydrodynamic size of about 7.7 nm.

Example 11: Synthesis of Gold Nano-Supports and Partial Coating of T.SUB.1 .Contrast Material

[0116] Gold nanoparticles were synthesized by reducing chloroauric acid (HAuCl.sub.4, Sigma, USA) in the presence of tetrakis(hydroxymethyl)phosphoniumchloride (THPC, Sigma, USA). Specifically, 4 L of THPC was dissolved in a basic aqueous solution, followed by magnetic stirring for a sufficiently long time. 29.4 mM HAuCl4 (0.6 mL) was added thereto, followed by again magnetic stirring for about 10 minutes. The synthesized gold nanoparticles were purified using a centrifugation membrane filter (MWCO 10,000, UltraCone, Millipore, USA). The synthesized gold nano-supports showed a dynamic diameter of about 7.8 nm. The purified gold nanoparticles were stirred together with an excessive amount of cystamine (Sigma, USA) at room temperature for 12 hours, to thereby introduce amine (NH.sub.2) functional groups to surfaces of the nanoparticles. FeCl.sub.2, FeCl.sub.3, and NaOH were respectively added at a molar ratio of 1:2:8 to the synthesized gold nanoparticles, followed by strong magnetic stirring for 30 minutes at room temperature, thereby introducing Fe.sub.3O.sub.4 as a T.sub.1 contrast material. The synthesized gold nanoparticles were purified using a centrifugation membrane filter (MWCO 10,000, UltraCone, Millipore, USA). The finally obtained gold nano-contrast agents showed a hydrodynamic size of about 22 nm.

Example 12: Stability of Protein Nano-Supports and Gold Nanoparticle Supports

[0117] In the protein support synthesized in example 10 and the gold nanoparticle support synthesized in example 11, the proportion for contrast material-bound functional groups in the overall functional groups was 19.38% and 30.08% for the cases, respectively, and these contrast agents showed a hydrodynamic size change of less than 10%, indicating high stability (FIG. 5). The proportion values of contrast material-bound functional groups are within a range in which the above-described dextran nano-supports showed high stability.

[0118] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.