AMPHIPHILIC POLYMER NANO MICELLE CONTAINING POLY-3,4-DIHYDROXYPHENYLALANINE CHELATED FERRIC IONS

Abstract

The disclosure discloses an amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions, in which ferric ions are chelated with a catechol structure on a side chain of a biodegradable poly-3,4-dihydroxyphenylalanine block. The disclosure also provides a method for preparing the above micelle, comprising: complexing an amphiphilic polymer containing poly-3,4-dihydroxyphenylalanine with a ferric ion compound, and obtaining the amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions through a solvent replacement method. The micelle prepared by the disclosure is used as a Fe.sup.3+ magnetic resonance Ti imaging contrast agent, which can avoid toxic or side effects caused by a traditional gadolinium reagent, has a longitudinal relaxation rate of 5.6 mM.sup.1.Math.s.sup.1, can cycle for 150 min in a mice body, and has an obvious imaging effect and a far higher comprehensive performance than that of a commercial gadolinium contrast agent, and as well as a promising application prospect.

Claims

1. An amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions, wherein a hydrophobic block in the amphiphilic polymer nano micelle is a poly-3,4-dihydroxyphenylalanine block; a catechol functional group on a side chain of a biodegradable poly-3,4-dihydroxyphenylalanine block chelates ferric ions, and a chelating bond is as shown in the following formula: ##STR00007##

2. The amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions according to claim 1, wherein a hydrophilic block in the amphiphilic polymer is a polysarcosine block, a polyethylene glycol block, a polyoligoethyleneglycol methacrylate block, a polyvinyl alcohol block, or a polyacrylic acid block.

3. The amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions according to claim 1, wherein the chain length of the hydrophilic block in the amphiphilic polymer is 11500; the chain length of poly-3,4-dihydroxyphenylalanine block is 1500.

4. The amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions according to claim 1, wherein the amphiphilic polymer is a diblock, triblock, multiblock, random, star-shaped, annular or grafted polymer containing the poly-3,4-dihydroxyphenylalanine block.

5. The amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions according to claim 1, wherein the amphiphilic polymer has any one of the following structure formulas (1)(5): ##STR00008## wherein, R.sub.1 is independently selected from alkyl, benzyl and a silyl group; R.sub.2 is independently selected from alkyl; m is an integer of 11500, n is an integer of 1500, and n.sub.1 is an integer of 1200.

6. The amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions according to claim 5, wherein the chain length of the poly-3,4-dihydroxyphenylalanine is between 5 and 50; the chain length of polysarcosine or polyethylene glycol is between 5 and 200.

7. Use of an amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions according to claim 1 as a magnetic resonance T.sub.1 imaging contrast agent in the field of magnetic resonance imaging.

8. The use according to claim 7, wherein the magnetic resonance contrast agent comprises the amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions, a pharmaceutical excipient, a diluent or ingredients.

9. The use according to claim 7, wherein the average particle size of the magnetic resonance contrast agent is 20200 nm; the amount of the chelated ferric ions is 101000 ppm.

Description

DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 shows proton nuclear magnetic resonance (NMR) spectra of poly-3,4-dihydroxyphenylalanine-polysarcosine block copolymer (A) and polysarcosine (B) prepared in example 1 of the disclosure.

[0034] FIG. 2 is a TEM diagram of an amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions prepared in example 1 of the disclosure.

[0035] FIG. 3 is a dynamic light scattering diagram of an amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions prepared in example 1 of the disclosure.

[0036] FIG. 4 shows in-vitro experimental results of a relationship between longitudinal relaxation time (T.sub.1) and ferric ion concentration of an amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions prepared in example 1 of the disclosure.

[0037] FIG. 5 is magnetic resonance angiography of an amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions as a Fe magnetic resonance contrast agent in a mice body prepared in example 1 of the disclosure.

[0038] FIG. 6 is a cytotoxicity test diagram of an amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions prepared in example 1 of the disclosure.

[0039] FIG. 7 is a proton NMR spectrum of a poly-3,4-dihydroxyphenylalanine-polyoligoethyleneglycol methacrylate grafted polymer prepared in example 3 of the disclosure.

[0040] FIG. 8 is a dynamic light scattering diagram of an amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions prepared in example 3 of the disclosure.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0041] In order to further understand the disclosure, the preparation and use of an amphiphilic polymer nano micelle containing poly-3,4-dihydroxyphenylalanine chelated ferric ions provided by the disclosure are described in detail in combination with embodiments. However, the disclosure is not limited to these embodiments. The non-essential improvements and adjustments made by those skilled in the art under the core guiding ideology of the disclosure still belong to the protective cope of the disclosure.

[0042] The characterization method involved in embodiments of the disclosure is briefly introduced as follows:

[0043] The NMR spectra were determined at 25 C. on Bruker Avarice DMX 00 superconducting NMR instrument. Deuterated DMSO was a solvent and tetramethylsilane (TMS) was an internal standard.

[0044] The hydrodynamic diameter of the polymer nano micelle in solution was detected by Zetasizer Nano series (Malvern instruments) detector. The wavelength was measured as 657 nm and a fixed angle was 90. Each sample was tested three times in parallel.

[0045] The particle size and morphology of the nano micelle were observed by HITACHI HT7700 transmission electron microscope, and an acceleration voltage was 100 KV.

[0046] The relaxation rate r.sub.1 of T.sub.1 weighted magnetic resonance imaging of metal chelated polymer nano particles was measured on a 3.0 T magnetic resonance imager (Aigna HDxt, GE Medical Systems, Milwaukee, Wis., USA).

[0047] The cytotoxicity test of metal chelating polymer nano particles was realized through MTT method. The test cells were mice embryonic fibroblasts (NIH 3T3), and the test results were obtained by ELIA SA (Thermo Fisher. Scientific (Waltham, Mass.).

Example 1

[0048] (1) The structural formula of poly-3,4-dihydroxyphenylalanine-poly(asarcosine) block copolymer (PDOPA-b-PSar) is as follows:

##STR00003##

[0049] wherein, R.sub.1 is benzyl, M=5200, n=550;

[0050] The specific synthesis steps include:

[0051] Sarcosine NCA was added into Schlenk bottle, dissolved with DMF, and then DMF solution of benzylamine was added. The molar ratio of sarcosine NCA to benzylamine was (5200):1, and the above materials reacted for 1 day at room temperature. Then DMF solution of DOPA NCA protected by benzyloxycarbonyl (CBZ) was added. The molar ratio of DOPA NCA to benzylamine was (550):1, and the above materials reacted for 1 day at room temperature. The polymer solution was poured into ether to be precipitated and filtered. The obtained polymer was dried in vacuum for 1 day to obtain the CBZ-protected poly-3,4-dihydroxyphenylalanine-polysarcosine block copolymer was obtained.

[0052] 300 mg of block copolymer was dissolved into 3 mL of trifluoroacetic acid, and 4-fold equivalent weight of hydrogen bromide acetic acid solution (33%) was added. After reaction for 3 hours, the reactant was precipitated with ether and filtered. The obtained polymer was dried in vacuum for 1 day to obtain poly-3,4-dihydroxyphenylalanine-polysarcosine block copolymer. The yield was 89%. The NMR spectrum of the polymer is shown in FIG. 1.

[0053] (2) 7.7 mg of weighed PDOPA-b-PSar was dissolved with DMF to be prepared into a solution, and then the DMF solution containing 1.69 mg of Fe(NO.sub.3).sub.3.9H.sub.2O was added slowly. After mixing evenly, the obtained mixture solution was dialyzed in deionized water for 48 hours to obtain the micelle solution. The micelle solution was subjected to metered volume and used after being filtered with a filter film having a pore size of 0.45 M. After conducting metered volume, the Fe.sup.3+ concentration was 94 mg/L.

[0054] The TEM of the micelles is shown in FIG. 2, and DLS test results are shown in FIG. 3. It can be seen from FIGS. 2 and 3 that the average particle size of the prepared micelle is 20 nm.

[0055] The in-vitro experimental results of the relationship between the longitudinal relaxation time (T1) of the micelle and the ferric ion concentration are shown in FIG. 4. It can be seen from FIG. 4 that the longitudinal relaxation rate of the micelle is 5.6 mM.sup.4.Math.s.sup.1, which is higher than that of the commercial gadolinium contrast agent (such as Gd-DTPA), showing excellent in-vitro magnetic resonance enhancement ability.

[0056] After the mice were anesthetized with isoflurane gas, the prepared micelle normal saline solution was injected through the tail vein of the mice. The magnetic resonance angiography of the micelle serving as the Fe.sup.3+ magnetic resonance contrast agent in the mice body is shown in FIG. 5. It can be seen from FIG. 5 that within 0-30 minutes after injection of the contrast agent, the signal intensity of the mice blood vessel rapidly rises and reaches a peak value, and the mice vascular structure can be clearly observed. Then, the signal intensity of the blood vessel gradually decreases, and the blood vessel is completely cleared when about 150 min after injection, which indicates that the circulation metabolism of the amphiphilic polymer nano micelle probe containing poly-3,4-dihydroxyphenylalanine (PDOPA) chelated ferric ions in the blood vessels is completed. Compared with the commercial Gd.sup.3+ reagent which cycles for less than 60 min in the body, the Fe.sup.3+ magnetic resonance contrast agent provided by the disclosure can make up the defect of short in-vivo circulation time.

[0057] The cytotoxicity of the micelle is determined by MTT method, and 5 parallel samples are set for each sample. The cytotoxicity test results are shown in FIG. 6, which shows that all samples show very small cytotoxicity at the concentration of 5-500 g/mL. When the concentration is greater than 50 g/mL, the cell survival rate slightly decreases with the increase of concentration, but all are kept to be above 85%, indicating that this Fe.sup.3+ magnetic resonance contrast agent has low biotoxicity and good hiocompatihility.

Example 2

[0058] (1) Other preparation conditions are the same as those in example 1. The difference is that amine-endcapped polyethylene glycol is used as a macromolecular initiator, and the structural formula of the prepared poly-3,4-dihydroxyphenylalanine-polyethylene glycol block copolymer (PDOPA-b-PEG) initiator is shown in the following formula:

##STR00004##

[0059] wherein, R.sub.2 is methyl; m=5200, and n=550.

[0060] (2) 9.7 mg of weighed PDOPA-b-PEG was dissolved with DMF to be prepared into a solution, and then DMF solution containing 3.27 mg of Fe(NO.sub.3).sub.3.9H.sub.2O was added slowly, and the above mixture solution was dialyzed in deionized water for 48 hours. The obtained micelle solution was subjected to metered volume and used after being filtered with a filter film having a pore size of 0.45 m.

[0061] Other performance test conditions are the same as those in example 1, and the micelle has an average particle size of 30 nm, and has an MRI in-vitro enhancement effect.

Example 3

[0062] (1) The structural formula of poly-3,4-dihydroxyphenylalanine-polyoligoethyleneglycol methacrylate grafted polymer (POEGMA-g-PDOPA) is as follows:

##STR00005##

[0063] wherein, R.sub.1 is n-butyl; m=5200, n.sub.1=550;

[0064] The specific synthesis steps include:

[0065] PDOPA was prepared by triggering ring opening polymerization and deprotection of CBZ-protected dopa NCA via n-butylamine, and the conditions are the same as those in example 1; polyoligoethyleneglycol methacrylate (POEGMA) was prepared through RAFT polymerization. 247.4 mg of POEGMA and 134.0 mg of PDOPA were dissolved in 1 mL of DMF, and reacted for 4 days in 35 C. oil bath. The polymer solution was poured into ether to be precipitated, filtered and dried in vacuum for 1 day, so as to obtain the poly-3,4-dihydroxyphenylatanine-polyoligoethyteneglycolmethacrylate grafted polymer. The proton NMR spectrum of the polymer is shown in FIG. 7.

[0066] (2) 22.7 mg of weighed POEGMA-g-PDOPA was dissolved with DMF to be prepared into a solution, and then the DMF solution containing 5.83 mg of Fe(NO.sub.3).sub.3.9H.sub.2O was added slowly, and the above mixture solution was dialyzed in deionized water for 48 hours. The obtained micelle solution was filtered with a filter film having a pore size of 0.45 pin and subjected to metered volume.

[0067] Other performance test conditions are the same as those in example 1. DLS test results are shown in FIG. 8. The micelle has an average particle size of 30 nm, and has an MRI in-vitro enhancement effect.

Example 4

[0068] (1) Other preparation conditions are the same as those in example 1. The difference is that a polypropylenimine tetramine dendrimer (generation 1) is used as an initiator, and the structural formula of the prepared poly-3,4-dihydroxyphenylalanine-polysarcosine star polymer (PDOPA-b-PSar star copolymer) is as follows:

##STR00006##

[0069] wherein, M=5200, and n=550.

[0070] (2) 10.1 mg of weighed PDOPA-b-PSar star copolymer was dissolved with DMF to be prepared into a solution, and then DMF solution containing 2.21 mg of Fe(NO.sub.3).sub.3.9H.sub.2O was added slowly, and the obtained mixture solution was dialyzed in deionized water for 48 hours. The micelle solution was filtered with a filter film having a pore size of 0.45 m and subjected to metered volume.

[0071] Other performance test conditions are the same as those in example 1, and the micelle has an average particle size of 35 nm, and has an MRI in-vitro enhancement effect.