Anti-tumor polypeptide Bax-BH3, fluorescent polymeric nanomicelle, preparation method and use thereof

Patent metadata

Field	Value
Publication number	US-11963997-B2
Application number	US-202217812934-A
Country	US
Kind code	B2
Filing date	Jul 15, 2022
Priority date	Jul 21, 2021
Publication date	Apr 23, 2024
Grant date	Apr 23, 2024

Abstract

The present disclosure provides an anti-tumor polypeptide Bax-BH3, a fluorescent polymeric nanomicelle, a preparation method and use thereof, belonging to the technical field of medicines. The anti-tumor polypeptide Bax-BH3 has an amino acid sequence set forth in SEQ ID No: 1; the fluorescent polymeric nanomicelle includes the anti-tumor polypeptide Bax-BH3 and a polymer carrier; and the polymer carrier is a block copolymer RGD-PHPMA-b-Poly(MMA-alt-(Rhob-MA)). In the present disclosure, the anti-tumor polypeptide Bax-BH3 has desirable biocompatibility and biological activity; and the fluorescent polymeric nanomicelle encapsulates the anti-tumor polypeptide Bax-BH3 by the block copolymer RGD-PHPMA-b-Poly(MMA-alt-(Rhob-MA)), with high encapsulation rate and drug loading, and good release performance.

First claim

What is claimed is:

1. A fluorescent polymeric nanomicelle, prepared by dissolving a block copolymer RGD-PHPMA-b-Poly(MMA-alt-(Rhob-MA)) in an organic solvent to obtain a block copolymer solution and adding the block copolymer solution dropwise into an aqueous solution of an anti-tumor polypeptide Bax-BH3 having an amino acid sequence set forth in SEQ ID No. 1, to obtain a fluorescent polymeric nanomicelle, the fluorescent polymeric nanomicelle comprising an anti-tumor polypeptide Bax-BH3 and a polymer carrier,
wherein the anti-tumor polypeptide Bax-BH3 has an amino acid sequence set forth in SEQ ID No. 1, and
wherein the polymer carrier is a block copolymer, the block copolymer is arginyl glycyl aspartic acid-poly(N-(2-hydroxypropyl)methacrylamide)-block polymethacryloyl rhodamine B-polymethyl methacrylate (“RGD-PHPMA-b-Poly(MMA-alt-(Rhob-MA))”).

2. A method for preparing a fluorescent polymeric nanomicelle, comprising the following steps:
1) dissolving a block copolymer RGD-PHPMA-b-Poly(MMA-alt-(Rhob-MA)) in an organic solvent to obtain a block copolymer solution; and
2) adding the block copolymer solution dropwise into an aqueous solution of an anti-tumor polypeptide Bax-BH3 having an amino acid sequence set forth in SEQ ID No. 1, to obtain a fluorescent polymeric nanomicelle.

3. The method according to claim 2 , wherein in step 1), a concentration of the block copolymer solution is in a range of 2 mg/mL to 3 mg/mL, and the organic solvent is tetrahydrofuran (“THF”).

4. The method according to claim 2 , wherein in step 2), a concentration of the aqueous solution of the anti-tumor polypeptide Bax-BH3 is in a range of 0.3 mg/mL to 0.7 mg/mL, wherein step 2) further comprises performing ultrasonic treatment on the aqueous solution of the polypeptide during the dropwise addition of the block copolymer solution, and wherein each drop of the block copolymer solution in the dropwise addition of the block copolymer solution has a volume of 5 μL to 15 μL.

5. The method according to claim 2 , wherein the block copolymer RGD-PHPMA-b-Poly(MMA-alt-(Rhob-MA)) in step 1) is prepared as follows:
1) reducing rhodamine B to synthesize reduced rhodamine B, and functional modifying the reduced rhodamine B with methacryloyl chloride to obtain methacryloyl-functionalized rhodamine B (“Rhob-MA”);
2) subjecting methyl methacrylate (“MMA”), 4-cyanopentanoic acid dithiobenzoate (“CPADB”), azobisisobutyronitrile (“AIBN”), and the Rhob-MA to a reaction under N 2 protection and using THF as a reaction solvent at 70° C. for 6 h after three times of freeze-thawing and degassing to obtain a product I, precipitating the product I with petroleum ether to obtain a precipitate I, and vacuum-drying the precipitate I to obtain a purified product polymethacryloyl rhodamine B-polymethyl methacrylate (“Poly(MMA-alt-(Rhob-MA))”);
3) subjecting hydroxypropyl methacrylate (“HPMA”), the AIBN, and the Poly(MMA-alt-(Rhob-MA)) to a reaction under N 2 protection and by using THF as a reaction solvent at 70° C. for 12 h after three times of freeze-thawing and degassing to obtain a product II, precipitating the product II with diethyl ether to obtain a precipitate II, and vacuum-drying the precipitate II to obtain a purified product PHPMA-b-Poly(MMA-alt-(Rhob-MA));
4) subjecting N-hydroxy succinimide (“NHS”), 1-ethyl-3-(−3-dimethylaminopropyl) carbodiimide hydrochloride (“EDC”), and the PHPMA-b-Poly(MMA-alt-(Rhob-MA)) to a reaction with a 2-(N-morpholino)ethanesulfonic acid (“MES”) buffer as a reaction solvent at 20° C. to 25° C. for 24 h to obtain a product III; drying the product Ill and filtering with THF, and collecting a filtrate to obtain a purified product NHS-PHPMA-b-Poly(MMA-alt-(Rhob-MA)); and
5) subjecting the NHS-PHPMA-b-Poly(MMA-alt-(Rhob-MA)) and arginyl glycyl aspartic acid (“RGDfK”) to a reaction with an MES buffer as a reaction solvent at 20° C. to 25° C. for 24 h to obtain a product IV; dialyzing and purifying the product IV by dialysis to obtain a purified product solution, and freeze-drying the purified product solution to obtain purified RGD-PHPMA-b-Poly(MMA-alt-(Rhob-MA)).

6. A method of treating a patient in need thereof comprising the steps of:
1) selecting a patient having a disease caused by abnormal expression of an anti-apoptotic protein in Bcl-2 family proteins selected from malignant tumors, hematological tumor-based diseases, neurodegenerative diseases, and autoimmune diseases; and 2) administering a therapeutically effective amount of a drug to the patient, the drug comprising a fluorescent polymeric nanomicelle, the fluorescent nanomicelle comprising an anti-tumor polypeptide Bax-BH3 and a polymer carrier prepared in accordance with the method of claim 2 ,
wherein the anti-tumor polypeptide Bax-BH3 has an amino acid sequence set forth in SEQ ID No. 1, and
wherein the polymer carrier is a block copolymer, the block copolymer is RGD-PHPMA-b-Poly(MMA-alt-(Rhob-MA)).

7. The method according to claim 6 , wherein the disease comprises one or more of a malignant tumor and an autoimmune disease.

Assignees

• Univ Jilin

Inventors

• Xu Li
• Guo Yi
• Huang Sijun
• Zhang Xi
• Zhang Mingming

Classifications

• A61K38/1709 Primary

  from mammals · CPC title

• A61K47/32

  Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers {, poly(meth)acrylates, or polyvinyl pyrrolidone} · CPC title

• A61K49/0082

  micelle, e.g. phospholipidic micelle and polymeric micelle · CPC title

• A61P35/00 Primary

  Antineoplastic agents · CPC title

• C07K14/4747 Primary

  Apoptosis related proteins · CPC title