CN103263674A - Tumor targeting material with tumor penetrating property, preparation method and applications thereof - Google Patents

Abstract

The invention relates to a targeting material with tumor penetrating properties, which is characterized in that the material is a linear block formed by covalently linking three parts of targeting polypeptide with tumor penetrating properties, polyethylene glycol and phospholipids Copolymer, wherein the molar ratio of targeting polypeptide, polyethylene glycol and phospholipid is 1:1:1. The invention also provides the preparation method and application of the targeting material. The targeting material protected by the invention can be used to prepare tumor-targeting liposome or micellar drug delivery system, endow it with tumor penetration performance, and improve the intratumoral delivery efficiency of the drug delivery system.

Description

Tumor targeting material with tumor penetrating performance, preparation method and application

technical field

The invention belongs to the field of pharmaceutical preparations, and relates to a tumor-targeting material, in particular to a tumor-penetrating polypeptide-modified targeting material, a preparation method and an application.

Background technique

Tumor is a major disease that threatens human health and life, and its incidence has shown an obvious upward trend in recent years. At present, the conventional clinical treatment for tumors is to perform systemic chemotherapy or radiotherapy after resection of the primary tumor and lymph node dissection. However, surgery cannot completely remove tumor cells and tumor metastatic lymph nodes, resulting in tumor recurrence; chemotherapy drugs are non-selective to tumor tissue after intravenous administration, and have serious systemic toxic and side effects; Skin reactions, blood changes, local mucosal reactions, etc. Tumor cells or tumor vascular endothelial cells express a series of specific receptors, and the tumor-targeting materials prepared by modifying biocompatible materials with their ligands are studied because they can actively target and localize to tumor tissues in vivo extensive attention of the audience. However, due to problems such as poor tumor targeting performance and ineffective improvement of drug efficacy, most tumor targeting materials have not yet entered clinical application. Therefore, it is imminent to research and develop high-performance tumor-targeting materials to improve the tumor-targeting performance and therapeutic effect.

RPAKPAR polypeptide is a straight-chain heptapeptide screened by phage display technology, which belongs to a kind of "CendR polypeptide", that is, it has an R/K-X-X-R/K structure at the C-terminus (wherein X represents any amino acid). This kind of peptide belongs to "tumor penetrating peptide", and many existing tumor targeting peptides conform to this structure, such as LyP-1, F3, iRGD and so on. Studies have shown that RPAKPAR is a specific ligand for glioma, showing dual affinity for tumor cells and tumor blood vessels. The affinity for tumor blood vessels can make it target the tumor blood vessel wall, while the tumor penetrability can make it penetrate through the tumor blood vessel wall and penetrate into the interior of tumor tissue, and the affinity for tumor cells can make it specific to tumor cells Therefore, as targeting molecules, such polypeptides have more obvious advantages compared with general tumor cell targeting molecules. It has been proven that RPAKPAR polypeptides have the ability to mediate phages to penetrate tumor blood vessels and enter tumor tissues, but there is no report on targeting materials modified by RPAKPAR polypeptides.

Contents of the invention

In order to overcome the deficiencies of the prior art, the present invention provides a tumor-targeting material with tumor-penetrating properties, a preparation method and an application.

A targeting material with tumor penetrating properties, characterized in that the material is a linear block copolymer formed by covalently linking three parts of a targeting polypeptide with tumor penetrating properties, polyethylene glycol and phospholipids, Wherein the molar ratio of targeting polypeptide, polyethylene glycol and phospholipid is 1: 1: 1.

The amino acid sequence of the targeting polypeptide with tumor penetration performance is RPAKPAR.

The weight average molecular weight of the polyethylene glycol is 2000-5000.

The phospholipids are distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), hydrogenated soybean phosphatidylethanolamine (HSPE), hydrogenated egg phosphatidylethanolamine , soybean phosphatidylethanolamine, and egg phosphatidylethanolamine.

The targeting polypeptide is a complex formed by linking polyethylene glycol-phospholipid through a covalent bond.

The targeting polypeptide provides a free sulfhydryl group, polyethylene glycol-phospholipid provides a maleimide group, and the two react specifically to form a covalent complex.

A method for preparing the above-mentioned targeting material, characterized in that the specific steps of the method are: using a solid-phase polypeptide synthesis method to synthesize a targeting polypeptide (the amino acid sequence is CRPAKPAR) with a C-terminal external cysteine (Cys), Dissolve it in phosphate buffered saline (PBS) at pH 7.0, and dissolve maleimide-polyethylene glycol-phospholipid complex (Mal-PEG-PE) in dimethylformamide (DMF), After the two are mixed, they are stirred and reacted until the Mal-PEG-PE reaction is complete, excess polypeptide and DMF are removed, and freeze-dried to obtain a targeting polypeptide-polyethylene glycol-phospholipid complex, that is, a targeting material with tumor penetration properties.

The application of the targeting material with tumor penetrating performance in the preparation of liposomes, micelles and other drug carriers.

The above-mentioned targeting materials can be used to prepare drug carriers such as liposomes and micelles with tumor-penetrating properties. After being injected into tumor-bearing mice, they can penetrate tumor blood vessels and penetrate into the interior of tumor tissues to achieve anti-tumor effects. Efficient targeted drug delivery. The invention uses the tumor-penetrating peptide RPAKPAR to modify the phospholipids to obtain the targeting material RPAKPAR-PEG-PE with tumor-penetrating performance, which can be used to prepare tumor-targeting liposomes or micelles.

The targeting material protected by the invention can be used to prepare tumor-targeting liposome or micellar drug delivery system, endow it with tumor penetration performance, and improve the intratumoral delivery efficiency of the drug delivery system.

Description of drawings

Accompanying drawing 1, the 1H-NMR spectrum of RPAKPAR-PEG-DSPE.

In the picture, A is the NMR spectrum of Mal-PEG-DSPE, and B is the NMR spectrum of RPAKPAR - PEG-DSPE. As can be seen from the figure, A shows the maleimide peak, while the peak in B disappears, and the rest The peak remained essentially unchanged, showing that the maleimide group in Mal-PEG-DSPE had reacted with CRPAKPAR.

Accompanying drawing 2, the photograph that the fluorescein-labeled liposome prepared by using RPAKPAR-PEG-DSPE is taken up by tumor cells.

Fluorescent micrographs of fluorescein-labeled liposomes prepared by using RPAKPAR-PEG-DSPE and ordinary fluorescein-labeled liposomes respectively interacted with U87 tumor cells for 2 hours at 37°C. The uptake of modified liposomes is much larger than that of ordinary liposomes.

Detailed ways

The following examples will help to further understand the present invention, but do not limit the content of the present invention.

Example 1: Synthesis, purification and characterization of targeting material RPARPAR-PEG-DSPE.

The tert-butoxycarbonyl-arginine (p-toluenesulfonyl)-p-acetamidobenzyl ester) oc-Arg(Tos)-PAM resin with a degree of substitution of 0.6mmol/g was treated with N,N-dimethylformamide DMF Swell, drain, then use trifluoroacetic acid TFA to remove Boc protecting group, remove TFA, wash with DMF; use benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate DMF solution of HBTU and N,N-diisopropylethylamine DIEA activate tert-butoxycarbonyl-alanine Boc-Ala to obtain Boc-Ala activation solution; add the resin washed with DMF to the obtained Boc-Ala activation solution , shake at 25°C for 25 minutes, remove the reaction solution, and wash the resin with DMF; repeat the above steps, and connect the remaining amino acids in sequence according to the sequence of CRPAKPAR; Add an appropriate amount of P-cresol to the peptide cutting tube, then pass through HF, and stir in an ice bath for 1 hour; after the reaction, remove the HF in the tube under reduced pressure, and precipitate the residue with an appropriate amount of ice ether, filter the precipitate and wash it with ice ether Precipitate is dissolved with TFA again, filters to obtain filtrate; Filtrate is precipitated in glacial ether again, filters, and filter residue is redissolved with water, freeze-dried to obtain CRPAKPAR pure product; Then get CRPAKPAR pure product 50mg and be dissolved in the 2ml PBS solution of pH7.0 , take the maleimide-polyethylene glycol-phospholipid complex Mal-PEG-DSPE (the molar equivalent is 0.8 times that of CRPAKPAR) and dissolve it in DMF, stir the reaction until the reaction of Mal-PEG-DSPE is complete, and remove excess CRPARPAR and DMF was freeze-dried to obtain RPARPAR-PEG-DSPE, and its structure was characterized by NMR. The results showed that the Mal-PEG-DSPE spectrum showed the characteristic peak of Mal at 6.67ppm, while this peak disappeared on the RPARPAR-PEG-DSPE spectrum, indicating that RPARPAR -PEG-DSPE was synthesized successfully.

Example 2: Synthesis, purification and characterization of targeting material RPARPAR-PEG-HSPE.

The tert-butoxycarbonyl-arginine (p-toluenesulfonyl)-p-acetamidobenzyl ester) oc-Arg(Tos)-PAM resin with a degree of substitution of 0.6mmol/g was treated with N,N-dimethylformamide DMF Swell, drain, then use trifluoroacetic acid TFA to remove Boc protecting group, remove TFA, wash with DMF; use benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate DMF solution of HBTU and N,N-diisopropylethylamine DIEA activate tert-butoxycarbonyl-alanine Boc-Ala to obtain Boc-Ala activation solution; add the resin washed with DMF to the obtained Boc-Ala activation solution , shake at 25°C for 25 minutes, remove the reaction solution, and wash the resin with DMF; repeat the above steps, and connect the remaining amino acids in sequence according to the sequence of CRPAKPAR; Add an appropriate amount of P-cresol to the peptide cutting tube, then pass through HF, and stir in an ice bath for 1 hour; after the reaction, remove the HF in the tube under reduced pressure, and precipitate the residue with an appropriate amount of ice ether, filter the precipitate and wash it with ice ether Precipitate is dissolved with TFA again, filters to obtain filtrate; Filtrate is precipitated in glacial ether again, filters, and filter residue is redissolved with water, freeze-dried to obtain CRPAKPAR pure product; Then get CRPAKPAR pure product 50mg and be dissolved in the 2ml PBS solution of pH7.0 , take the maleimide-polyethylene glycol-phospholipid complex Mal-PEG-HSPE (the molar equivalent is 0.8 times that of CRPAKPAR) and dissolve it in DMF, stir the reaction until the reaction of Mal-PEG-HSPE is complete, remove excess CRPARPAR and DMF was freeze-dried to obtain RPARPAR-PEG-HSPE, and its structure was characterized by NMR. The results showed that the Mal-PEG-HSPE spectrum showed the characteristic peak of Mal at 6.67ppm, while this peak disappeared on the RPARPAR-PEG-HSPE spectrum, indicating that RPARPAR -PEG-HSPE was synthesized successfully.

Example 3: Synthesis, purification and characterization of targeting material RPARPAR-PEG-DOPE.

The tert-butoxycarbonyl-arginine (p-toluenesulfonyl)-p-acetamidobenzyl ester) oc-Arg(Tos)-PAM resin with a degree of substitution of 0.6mmol/g was treated with N,N-dimethylformamide DMF Swell, drain, then use trifluoroacetic acid TFA to remove Boc protecting group, remove TFA, wash with DMF; use benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate DMF solution of HBTU and N,N-diisopropylethylamine DIEA activate tert-butoxycarbonyl-alanine Boc-Ala to obtain Boc-Ala activation solution; add the resin washed with DMF to the obtained Boc-Ala activation solution , shake at 25°C for 25 minutes, remove the reaction solution, and wash the resin with DMF; repeat the above steps, and connect the remaining amino acids in sequence according to the sequence of CRPAKPAR; Add an appropriate amount of P-cresol to the peptide cutting tube, then pass through HF, and stir in an ice bath for 1 hour; after the reaction, remove the HF in the tube under reduced pressure, and precipitate the residue with an appropriate amount of ice ether, filter the precipitate and wash it with ice ether Precipitate is dissolved with TFA again, filters to obtain filtrate; Filtrate is precipitated in glacial ether again, filters, and filter residue is redissolved with water, freeze-dried to obtain CRPAKPAR pure product; Then get CRPAKPAR pure product 50mg and be dissolved in the 2ml PBS solution of pH7.0 , take the maleimide-polyethylene glycol-phospholipid complex Mal-PEG-DOPE (the molar equivalent is 0.8 times that of CRPAKPAR) dissolved in DMF, stir the reaction until the reaction of Mal-PEG-DOPE is complete, remove excess CRPARPAR and DMF was freeze-dried to obtain RPARPAR-PEG-DOPE, and its structure was characterized by NMR. The results showed that the Mal-PEG-DOPE spectrum showed the characteristic peak of Mal at 6.67ppm, while this peak disappeared on the RPARPAR-PEG-DOPE spectrum, indicating that RPARPAR -PEG-DOPE was synthesized successfully.

Example 4: Synthesis, purification and characterization of targeting material RPARPAR-PEG-DPPE.

The tert-butoxycarbonyl-arginine (p-toluenesulfonyl)-p-acetamidobenzyl ester) oc-Arg(Tos)-PAM resin with a degree of substitution of 0.6mmol/g was treated with N,N-dimethylformamide DMF Swell, drain, then use trifluoroacetic acid TFA to remove Boc protecting group, remove TFA, wash with DMF; use benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate DMF solution of HBTU and N,N-diisopropylethylamine DIEA activate tert-butoxycarbonyl-alanine Boc-Ala to obtain Boc-Ala activation solution; add the resin washed with DMF to the obtained Boc-Ala activation solution , shake at 25°C for 25 minutes, remove the reaction solution, and wash the resin with DMF; repeat the above steps, and connect the remaining amino acids in sequence according to the sequence of CRPAKPAR; Add an appropriate amount of P-cresol to the peptide cutting tube, then pass through HF, and stir in an ice bath for 1 hour; after the reaction, remove the HF in the tube under reduced pressure, and precipitate the residue with an appropriate amount of ice ether, filter the precipitate and wash it with ice ether Precipitate is dissolved with TFA again, filters to obtain filtrate; Filtrate is precipitated in glacial ether again, filters, and filter residue is redissolved with water, freeze-dried to obtain CRPAKPAR pure product; Then get CRPAKPAR pure product 50mg and be dissolved in the 2ml PBS solution of pH7.0 , take the maleimide-polyethylene glycol-phospholipid complex Mal-PEG-DPPE (the molar equivalent is 0.8 times that of CRPAKPAR) and dissolve it in DMF, stir the reaction until the reaction of Mal-PEG-DPPE is complete, remove excess CRPARPAR and DMF was freeze-dried to obtain RPARPAR-PEG-DPPE, and its structure was characterized by NMR. The results showed that the Mal-PEG-DPPE spectrum showed the characteristic peak of Mal at 6.67ppm, while this peak disappeared on the RPARPAR-PEG-DPPE spectrum, indicating that RPARPAR -PEG-DPPE was synthesized successfully.

Example 5: Preparation of liposome and its in vitro tumor cell uptake experiment.

The composition of the liposome membrane material formulation is HSPC (hydrogenated soybean phospholipid)/Chol (cholesterol)/mPEG-DSPE (polyethylene glycol-distearoylphosphatidylethanolamine complex) (55: 45: 2, mol/mol) , the RPARPAR modified PEG liposome membrane material was formulated as HSPC/Chol/mPEG-DSPE/RPARPAR-PEG-DSPE (55: 45: 2: 0.5, mol/mol). Weigh the above membrane material and dissolve it in chloroform, remove the organic solvent by rotary evaporation under reduced pressure to obtain a uniform lipid membrane, and dry it in vacuum for 24 hours. Add FAM aqueous solution for hydration, and shake in a water bath at 60°C for 2 hours to obtain a liposome suspension. In a water bath at 60°C, the liposomes were sequentially extruded through 400, 200, 100 and 50 nm nuclear pore membranes using a micro-extruder to reduce the particle size. Then use physiological saline as the eluent to separate and remove unencapsulated FAM through Sephadex G-50 chromatography column to obtain liposomes. The two kinds of liposomes were co-incubated with tumor cells for 4 hours, the supernatant was discarded, washed with PBS, and the uptake of liposomes by cells was observed by laser confocal microscope. The results showed that ordinary liposomes were hardly taken up, while RPARPAR modified liposomes were taken up in large quantities, indicating that RPARPAR-modified targeting material RPARPAR-PEG-DSPE endowed liposomes with in vitro targeting of tumor cells. the

Claims (8)

1. targeting material with tumor penetration performance, it is characterized in that, this material be by the target polypeptide with tumor penetration performance, Polyethylene Glycol and phospholipid three parts by the covalently bound linear block copolymers that forms, wherein the mol ratio of target polypeptide, Polyethylene Glycol and phospholipid is 1:1:1.

2. according to the described targeting material with tumor penetration performance of claim 1, it is characterized in that described aminoacid sequence with target polypeptide of tumor penetration performance is RPAKPAR.

3. according to the described targeting material with tumor penetration performance of claim 1, it is characterized in that the weight average molecular weight of described Polyethylene Glycol is 2000 ~ 5000.

4. according to the described targeting material with tumor penetration performance of claim 1, it is characterized in that described phospholipid is a kind of in DSPE (DSPE), two palmityl PHOSPHATIDYL ETHANOLAMINE (DPPE), DOPE (DOPE), hydrogenated soya phosphatide acyl ethanolamine (HSPE), hydrogenation egg phosphatide acyl ethanolamine, soybean phospholipid acyl ethanolamine, the egg phosphatide acyl ethanolamine.

5. according to the described targeting material with tumor penetration performance of claim 1, it is characterized in that the complex that described target polypeptide is formed by connecting by covalent bond form and Polyethylene Glycol-phospholipid.

6. according to the described targeting material with tumor penetration performance of claim 1, it is characterized in that, described target polypeptide provides a free sulfhydryl groups, and Polyethylene Glycol-phospholipid provides a dimaleoyl imino, and both specific reaction take place and connect into covalent complex.

7. one kind prepares according to claim 1,2,3,4,5, the method of 6 each described targeting materials, it is characterized in that, the concrete steps of this method are: adopt the synthetic C of solid-phase polypeptide synthetic method to hold the target polypeptide (aminoacid sequence is CRPAKPAR) of external cysteine (Cys), it is dissolved in the phosphate buffer (PBS) of pH7.0, get maleimide-Polyethylene Glycol-phosphatide complexes (Mal-PEG-PE) and be dissolved in dimethyl formamide (DMF), both mix the back stirring reaction and react completely to Mal-PEG-PE, remove excessive polypeptide and DMF, lyophilization, obtain target polypeptide-Polyethylene Glycol-phosphatide complexes, namely have the targeting material of tumor penetration performance.

8. according to the claim 1-6 described application of targeting material in pharmaceutical carriers such as preparation liposome, micelle with tumor penetration performance arbitrarily.

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