CN116410465A

CN116410465A - Acid-responsive block copolymer, responsive micelle, preparation method and application thereof

Info

Publication number: CN116410465A
Application number: CN202310227169.XA
Authority: CN
Inventors: 聂广军; 李素萍; 戚飞龙; 李博朝
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2023-03-10
Filing date: 2023-03-10
Publication date: 2023-07-11

Abstract

The invention provides a targeting peptide modified acid response block copolymer, a response micelle, a preparation method and application thereof. The fulvestrant micelle new dosage form can respectively load fulvestrant and another chemotherapeutic drug into the hydrophobic inner core of the micelle with higher encapsulation efficiency and drug loading, the micelle has good stability, can target and co-deliver fulvestrant and the other chemotherapeutic drug to tumor tissues, responds to a slightly acidic environment to release the drug, and can specifically realize high-efficiency killing of tumor cells, simultaneously avoid toxic and side effects on normal tissues, has good biocompatibility and has very wide application prospect. Can be used for treating breast cancer positive for estrogen receptor.

Description

Acid-responsive block copolymer, responsive micelle, preparation method and application thereof

Technical Field

The invention belongs to the field of nano-drug preparations, and particularly relates to a targeting peptide modified acid response block copolymer, a response micelle, a preparation method and application thereof.

Background

Breast cancer is one of the most common malignant tumors worldwide, which is caused by the unlimited proliferation of mammary epithelial cells. According to the latest published data of the world health organization, the new occurrence of breast cancer reaches 230 ten thousand in 2020, and the new occurrence of breast cancer accounts for 24.5% of the new occurrence of female cancer, and the new occurrence of breast cancer has become the first cancer worldwide instead of lung cancer. But the incidence of breast cancer in China is more optimistic, and new cases and death cases are the first in the world. Breast cancer can be divided into different subtypes, with estrogen receptor (Estrogen Receptor, ER) positive breast cancer being the most common, accounting for over 70% of breast cancer patients, with lower survival rates 5 years after metastasis, and thus being particularly important for the treatment of such breast cancer.

At present, the treatment of ER-positive breast cancer clinically at home and abroad mainly adopts medicines represented by fulvestrant and the like for endocrine treatment. Fulvestrant is a competitive antagonist of ER, effectively shielding the pro-proliferative effect of estrogen on cancer cells and down regulating ER expression. However, prolonged use of fulvestrant can lead to secondary resistance in almost all patients, and some patients who have never been treated with fulvestrant can also develop primary resistance, further reducing the effectiveness of endocrine therapy. On the other hand, fulvestrant has poor solubility and must be dissolved in a mixed solvent of ethanol, benzyl alcohol and castor oil, and can be administered by hip intramuscular injection. When the medicine is administrated, two 5ml injections are required to be continuously and slowly injected, the duration of each injection is not shorter than 2 minutes, the process can cause serious pain, and the medication compliance of patients is obviously reduced. On the other hand, the clinical requirement on the professional skill for the intramuscular injection with large volume is extremely high, and the success rate of the intramuscular injection is only 32-52 percent according to statistics. Moreover, the existence of castor oil makes the injection very viscous, causes swelling and nodule at the injection site, and even necrosis and ulcer can occur when severe, and the toxic and side effects of the medicine further reduce the compliance of patients. Thus, endocrine therapy employed clinically against ER-positive breast cancer still suffers from the disadvantages of easy drug resistance and poor compliance, and development of new dosage forms or administration strategies is urgently needed.

Research shows that the molecular mechanism of fulvestrant resistance has very close relation with cell cycle. In particular, CDK4 and CDK6 play a critical role in promoting proliferation and drug resistance formation of breast cancer cells, a phenomenon which occurs in most breast cancer patients (. Gtoreq.50%), and thus inhibition of CDK4/CDK6 activity is an emerging strategy to overcome fulvestrant resistance. Clinical experiments also show that the combination of the CDK4/CDK6 inhibitor on the basis of fulvestrant can improve the treatment effect of fulvestrant drug-resistant patients and remarkably prolong the progression-free survival time of the patients. Currently, the latest generation of CDK4/CDK6 inhibitors is abetacilib (Abemacilib), which in combination with fulvestrant can exert a good breast cancer inhibiting effect, whereas the water solubility of abberacil and fulvestrant severely limits the bioavailability and efficacy of the co-administration strategy.

In recent years, the rise of nanotechnology provides a new thought for solving the problems, but clinical treatment means of ER positive breast cancer mainly comprise traditional modes such as surgical excision, radiotherapy and chemotherapy, endocrine treatment and the like, and an intelligent response type nano drug delivery strategy has not been developed in breakthrough. Currently, there are few reports on the use of nanosystems for delivering fulvestrant. For example, polylactic acid glycolic acid-polyethylene glycol and polycaprolactone-polyethylene glycol can encapsulate fulvestrant and self-assemble into nano particles, but the drug release is incomplete and very slow, and only 30% of fulvestrant is released after 50 days, and in vitro cell experiments prove that the polymer delivery system has anti-tumor activity. Mesoporous silica can also be used for entrapment of fulvestrant, but it has not been evaluated for any antitumor effect. The only few studies described above focus mainly on the construction and characterization of the vector, and the biological functions of the vector are less focused, lack or are only subjected to simple drug effect evaluation at the cellular level, and drug effect studies are not performed at the living level, and the problem of pain caused by intramuscular injection is not focused, which is far from true clinical application. Therefore, how to find an effective breast cancer treatment strategy by utilizing the nanotechnology can bring hopes to a plurality of breast cancer patients, has huge beneficiary, and has great social significance.

Disclosure of Invention

Therefore, the invention aims to overcome the defects in the prior art and provide a targeting peptide modified acid response block copolymer, a response micelle, a preparation method and application thereof, so as to solve the defects of poor solubility of fulvestrant, effective curative effect, easy drug resistance, large toxic and side effects, low compliance and the like. By virtue of the advantages of the nanotechnology, a new fulvestrant dosage form capable of being injected intravenously is designed and constructed, and the nano medicament can be actively targeted to enrich tumor tissues and respond to the disintegration of a slightly acidic environment so as to release medicament at fixed points, thereby enhancing the tumor killing effect and reducing toxic and side effects.

Before setting forth the present disclosure, the terms used herein are defined as follows:

the term "PD-1/PD-L1 mab" refers to: programmed death receptor 1 or programmed death receptor ligand 1.

The term "RGD motif" refers to: RGD fragments.

The term "PAE" refers to: poly-beta amino esters.

The term "PEG" refers to: polyethylene glycol.

The term "Fmoc" refers to: 9-fluorenylmethoxycarbonyl.

The term "Fmoc-PEG-OH" refers to: 9-fluorenylmethoxycarbonyl-polyethylene glycol-hydroxy.

The term "Fmoc-PEG-PAE" refers to: 9-fluorenylmethoxycarbonyl-polyethylene glycol-poly beta-amino ester.

The term "NHS-C6-MAL" refers to: n- (6-maleimidocaprooic acid) succinimide.

The term "PEG-PAE" refers to: polyethylene glycol-poly beta amino ester

The term "NHS-PEG-OH" refers to: succinimidyl ester-polyethylene glycol-hydroxy.

The term "PAE-PEG-NHS" refers to: poly beta amino ester-polyethylene glycol-succinimidyl ester term "PAE-PEG-NH ₂ "means: poly beta amino ester-polyethylene glycol-amino.

The term "PAE-PEG-MAL" refers to: poly beta amino ester-polyethylene glycol-maleimide.

The term "Michael addition reaction" refers to: michael addition reaction, a conjugated addition reaction of electrophilic conjugated systems (electron acceptors) with nucleophilic carbanions (electron donors).

The term "PAE-PEG-cRGD" refers to: targeting peptide modified acid-responsive block copolymers.

The term "sample" refers to: physiological saline.

The term "ER" refers to: estrogen receptor (Estrogen Receptor).

The term "PPFA" refers to: and carrying the fulvestrant and abbe cily acid-responsive micelle.

The term "PPFA-mRGD" refers to: and the acid-responsive micelle is loaded with fulvestrant and abbe-cilia and has mutant peptide modification.

The term "FUL+ABE" means: free fulvestrant and arbekiln.

The term "PPAE-F-cRGD" refers to: acid-responsive micelles carrying fulvestrant and having cRGD tumor-targeting peptide modifications

The term "PPAE-A-cRGD" refers to: an acid-responsive micelle carrying abbe-cili and having a cRGD tumor-targeting peptide modification.

The term "PPAE-cRGD" refers to: empty and cRGD tumor-targeting peptide modified acid-responsive micelles.

The term "PPFA-cRGD" refers to: an acid-responsive micelle carrying fulvestrant and abbe-cili and modified by cRGD tumor targeting peptide.

To achieve the above object, a first aspect of the present invention provides a targeting peptide modified acid responsive block copolymer comprising a tumor targeting peptide and a block copolymer segment; wherein, the liquid crystal display device comprises a liquid crystal display device,

the tumor targeting peptide is an integrin receptor targeting peptide, and the block copolymer segment comprises a hydrophilic segment and an acid response segment.

The targeting peptide modified acid responsive block copolymer according to the first aspect of the invention, wherein,

the integrin receptor targeting peptide is a cyclic peptide, preferably a single-ring active peptide which contains a D-type amino acid and is connected with peptide bonds to form a ring from the head to the tail, and more preferably a cyclic pentapeptide;

the hydrophilic segment is selected from one or more of the following: polyethylene glycol, polyvinyl alcohol, polyethylenimine, polyvinylpyrrolidone, polyethylene oxide, polyacrylic acid, preferably selected from one or more of the following: polyethylene glycol, polyvinyl alcohol, polyethylene imine, more preferably polyethylene glycol or polyethylene imine, most preferably polyethylene glycol; and/or

The acid responsive segment is a polymer segment containing an acid responsive group or chemical bond, preferably selected from one or more of the following: polymers containing schiff base structures, polymers containing protonatable groups, polymers containing β -carboxylic acid amide linkages, polymers containing ketal or acetal structures, polymers containing orthoester linkages, more preferably selected from one or more of the following: polymers containing quaternary ammonium groups, polymers containing a benzamide linkage, polymers containing an acylhydrazone linkage, polymers containing 2, 3-dimethylmaleic anhydride, poly-beta amino esters, polyhistidine, polymethacrylic acid, most preferably poly-beta amino esters;

preferably, the sequence of the cyclic pentapeptide is selected from one or more of the following: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, more preferably selected from one or more of the following: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, further preferably SEQ ID NO.1 or SEQ ID NO.2, most preferably SEQ ID NO.1.

the targeting peptide modified acid-responsive block copolymer has a structure represented by the general formula I:

in formula I:

x is the number of hydrophilic segment monomers, preferably 1 to 1000, more preferably 1 to 500, still more preferably 10 to 100;

y is the number of acid-responsive segment monomers, preferably 1 to 1000, more preferably 1 to 500, still more preferably 10 to 50;

preferably, the tumor targeting peptide is covalently linked to the hydrophilic segment portion of the block copolymer segment by a chemical bond;

more preferably, the tumor targeting peptide is covalently linked to maleimide-modified polyethylene glycol by an affinity addition reaction between a thiol group and maleimide to form a stable thioether bond.

In a second aspect, the present invention provides a fulvestrant acid-responsive micelle comprising: nanoparticles formed from the targeting peptide-modified acid-responsive block copolymer of the first aspect and a drug; wherein, the liquid crystal display device comprises a liquid crystal display device,

the medicine comprises fulvestrant and other antitumor medicines except fulvestrant;

preferably, the other antitumor drug is selected from one or more of the following: alkylating agents, antitumor antibiotics, hormonal agents, metallic platinum agents, bioactive peptides, cytokines, antibodies, antimetabolites, more preferably selected from one or more of the following: abeli, piperaquine Bai Xili, dareli, rabocicli, anastrozole, apilimus, everolimus, PD-1/PD-L1 mab, further preferably selected from one or more of the following: abeli, piperaquine Bai Xili, dareli, rabocicli, anastrozole, apirism, even more preferably selected from one or more of the following: abeli, piperaquine Bai Xili, dareli, rebaudil, most preferably Abeli.

The fulvestrant acid-responsive micelle according to the second aspect of the present invention has a particle size of 10 to 1000nm, preferably 10 to 500nm, more preferably 10 to 250nm, and even more preferably 50 to 200nm.

A third aspect of the present invention provides a method of preparing the targeting peptide modified acid responsive block copolymer of the first aspect, the method comprising the steps of:

(1) Preparation of PAE-PEG-NH ₂ ；

(2) Preparing a polymer PAE-PEG-MAL;

(3) And (3) reacting the polymer PAE-PEG-MAL prepared in the step (2) with a tumor targeting peptide to obtain the targeting peptide modified acid response block copolymer.

The method according to the third aspect of the present invention, wherein the step (1) further includes: fmoc-PEG-OH is taken as a raw material to react with acryloyl chloride, then a reagent is added into an intermediate product to react to obtain Fmoc-PEG-PAE, and finally Fmoc is removed to obtain PAE-PAE-NH ₂ ；

Preferably, the Fmoc group is a protecting group; and/or

Preferably, the reagent is 1, 6-bis (acryloyloxy) hexane and/or 1, 3-bis (4-piperidine) propane.

The method according to the third aspect of the present invention, wherein,

the step (2) further comprises: introducing maleimide group for modification to obtain a polymer PAE-PEG-MAL; preferably, the modifying agent is selected from one or more of the following: NHS-C6-MAL, NHS-C7-MAL, NHS-C8-MAL, more preferably NHS-C6-MAL or NHS-C7-MAL, most preferably NHS-C6-MAL; and/or

In the step (3): the reaction is an addition reaction, preferably a Michael addition reaction between a thiol group and a maleimide.

A fourth aspect of the present invention provides a method of preparing fulvestrant acid-responsive micelles according to the second aspect, the method comprising:

(4) Forming micelle by hydrophobic interaction between the targeting peptide modified acid response block copolymer obtained in the step (3) and the drug under ultrasonic condition, and packaging the drug in the interior;

preferably, the time of the ultrasonic wave is 0.5 to 30min, more preferably 0.5 to 20min, still more preferably 0.5 to 10min.

The fifth aspect of the invention provides the use of the targeting peptide modified acid responsive block copolymer of the first aspect or the fulvestrant acid responsive micelle of the second aspect in the preparation of a medicament for treating breast cancer;

preferably, the breast cancer is hormone receptor positive breast cancer;

more preferably, the breast cancer is hormone receptor positive human epidermal growth factor receptor 2 negative breast cancer;

further preferably, the breast cancer is a hormone receptor positive HER 2 negative locally advanced or metastatic breast cancer.

The invention provides a tumor targeting peptide which is cyclic pentapeptide, and the amino acid sequence of the tumor targeting peptide is shown as SEQ ID NO. 1-6:

SEQ ID NO.1: the standard format of cyclo (Arg-Gly-Asp-DPhe-Cys), i.e., c (RGDfC) (the standard format of this sequence listing is not cyclic (cyclo) and therefore SEQ ID NO.1 is based on the sequence information described herein).

SEQ ID NO.2: cyclo (Arg-Gly-Asp-DTyr-Cys), i.e. c (RGDyC) (because the standard format of the sequence listing attached to the present application cannot embody a loop (cyclo), SEQ ID NO.2 is based on the sequence information described herein).

SEQ ID NO.3: cyclo (Arg-Gly-Asp-DPhe-Lys), i.e., c (RGDfK) (because the standard format cannot embody a loop (cyclo) in the sequence listing attached to the present application, SEQ ID NO.3 controls the sequence information described herein).

SEQ ID NO.4: cyclo (Arg-Gly-Asp-DTyr-Lys), i.e. c (RGDyK) (because the standard format of the sequence listing attached to the present application cannot embody a loop (cyclo), SEQ ID NO.4 is based on the sequence information described herein).

SEQ ID NO.5: cyclo (Arg-Gly-Asp-DPhe-Glu), i.e., c (RGDfE) ((because the standard format cannot represent a cyclic ring (cyclo) in the sequence listing attached to the present application, SEQ ID NO.5 controls the sequence information described herein).

SEQ ID NO.6: cyclo (Arg-Gly-Asp-DTyr-Glu), i.e. c (RGDyE) ((because the standard format cannot represent a cyclic ring (cyclo) in the sequence listing attached to the present application, SEQ ID NO.6 controls the sequence information described herein).

According to a specific embodiment of the invention, the first aspect of the invention provides a targeting peptide modified acid responsive block copolymer and a method for synthesizing the same, wherein the block copolymer comprises a tumor targeting peptide and a block copolymer segment.

Preferably, the tumor targeting peptide is an integrin receptor targeting peptide.

Preferably, the integrin receptor targeting peptide is a cyclic peptide.

Preferably, the cyclic integrin receptor targeting peptide is cyclic pentapeptide, i.e., a monocyclic active peptide with peptide bond ring formed by connecting head and tail, which contains a D-type amino acid, and the specific sequence is cyclo (Arg-Gly-Asp-DPhe-Cys), i.e., c (RGDfC).

Preferably, the block copolymer comprises a polyethylene glycol ((poly (ethylene glycol), PEG)) as the hydrophilic moiety and the acid-responsive segment is a polymer segment comprising an acid-responsive group or chemical bond.

Preferably, the acid responsive segment is poly (β -amino ester), (PAE), which is hydrophobic in a medium-sized environment, such that a micellar system is formed to encapsulate fulvestrant with another drug inside, whereas in a tumor slightly acidic environment, protonation can occur, such that the PAE moiety turns hydrophilic, the micelle breaks, releasing the carried drug.

Preferably, c (RGDfC) is covalently linked to the polyethylene glycol moiety of the amphiphilic polymer fragment by a chemical bond.

Preferably, c (RGDfC) is covalently linked to maleimide-modified polyethylene glycol by an affinity addition reaction between a thiol group and maleimide to form a stable thioether linkage.

Preferably, the specific structure of the targeting peptide modified acid response block copolymer is PAE-PEG-cRGD.

Preferably, the molecular weight of the PAE and the PEG part in the PAE-PEG-cRGD is 1 KD-20 KD.

Preferably, the molecular weights of the PAE and PEG moieties in the PAE-PEG-cRGD are 5KD and 10KD, respectively.

Preferably, the method for synthesizing the targeting peptide modified acid-responsive block copolymer is as follows:

wherein X is the number of ethylene glycol monomers, and Y is the number of beta-amino ester monomers.

In a second aspect, the present invention provides a method of preparing a targeted peptide modified acid-responsive block copolymer according to the first aspect for self-assembly into a nano-drug delivery system.

In the invention, an emulsification method is adopted, so that micelle nano particles loaded with two medicines are formed.

Preferably, the two drugs include fulvestrant and another anti-tumor drug, which includes a chemotherapeutic drug or an immune drug, and the like.

Preferably, the two medicaments in the invention are fulvestrant and abbe-cilia

Preferably, the fulvestrant polymer micelle has a particle size of 10 1000nm, for example 10nm, 50nm, 100nm, 150nm, 200nm, 500nm, 1000nm or the like.

Preferably, the self-assembly process of the micelle requires dissolving the polymer material and the drug in methylene dichloride, dispersing and emulsifying the micelle in water by ultrasonic, wherein the ultrasonic power is 10-200W, and the duration is 0.5-30 minutes.

Preferably, the methylene chloride involved in the emulsification process is removed by spin evaporation after micelle formation.

The preparation method provided by the invention has the advantages of simple synthesis, large drug loading, lasting drug release and uniform particle size.

In a third aspect, the present invention provides the use of fulvestrant polymer micelles according to the second aspect in the treatment of breast cancer.

According to another specific embodiment of the invention, the invention provides fulvestrant acid-responsive micelle, which comprises nano-particles formed by acid-responsive material monomers modified by tumor targeting peptides and medicines;

the acid-responsive material comprises a hydrophilic segment and an acid-responsive segment;

the acid response fragment is poly (beta-amino ester), PAE;

the hydrophilic segment is polyethylene glycol (poly (ethylene glycol), PEG);

the tumor targeting peptide is an integrin receptor targeting peptide, and the integrin receptor targeting peptide is a cyclic peptide with a sequence;

arginine glycine aspartic acid-D phenylalanine-cysteine;

the structural formula of the targeting peptide modified acid response polymer is shown in the following figure:

the fulvestrant and another drug are delivered to tumor tissues together in a targeting way by using a micelle system formed by an acid response polymer modified by a tumor targeting peptide, and the drug is released in response to a tumor micro-acid environment, so that an anti-tumor effect is exerted.

The preparation method of the fulvestrant polymer micelle comprises the following steps of:

(1) Fmoc-PEG-OH is taken as a raw material, the Fmoc-PEG-OH is reacted with acryloyl chloride, then 1, 6-bis (acryloyloxy) hexane and 1, 3-bis (4-piperidine) propane are added into an intermediate product to react to obtain Fmoc-PEG-PAE, and finally Fmoc is removed to obtain PAE-PEG-NH ₂ ；

(2) Further introducing maleimide group for modification to obtain a polymer PAE-PEG-MAL;

(3) The PAE-PEG-MAL further performs addition reaction with cRGD cyclic peptide, so that an amphiphilic block polymer PAE-PEG-cRGD with acid response modified by the cRGD targeting peptide is obtained;

(5) And (3) forming micelle by the PAE-PEG-cRGD, fulvestrant and another antitumor drug obtained in the step (3) under the ultrasonic condition under the help of hydrophobic effect, and coating the drug inside.

The protecting group in step (1) is an Fmoc group.

The coupling reagent in the step (2) is NHS-C6-MAL.

The coupling reaction in the step (3) is a Michael addition reaction between a mercapto group and maleimide.

The ultrasonic time in the step (4) is 1-30min, and the medicine is fulvestrant and another anti-tumor medicine.

The invention also provides application of fulvestrant micelle in preparation of an anti-breast cancer drug.

In the invention, the acid response micelle system can be used as a delivery platform to realize the combined delivery of fulvestrant and other various medicines, realize the synergistic effect of the two medicines, and prepare the medicine composition for treating tumor to play an anti-tumor effect.

The invention provides a new fulvestrant administration dosage form, a preparation method and application thereof. The invention provides a fulvestrant acid responsive micelle delivery system for treatment of estrogen receptor positive breast cancer. The delivery system includes: a drug and an acid responsive material. The medicine is fulvestrant and other various antitumor medicines represented by Abeli; the acid-responsive material includes one or more of materials having acid-responsive ability such as poly-beta amino esters. The fulvestrant micelle new dosage form can respectively load fulvestrant and another chemotherapeutic drug into the hydrophobic inner core of the micelle with higher encapsulation efficiency and drug loading, the micelle has good stability, can target and co-deliver fulvestrant and the other chemotherapeutic drug to tumor tissues, responds to a slightly acidic environment to release the drug, and can specifically realize high-efficiency killing of tumor cells, simultaneously avoid toxic and side effects on normal tissues, has good biocompatibility and has very wide application prospect.

Integrins act as a ubiquitous class of cell adhesion factors, the heterodimeric transmembrane glycoprotein receptors on the cell surface. Studies have shown that integrins, in particular alpha _v β ₃ The over-expression in various tumor cells plays a vital role in physiological processes such as proliferation, invasion, metastasis and the like of tumors, and is an ideal target for tumor diagnosis and treatment. Due to integrin alpha _v β ₃ Arginine-glycine-aspartic acid (Arg-Gly-Asp, RGD) sequences can be specifically recognized, and thus targeting peptides containing RGD sequences have been developed for targeting integrin αvβ3. Such RGD targeting peptides can be classified into linear and cyclic based on structure, both of which have good integrin targeting ability, but cyclic RGD polypeptides have stronger targeting ability. Moreover, compared with the linear peptide, on the one hand, the rigidity of the molecular structure of the cyclic polypeptide is enhanced, and on the other hand, the stability of the cyclic polypeptide is significantly higher than that of the linear polypeptide because the salt bridge between the guanidyl group in arginine and the carboxyl group of aspartic acid can prevent the polypeptide from being degraded. The inventor further replaces phenylalanine or tyrosine in RGD cyclic peptide with D-type amino acid, which can improve the stability of the cyclic RGD polypeptide again.

The fulvestrant acid-responsive micelles of the invention may have, but are not limited to, the following benefits:

the invention starts from the practical problems (easy drug resistance, low patient compliance, poor pharmacokinetics and the like) existing in the clinical application of fulvestrant, selects a nanomaterial with better biocompatibility, develops a nano drug-carrying system capable of effectively improving the curative effect of fulvestrant, and aims to develop a new formulation of fulvestrant drug administration. In the invention, poly (beta-amino ester) capable of responding to the tumor micro-acid environment, PAE (poly (beta-amino ester)) is used as a hydrophobic part, polyethylene glycol (poly (ethylene glycol), PEG) is used as a hydrophilic part, an acid response block copolymer (PAE-PEG) is constructed, and a tumor targeting peptide (cRGD cyclic peptide) is modified, so that the block copolymer (PAE-PEG-cRGD) with acid response characteristics and tumor cell targeting capability is obtained. The polymer can be self-assembled into a micelle through a hydrophobic effect, and fulvestrant and Abbe Li Bao are loaded inside to obtain the drug-loaded nano drug (PPFA-cRGD). The cRGD on the surface of the nano-drug provided by the invention can be specifically combined with integrin on the surface of tumor cells, so that the nano-drug is enriched in tumor tissues. When entering tumor tissue, it can respond to the slightly acidic environment, the PAE part is protonated, the PAE part is converted from hydrophobicity to hydrophilicity, and the micelle is disintegrated and the drug molecule is released rapidly. On the one hand, the co-delivery of fulvestrant and arbekiln can avoid the generation of drug resistance and synergistically increase the effect; on the other hand, compared with the long-term low-dose drug action, the short-term high-dose drug burst induced by the disintegration of the nano-drug can be eliminated before the tumor cells form drug resistance. Compared with the prior study, the invention carries out antitumor effect evaluation on the living body level of the tumor-bearing mice for the first time on the basis of carrying out curative effect verification on the cellular level. The nano-drug provided by the invention can be administered by intravenous injection, so that severe pain caused by intramuscular injection is avoided, a brand new administration way is provided for fulvestrant, and the defects of easiness in drug resistance, low compliance, poor pharmacokinetics and the like of fulvestrant are effectively overcome. The invention has important clinical application value or provides a new strategy for clinical treatment of estrogen receptor positive breast cancer.

Drawings

Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

figure 1 shows the morphological characterization of fulvestrant acid response micelles PPFA-cRGD of the invention (scale 100 nm).

FIG. 2 shows the drug release profile of the synthesized PPFA-cRGD of the present invention; wherein, FIG. 2a is a release curve of fulvestrant showing that PPFA-cRGD can release fulvestrant in response to a slightly acidic environment; fig. 2b is a release profile of arbelii showing the pH-dependent release behavior of arbelii.

FIG. 3 shows the killing ability of PPFA-cRGD tumor cells of the present invention.

FIG. 4 shows the targeting ability of the PPFA-cRGD living tumor tissue of the present invention.

FIG. 5 shows the therapeutic effect of the PPFA-cRGD of the present invention in a human tumor xenograft (Patient derived tumor xenograft, PDX) model.

FIG. 6 shows a schematic representation of fulvestrant acid responsive micelles PPFA-cRGD; wherein, FIG. 6a shows the preparation process of PPFA-cRGD micelle; FIG. 6b shows the mechanism of action of PPFA-cRGD in killing tumor cells in response to release of drug in a slightly acidic environment after targeting tumor tissue.

Detailed Description

The invention is further illustrated by the following specific examples, which are, however, to be understood only for the purpose of more detailed description and are not to be construed as limiting the invention in any way.

This section generally describes the materials used in the test of the present invention and the test method. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. It will be apparent to those skilled in the art that in this context, the materials and methods of operation used in the present invention are well known in the art, if not specifically described.

The reagents and instrumentation used in the following examples were as follows:

reagent:

abeli and fulvestrant, all available from Selleck Biotechnology Inc.

Cetyl trimethylammonium bromide (CTAB), available from Shanghai Michelin Biochemical technologies Co., ltd.

Dichloromethane, uranium acetate, dialysis bags, PBS (phosphate buffer), all purchased from shanghai ji to biochemical technologies limited.

Cell Counting Kit-8 (CCK) solution was purchased from the Japan same Combo chemical institute.

NOD-SCID mice and nude mice were purchased from Peking Violet laboratory animal technologies Inc.

Instrument:

cell ultrasonic breaker model JY 92-IID, available from Ningbo Xinzhi biotechnology Co.

Rotary evaporator, model RE52CS, available from Shanghai asia biochemical instrumentation factory.

Transmission electron microscope, model HT7700, available from HITACHI, japan.

High performance liquid chromatograph, model LC-20AT, available from Shimadzu corporation, japan.

A low temperature, high speed centrifuge, model Multifuge X1 Pro, available from Sieimer's Fisher (Thermo Fisher) technologies, inc. of America.

Small animal optical 3D in vivo imaging system, model IVIS Spectrum, available from Perkinelmer, U.S. Perkinelmer.

Example 1

This example is presented to illustrate the preparation of fulvestrant acid responsive micelles PPFA-cRGD.

The cyclic pentapeptide used in this example is the sequence shown in SEQ ID NO.1: cyclo (Arg-Gly-Asp-DPhe-Cys), i.e. c (RGDfC).

2.0g of Fmoc-PEG-OH is weighed and dissolved in 20ml of chloroform, acryloyl chloride and triethylamine are added, and the mixture is stirred and reacted for 12 hours at room temperature to obtain Fmoc-PEG-propylene. 2.0g of Fmoc-PEG-propylene was weighed and dissolved in 20ml of chloroform, 1, 6-bis (acryloyloxy) hexane and 1, 3-bis (4-piperidine) propane were added, and the mixture was stirred and reacted at 55℃for 48 hours, followed by purification to obtain Fmoc-PEG-PAE. Removing Fmoc protecting group to obtain PAE-PEG-NH ₂ . Dissolving the PAE-PEG-MAL and NHS-6-MAL in chloroform, adding triethylamine, reacting at room temperature for 12h, and purifying to obtain PAE-PEG-MAL.100mg of PAE-PEG-MAL is dissolved in 5mL of DMF, cRGD is added for complete dissolution, the reaction is carried out for 12h at room temperature, after dialysis for 24h (molecular weight cut-off 3500 Da), the PAE-PEG-cRGD is obtained after freeze drying.

120mg of PEG-PAE and 60mg of PAE-PEG-cRGD were dissolved in 2mL of methylene chloride, respectively. Abeli 6mg and fulvestrant 3mg were added, and deionized water 7.5mL was added. Emulsifying (100 w, 5s for working and 5s for intermittence) after 1min of action by a probe ultrasonic instrument. The organic solvent was then removed by rotary evaporation at 37 ℃ for 30min to give drug-loaded polymer micelles (PPFA-cRGD). Then, the nanoparticles were collected by centrifugation at 15000g for 5 minutes and washed 3 times with pure water. The solution was sterilized with a 0.22 μm filter before use.

Example 2

The method steps of this example are the same as those of example 1, except that the cyclic pentapeptide used in this example is the sequence shown in SEQ ID NO.2: cyclo (Arg-Gly-Asp-DTyr-Cys), i.e. c (RGDyC).

Example 3

The cyclic pentapeptide used in this example is the sequence shown in SEQ ID NO.3: cyclo (Arg-Gly-Asp-DPhe-Lys), i.e. c (RGDfK).

Weighing 2.0g of NHS-PEG-OH, dissolving in 20ml of chloroform, adding acryloyl chloride and triethylamine, and stirring at room temperature for reaction for 12 hours to obtain NHS-PEG-propylene. 2.0g of NHS-PEG-propylene is weighed and dissolved in 20ml of chloroform, 1, 6-bis (acryloyloxy) hexane and 1, 3-bis (4-piperidine) propane are added, and stirred and reacted for 48 hours at 55 ℃, and then NHS-PEG-PAE is obtained after purification. 100mg of PAE-PEG-NHS is dissolved in 5mL of DMF, cRGD is added for complete dissolution, the reaction is carried out for 12h at room temperature, after dialysis for 24h (molecular weight cut-off 3500 Da), the PAE-PEG-cRGD is obtained after freeze drying.

120mg of PAE-PEG and 60mg of PAE-PEG-cRGD were dissolved in 2mL of methylene chloride, respectively. Abeli 6mg and fulvestrant 3mg were added, and deionized water 7.5mL was added. Emulsifying (100 w, 5s for working and 5s for intermittence) after 1min of action by a probe ultrasonic instrument. The organic solvent was then removed by rotary evaporation at 37 ℃ for 30min to give drug-loaded polymer micelles (PPFA-cRGD). Then, the nanoparticles were collected by centrifugation at 15000g for 5 minutes and washed 3 times with pure water. The solution was sterilized with a 0.22 μm filter before use.

Example 4

The method steps of this example are the same as those of example 3, except that the cyclic pentapeptide used in this example is the sequence shown in SEQ ID NO.4: cyclo (Arg-Gly-Asp-DTyr-Lys), i.e. c (RGDyK).

Example 5

The cyclic pentapeptide used in this example is the sequence shown in SEQ ID NO.5: cyclo (Arg-Gly-Asp-DPhe-Glu), i.e. c (RGDfE).

Synthesis and purification of PAE-PEG-NH according to the conditions in example 1 ₂ After that, 100mg of PAE-PEG-NH ₂ Dissolving in 5mL DMF, adding the target peptide, dissolving completely, reacting at room temperature for 12h, dialyzing for 24h (retention fraction)Molecular weight 3500 Da), and freeze-drying to obtain the corresponding PAE-PEG-cRGD. Drug-loaded polymer micelles (PPFA-cRGD) were further prepared in the same manner as in example 1

Example 6

The procedure of this example is the same as in example 5, except that the cyclic pentapeptide used in this example is the sequence shown in SEQ ID NO.6: cyclo (Arg-Gly-Asp-DTyr-Glu), i.e. c (RGDyE).

Fulvestrant acid-responsive micelles prepared in examples 1 to 6, in which the presence of cysteine or lysine in the RGD cyclic peptides shown in SEQ ID No.1 to SEQ ID No.6 imparts a free thiol or amino group to the system, allowing the 6 cyclic targeting peptides to be coupled to a variety of functional molecules conveniently and efficiently, were subjected to the following test only by way of example to select fulvestrant acid-responsive micelles prepared in example 1, but it will be appreciated by those skilled in the art that the test results of examples 7 to 10 are equally applicable to examples 2 to 6.

Example 7

This example is used to illustrate the characterization of fulvestrant acid response micelle PPFA-cRGD and the pH-responsive release of drugs.

(1) Characterization of the PPFA-cRGD obtained in example 1

Morphology characterization: after the fulvestrant polymer micelle is dyed by uranium acetate, the morphology of the fulvestrant polymer micelle is characterized by adopting a transmission electron microscope, and as shown in figure 1, the nano micelle is spherical, has uniform particle size and has average particle size of about 100nm.

(2) pH-responsive release of drug: in vitro release experiments of fulvestrant and arbelide were performed in Phosphate Buffer (PBS) containing 0.1% cetyl trimethylammonium bromide (CTAB) at pH 5.0, 6.5 and 7.4, respectively. The prepared PPFA-cRGD was dialyzed with 10mL PBS in a dialysis bag, the molecular weight cut-off was 10kD,37℃and shaken at 100 rpm. Dialysate (100 μl) samples were collected at designated time points and equal amounts of PBS were added. The concentration of released drug was determined by high performance liquid chromatography and quantified against a standard curve. The resulting drug release profile is shown in figure 2. Fig. 2 illustrates that the fulvestrant polymer micelle provided by the invention can respond to a slightly acidic environment to rapidly release the fulvestrant and the arbelide carried by the fulvestrant polymer micelle, and has the pH-dependent and time-dependent drug release characteristics.

Example 8

The method is used for evaluating the killing effect of PPFA-cRGD on estrogen receptor positive breast cancer cells, and comprises the following specific steps:

MCF-7 cells were seeded into 96-well plates (3000 cells/well) and different groups of drugs were added to the plates during the logarithmic phase of growth. After 24h of treatment, the medium was aspirated, 100. Mu.L of a solution containing 10% Cell Counting Kit-8 (CCK) was added, and after about 1 hour of incubation, the absorbance was measured at 450 nm. As can be seen from FIG. 3, PPFA-cRGD has a strong killing ability against MCF-7 cells.

Example 9

The embodiment is used for evaluating the living tumor targeting capability of the nano-drug PPFA-cRGD, and comprises the following specific steps:

cy5.5-labeled PPFA (no targeting peptide modified group), PPFA-mRGD (mutant peptide modified group) PPFA-cRG (targeting peptide modified group) and free Cy5.5 solution were intravenously injected into MCF-7 breast cancer model mice (n=3). At a specific time point after injection, the mice were anesthetized and then subjected to whole-body fluoroscopic imaging using a living imaging system. The results obtained are shown in FIG. 4, in which the PPFA-cRGD group has a very remarkable tumor tissue targeting ability, thereby specifically delivering drugs to tumor tissues.

Example 10

In this example, the living antitumor efficacy of PPFA-cRGD obtained in example 1 was evaluated by constructing a human breast cancer patient-derived xenograft tumor (PDX) model, and the specific procedure was as follows:

and taking estrogen receptor positive tumor tissues, transplanting the tumor tissues into a mammary fat pad of a NOD-SCID mouse, collecting the tumor tissues after the tumor tissues grow up, and continuously inoculating the tumor tissues into the mammary fat pad of the nude mouse, thereby successfully constructing a PDX model. When the tumor volume is about 100mm ³ At this time, the mice were randomly divided into 7 groups (n=6). Physiological saline, PPAE-cRGD, FUL+ABE, PPAE-F-cRGD, PPAE-A-cRGD, PPFA-mRGD and PPFA-cRGD were administered respectively for 5 times, and the tumor size was monitored every 2 days with vernier calipers. As shown in FIG. 5, PPFA-cRGD was able to effectively inhibit tumor growth compared to the other groups.

The applicant states that the invention is illustrated by the above examples as well as methods of making and using the same, but the invention is not limited to, i.e., does not necessarily rely on, the above process steps to practice the invention. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes may be made in the individual conditions without departing from the spirit and scope of the invention. It is to be understood that the invention is not to be limited to the described embodiments, but is to be given the full breadth of the claims, including equivalents of each of the elements described.

Claims

1. A targeting peptide modified acid-responsive block copolymer, wherein the targeting peptide modified acid-responsive block copolymer comprises a tumor targeting peptide and a block copolymer segment; wherein, the liquid crystal display device comprises a liquid crystal display device,

2. The targeting peptide modified acid responsive block copolymer of claim 1, wherein:

3. The targeted peptide modified acid-responsive block copolymer of claim 1 or 2, wherein the targeted peptide modified acid-responsive block copolymer has a structure represented by general formula I:

in formula I:

4. A fulvestrant acid-responsive micelle, characterized in that the fulvestrant acid-responsive micelle comprises: the targeted peptide modified acid-responsive block copolymer of claim 1 formed with a drug; wherein, the liquid crystal display device comprises a liquid crystal display device,

5. Fulvestrant acid-responsive micelle according to claim 4 having a particle size of 10 to 1000nm, preferably 10 to 500nm, more preferably 10 to 250nm, even more preferably 50 to 200nm.

6. A method of preparing a targeted peptide modified acid-responsive block copolymer as claimed in any one of claims 1 to 3, characterized in that the method comprises the steps of:

(1) Preparation of PAE-PEG-NH ₂ ；

(2) Preparing a polymer PAE-PEG-MAL;

7. The method of claim 6, wherein the step (1) further comprises: fmoc-PEG-OH is taken as a raw material to react with acryloyl chloride, then a reagent is added into an intermediate product to react to obtain Fmoc-PEG-PAE, and finally Fmoc is removed to obtain PAE-PAE-NH ₂ ；

Preferably, the Fmoc group is a protecting group; and/or

8. The method according to claim 6 or 7, characterized in that:

9. A method of preparing fulvestrant acid-responsive micelles according to claim 4 or 5, wherein the method comprises:

10. Use of the targeting peptide modified acid responsive block copolymer of any one of claims 1 to 3 or the fulvestrant acid responsive micelle of claim 4 or 5 in the manufacture of a medicament for use in the treatment of breast cancer;

preferably, the breast cancer is hormone receptor positive breast cancer;