CN110483785B

CN110483785B - Triblock polymer, drug-loaded nano micelle, nano drug, and preparation method and application thereof

Info

Publication number: CN110483785B
Application number: CN201910584048.4A
Authority: CN
Inventors: 帅心涛; 何进; 肖洪; 王勇
Original assignee: National Sun Yat Sen University
Current assignee: National Sun Yat Sen University
Priority date: 2019-07-01
Filing date: 2019-07-01
Publication date: 2020-05-19
Anticipated expiration: 2039-07-01
Also published as: CN110483785A

Abstract

The invention relates to a triblock polymer, a drug-loaded nano micelle, a nano drug, and a preparation method and application thereof. The triblock polymer comprises a hydrophilic polyethylene glycol block, a middle poly (methacryloyl lysine-g- (cyclodextrin polypeptide)) block and a hydrophobic poly (aspartic acyl-N, N-dibutyl propylamine) block. The triblock polymer comprises a pH sensitive chain segment and an MMP-2 enzyme sensitive middle segment, and can be self-assembled into a nano micelle; the nano micelle obtained by assembly takes the pH sensitive chain segment as a core, takes the MMP-2 enzyme sensitive chain segment as an intermediate layer, takes methoxy polyethylene glycol as an outermost layer, has smaller particle size, higher drug loading rate and good pH and MMP enzyme sensitivity, and has small toxicity to cells; not only can load chemotherapeutic drugs and anti-angiogenesis drugs at the same time, but also can realize the micro-environment responsive release of the drugs, thereby realizing the targeted drug therapy of different cells at the tumor part, and having greater innovation and application value.

Description

Triblock polymer, drug-loaded nano micelle, nano drug, and preparation method and application thereof

Technical Field

The invention belongs to the field of nano-medicine, and particularly relates to a triblock polymer, a drug-loaded nano-micelle, a nano-drug, and a preparation method and application thereof.

Background

Solid tumors have the following: low pH, high intracellular reduced Glutathione (GSH) concentrations, and a complex physiological microenvironment such as the anaerobic environment inside the tumor. While the cellular composition of solid tumor tissue is quite complex. The non-malignant cells in the tumor tissue except cancer cells are closely related to the growth, proliferation, infiltration, metastasis, angiogenesis and other processes of the tumor.

The complex composition and rapid proliferation behavior of tumor tissue results in heterogeneity of tumor tissue structure and complex tumor microenvironment. Chemotherapeutic strategies directed only at this single target of cancer cells often do not completely destroy the tumor. To solve the problem of limited curative effect of single treatment, the current research direction mainly focuses on the following two aspects. The first is to directly aim at tumor cells and carry out cooperative treatment on a plurality of pathogenic targets of the tumor cells, such as combining chemotherapy drug adriamycin (DOX) with siRNA or other small molecule inhibitors, so as to achieve the purpose of cooperative treatment of the tumor cells. On the other hand, the tumor micro-environment therapy is targeted respectively aiming at tumor cells and other stromal cells at the tumor part, different drugs are respectively targeted to different cells, and the multi-cell target point cooperation or combination therapy is carried out to treat the tumor. The targeted tumor microenvironment treatment strategy is to effectively block the connection and interaction between tumor cells and the tumor microenvironment, thereby preventing the related processes of tumor proliferation, metastasis and the like. After the determination of the therapeutic target is completed, the problem to be considered is how to combine drug molecules with different action targets and action mechanisms according to different requirements, so that the regulation and control mechanism of the microenvironment on tumorigenesis, development and metastasis needs to be deeply researched; in addition, how to accurately deliver the multi-target drugs to the specific positions of the corresponding tumor sites is an urgent problem to be solved in the combination therapy of the multi-target drugs for tumors.

Disclosure of Invention

The invention aims to overcome the problems and defects that multi-target drugs cannot be accurately delivered to specific positions of corresponding tumor sites in the prior art, and provides a triblock polymer. The triblock polymer provided by the invention comprises a pH sensitive chain segment and an MMP-2 enzyme sensitive middle segment, and can be self-assembled into a nano micelle; the nano micelle obtained by assembly takes the pH sensitive chain segment as a core, takes the MMP-2 enzyme sensitive chain segment as an intermediate layer, takes methoxy polyethylene glycol as an outermost layer, has smaller particle size, higher drug loading rate and good pH and MMP enzyme sensitivity, and has small toxicity to cells; not only can load chemotherapeutic drugs and anti-angiogenesis drugs at the same time, but also can realize the micro-environment responsive release of the drugs, thereby realizing the targeted drug therapy of different cells at the tumor part, and having greater innovation and application value.

Another object of the present invention is to provide a process for the preparation of the above triblock polymer.

The invention also aims to provide a drug-loaded nano-micelle with tumor microenvironment response.

The invention also aims to provide application of the drug-loaded nano-micelle with tumor microenvironment response in drug loading.

Another object of the present invention is to provide a pH and MMP dual-responsive nano-drug.

The invention also aims to provide a preparation method of the nano-medicament with dual response of pH and MMP.

In order to achieve the purpose, the invention adopts the following technical scheme:

a triblock polymer comprising a hydrophilic block of polyethylene glycol, a middle block of poly (methacryloyllysine-g- (cyclodextrin polypeptide)) and a hydrophobic block of poly (asparaginyl-N, N-dibutylpropylamine); the molecular weight of the polyethylene glycol is 2-10 kDa, the molecular weight of the poly (methacryloyl lysine-g- (cyclodextrin polypeptide)) is 2-12 kDa, and the molecular weight of the poly (asparaginyl-N, N-dibutylpropylamine) is 2-6 kDa.

The invention provides an amphiphilic triblock polymer, which is characterized in that an acid-sensitive chain segment and an MMP enzyme-sensitive chain segment are simultaneously introduced through structural design, so that a micelle with pH and MMP enzyme double sensitivity can be formed after self-assembly, a hydrophobic inner core of the micelle can be loaded with a hydrophobic drug PTX, and meanwhile, the hydrophobic inner core can be loaded in a hydrophobic cavity of cyclodextrin through a special 'host-guest' action between the cyclodextrin and a Sunitinib molecule. The therapeutic strategy of simultaneously entrapping two drug molecules with different action targets by using different modes and respectively releasing the drug molecules to act on different cells at the tumor part under the action of pH and MMP enzyme can effectively solve the problem that the existing mainstream multi-target nano-carrier is difficult to realize the fixed-point release and the respective targeting of the drugs. On the other hand, the biodegradable polyamino acid material is taken as a main chain, the possibility of long-term in vivo application of the nano material is given, and the design of combining one-step initiation and aminolysis and click chemistry also enables the material to be easily controllably synthesized and the structure to be accurately regulated and controlled.

Preferably, the polyethylene glycol has a molecular weight of 5 kDa; the poly (methacrylyl lysine-g- (cyclodextrin polypeptide)) has a molecular weight of 10 kDa; the poly (asparaginyl-N, N-dibutylpropylamine) has a molecular weight of 5 kDa.

The molecular weight of each chain segment in the triblock polymer is regulated, the size of the nano micelle obtained by self-assembly of the triblock polymer can be regulated, and the size of the nano micelle loaded with the drug can be regulated. Under the condition of the molecular weight, the size of the obtained nano-drug is less than 200nm, and the nano-drug has higher drug loading rate and stability.

The preparation method of the triblock polymer comprises the following steps:

s1: synthesis of aminated mPEG-NH by taking mPEG-OH as raw material₂；

S2: with mPEG-NH₂Sequentially initiating the ring-opening polymerization of a methacrylyl Lysine MA-Lysine-NCA monomer and an aspartic acid cyclic anhydride BLA-NCA monomer to synthesize a triblock polymer main chain mPEG-b-PLLMA-b-PBLA as raw materials;

s3: then performing ammonolysis reaction, and converting the hydrophobic PBLA block of the mPEG-b-PLLMA-b-PBLA in S2 into a polyaspartic acid block by using N, N-dibutyl propylene diamine DBP to obtain a polymer mPEG-b-PLLMA-b-PASp (DBP);

s4: the cyclodextrin polypeptide conjugate CD-Peptide-SH is connected to the double bond of the PLLMA chain segment in the polymer mPEG-b-PLLMA-b-PASp (DBP) by utilizing the click reaction of thiol-double bond thiol-ene, and the triblock polymer is obtained.

The triblock polymer can be abbreviated as: PEG-b-PLLMA (CD-Peptide-SH) -b-PASp (DBP).

Preferably, the cyclodextrin polypeptide conjugate CD-Peptide-SH in S4 is prepared by the following process:

s401, β -cyclodextrin with p-toluenesulfonyl chloride to obtain CD-OTs;

s402: alkynylating CD-OTs to obtain alkynylated cyclodextrin CD-Yne;

s403: and (3) coupling alkynyl cyclodextrin CD-Yne and MMP-2 enzyme sensitive Peptide to obtain the cyclodextrin polypeptide conjugate CD-Peptide-SH.

A drug-loaded nano-micelle with tumor microenvironment response is formed by self-assembly of the triblock polymer.

The drug-loaded nano-micelle provided by the invention is a novel pH and MMP-2 enzyme responsive polymer nano-drug delivery system, and is designed aiming at the specific microenvironment of tumor sites. The core is a pH sensitive chain segment, the middle layer is an MMP-2 enzyme sensitive chain segment, and the outermost layer is methoxy polyethylene glycol, so that the core has a small particle size, a high drug loading rate and good pH and MMP enzyme sensitivity. The drug-loaded nano micelle has low toxicity to cells, can load chemotherapeutic drugs and anti-angiogenesis drugs at the same time, and can realize microenvironment responsive release of the drugs, thereby realizing targeted drug therapy on different cells at tumor parts, and having great innovation and application value.

the specific action principle is that a tumor angiogenesis inhibitor Sunitiib is loaded into a barrel-shaped hydrophobic cavity of β -CD by utilizing the interaction between a host and an object of β -cyclodextrin and an anti-angiogenesis small molecule drug Sunitiib, cyclodextrin molecules are connected to a polymer molecule main chain through an MMP-2 enzyme sensitive polypeptide chain segment, meanwhile, a polymer main chain molecule is used as an amphiphilic block polymer and can be self-assembled to form a nano micelle, the hydrophilic and hydrophobic effects are utilized to wrap chemotherapeutic paclitaxel PTX in a hydrophobic core of the nano particle, the formed nano particle loaded with the two drugs is passively targeted through an Enhanced Permeation and Retention (EPR) effect and can be accumulated at a tumor part through blood circulation, after the nano drug reaches the tumor part, the nano particle firstly carries the Sunitiib cyclodextrin enzyme digestion side chain under the action of the MMP-2 enzyme highly expressed in a tumor cell extracellular matrix (ECM), the nano particle releases the Sunitinib in the tumor extracellular matrix (ECM), and can preferentially act on an endothelial cell of the tumor cell to inhibit the tumor angiogenesis targeting lysosome tumor cells to generate the tumor cell nucleus through the synergistic effect of the Sunitinib and the intracellular targeting chemotherapeutic drug to realize the synergistic tumor cell targeting chemotherapy.

The application of the drug-loaded nano-micelle in drug loading is also in the protection scope of the invention.

Preferably, the drug is one or more of paclitaxel PTX and Sunitinib.

The pH and MMP dual-responsiveness nano-drug is obtained by simultaneously encapsulating Sunitinib and PTX by the drug-loaded nano-micelle.

The wrapping capacity of the medicine can be adjusted according to actual needs.

Preferably, the wrapping amount of the PTX accounts for 2.11-5.34% of the total mass of the micelle, and the wrapping amount of the drug Sunitinib accounts for 0.5-1.6% of the total mass of the micelle.

More preferably, the PTX loading is 5.34% of the total mass of the micelle.

The drug-loaded nano-micelle is sensitive to pH; at a pH value of 7.4 or so, the pH sensitive side chain is in a hydrophobic state and can load a hydrophobic anti-tumor drug, such as paclitaxel PTX, and in a tumor lysosome environment with a pH value of 5.0, the micelle is disintegrated, the anti-tumor drug PTX is released, and tumor cells are killed; when the micelle was loaded with the drug PTX, the maximum loading of the drug was 5.34% of the mass of the micelle.

More preferably, the loading of Sunitinib is 1.12% of the total mass of the micelle.

Polymeric micelles are sensitive to MMP-2 enzyme: the small molecule drug Sunitinib loaded on the micellar enzyme sensitive segment is slowly released in PBS with pH of 7.4 along with the prolonging of time, but the cumulative release amount is not more than 15% within 36 h; under the same condition, 10nM MMP-2 enzyme is added, the accumulative release amount within 12h exceeds 60%, after 36h, the Sunitinib is basically and completely released, and better time and concentration dependent MMP-2 response release is shown; when the micelle is loaded with the Sunitinib drug, the loading amount of the drug accounts for 1.1% of the mass of the micelle.

The preparation method of the pH and MMP dual-responsiveness nano-drug comprises the following steps:

s5: dissolving a triblock polymer and PTX in an organic solvent to obtain a solution, dropwise adding the solution into water under ultrasonic oscillation, and removing the organic solvent to obtain a micellar solution;

s6: and dissolving Sunitinib in an organic solvent, adding the Sunitinib into the micelle solution, stirring the mixture in the dark, removing the organic solvent, filtering the mixture, and concentrating the filtered mixture to obtain the pH and MMP dual-responsiveness nano-drug.

Preferably, the organic solvent in S5 and S6 is one or more of DMF, DMAc or DMSO.

Preferably, the organic solvent is removed by dialysis in S5 and S6.

Compared with the prior art, the invention has the following beneficial effects:

the triblock polymer provided by the invention comprises a pH sensitive chain segment and an MMP-2 enzyme sensitive middle segment, and can be self-assembled into a nano micelle; the nano micelle obtained by assembly takes the pH sensitive chain segment as a core, takes the MMP-2 enzyme sensitive chain segment as an intermediate layer, takes methoxy polyethylene glycol as an outermost layer, has smaller particle size, higher drug loading rate and good pH and MMP enzyme sensitivity, and has small toxicity to cells; not only can load chemotherapeutic drugs and anti-angiogenesis drugs at the same time, but also can realize the micro-environment responsive release of the drugs, thereby realizing the targeted drug therapy of different cells at the tumor part, and having greater innovation and application value.

Drawings

FIG. 1 is a synthetic route for cyclodextrin polypeptide conjugates;

FIG. 2 is a scheme for the synthesis of related polymers;

FIG. 3 is a representation of the correlation of cyclodextrin polypeptide conjugates¹H-NMR：(A)CD-OTs；(B)CD-Yne；(C)CD-Peptide-SH；

FIG. 4 is a nuclear magnetic map of the relevant block polymer (A) mPEG-PLLMA; (B) mPEG-PLLMA-PBLA; (C) mPEG-PLLMA (CD-Peptide-SH) -PASp (DBP);

FIG. 5 is a gel permeation chromatography GPC chart for a polymer;

FIG. 6 shows blank micelle pep-CD of DLS particle size (A) and MMP-2 enzyme sensitive double drug combination PTX & Sunitinib drug-loaded micelle of micelle and corresponding transmission electron microscope (C) blank micelle (D) PTX & Sunitinib drug-loaded micelle;

FIG. 7 is a transmission electron microscope image of drug-loaded micelles after (A) acid treatment at pH 5.0 and (B) MMP-2 enzyme treatment at 10nM of drug-loaded nanoparticles;

FIG. 8 is a graph of pH and MMP-2 enzyme sensitive drug in vitro simulated release behavior of drug-loaded nanoparticles.

Detailed Description

The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples below, generally according to conditions conventional in the art or as suggested by the manufacturer; the raw materials, reagents and the like used are, unless otherwise specified, those commercially available from the conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.

Example 1 Synthesis of Cyclodextrin polypeptide conjugates

The synthesis of cyclodextrin polypeptide conjugates is shown in fig. 1, and is divided into the following three parts:

firstly, weighing 30g of beta-cyclodextrin, dissolving the beta-cyclodextrin in 300mL of 1mol/L NaOH solution, adding 6g of p-toluenesulfonyl chloride under the condition of ice-water bath, continuing to magnetically stir at 0-5 ℃ for 5 hours, after the reaction is finished, performing suction filtration, removing solid insoluble substances, adjusting the pH of filtrate to be about 7 by using 10% hydrochloric acid, generating a large amount of white precipitate, putting the white precipitate into a refrigerator at 4 ℃ overnight, fully precipitating the precipitate, then performing suction filtration, washing the obtained solid with a small amount of cold deionized water for three times, and recrystallizing the solid in hot water to obtain a product CD-OTs shown in figure 3A, wherein the nuclear magnetic resonance spectrum of the solid in deuterated DMSO is (the nuclear magnetic resonance spectrum of the solid in deuterated DMSO is shown in the figure 3¹H-NMR) chemical shift cases were as follows: 2.43ppm (singlet, 3H, -CH)₃) 3.80-3.15ppm (multiplet, 42H, overlap with water peak, H₂,H₃,H₄,H₅,H_6a,H_6b) 4.60-4.41ppm (multiplet, 6H, OH)₆) 4.91-4.75ppm (multiplet, 7H, H)₁) 5.85-5.65ppm (multiplet, 14H, OH)_2,3) 7.48ppm (doublet, 2H, ArH),7.79ppm (doublet, 2H, ArH).

Then, N₂Under protection, 1g of CD-OTs and 124mg of propynylamine are dissolved in 15mL of DMF and reacted at 70 ℃ for 24 h. After the reaction, the reaction mixture was cooled to room temperature, precipitated in 50mL of acetone, the solid was centrifuged, dried in vacuo, dissolved in 30mL of a mixed solvent of methanol/water (1: 1; volume ratio), and then precipitated in a large amount of acetone, and the process was repeated twice to remove impurities. Then heating and recrystallizing in water and acetonitrile to obtain the alkynylated cyclodextrin CD-Yne. The NMR spectrum is shown in FIG. 3B, and we found that the NMR peaks of p-toluenesulfonic acid at 2.43ppm, 7.48ppm and 7.79ppm disappeared and propargylamine characteristic peaks appeared, compared with CD-OTs: 2.75ppm (HC ≡ C-CH)₂-)、3.03ppm(HC≡C-CH₂-)、1.96ppm(HC≡C-CH₂NH-)。

Finally, 100mg of CD-Yne, 20mg of sodium ascorbate, 100mg of MMP-2 enzyme sensitive peptide were dissolved in 10mL of anhydrous DMF and introduced into a 100mL Schlenk tube, and after three freeze/thaw cycles, air in the system was completely removed, followed by N-charging₂Under the condition, 5mg of CuSO is added₄·5H₂O, sealing the reaction bottle, and reacting for 24 hours in an oil bath at 40 ℃. After the reaction, 100. mu.L of Pentamethyldiethylenetriamine (PMDETA) was added, and then the mixture was precipitated into ether, centrifuged again, dried in vacuo and dissolved in 5mL of deionized water, and the solution was filled into a dialysis bag (MWCO 1000Da) and dialyzed against 3X 1L of deionized water for 2 days. And finally, freeze-drying to obtain white solid powder. Its nuclear magnetic resonance hydrogen spectrum (¹H-NMR) as shown in fig. 3C, we can see not only nuclear magnetic shift peaks characteristic of cyclodextrins: 3.35-3.15ppm (multiplet, 42H, overlap with water peak, H₂,H₃,H₄,H₅,H_6a,H_6b) 4.60-4.41ppm (multiplet, 6H, OH)₆) 4.91-4.75ppm (multiplet, 7H, H)₁) 5.85-5.65ppm (multiplet, 14H, OH)_2,3) New nuclear magnetic peaks also appear, and the specific assignments of these peaks are as follows: 0.80-0.92ppm (CH)₃-Leu, belonging to the polypeptide sequence); 1.1-1.7ppm(-CH₂-, -CH-belongs to the methylene and methine peaks of the side chains in the polypeptide sequence); 4.0-4.5ppm (-CONHCH-NH, methylene or methine peak connected with amido bond on the polypeptide skeleton); 7.37ppm (N)₃Characteristic peak of five-membered triazole ring formed by reaction with alkynyl Click).

Example 2 Synthesis of related Polymer

As shown in fig. 2, the synthesis of the polymer (i.e., triblock polymer) is divided into the following steps:

first, 1g mPEG_5k-NH₂(95% conversion of amino group) was charged into a 100mL Schlenk tube, dried under vacuum at 70 ℃ for 2h, and then cooled to room temperature. Ice water bath, filling N₂Under protection, 20mL freshly distilled CHCl was added₃0.5g MA-Lysine-NCA was dissolved in 5mL of ultra-dry DMF and added to the reaction flask in one portion. The reaction was sealed and magnetically stirred for 48h at 35 ℃ (at which time samples were taken for nuclear magnetic measurements to determine PLLMA repeat number) and then N₂1g BLA-NCA monomer dissolved in 5mL ultra dry DMF under protection was added to the reaction flask and 10mL freshly distilled CHCl was added₃Sealing and continuing the reaction at 35 ℃ for 48 h. After the reaction is finished, repeatedly precipitating in anhydrous ether, centrifuging, and drying in vacuum to obtain the product. The main chain of the block polymer is formed by mPEG-NH₂Respectively initiating the ring-opening polymerization of MA-Lysine-NCA and BLA-NCA to obtain the product. Firstly, a two-block polymer PEG-PLLMA is obtained, the nuclear magnetism of which is represented as figure 4A, and the nuclear magnetism of which is 3.5ppm (-OCH)₂CH₂-), 5.2ppm and 5.68ppm (s, -C (CH)₃)＝CH₂)，1.8ppm(s，-C(CH₃)＝CH₂)，1.1-1.8ppm(m,-(CH₂)₃CH₂NHCO-). The number of the repeating units of the PLLMA section is calculated to be 15 through nuclear magnetic integration, and is basically consistent with the designed number of the repeating units. Then mPEG-PLLMA is used as a macroinitiator to continuously initiate BLA-NCA ring-opening polymerization to obtain a triblock polymer mPEG-PLLMA-PBLA, and the nuclear magnetism diagram 4B shows that the nuclear magnetism peaks of mPEG section and PLLMA section and the characteristic nuclear magnetism displacement of PBLA appear: 5.0ppm (-COOCH)₂C₆H₅)，7.2ppm(-COOCH₂C₆H₅) PBLA segments also calculated by nuclear magnetic integral analysisThe number of repeating units (DP) was 20.

Then the polymer mPEG-b-PLLMA-b-PBLA obtained in the step (b) is subjected to ammonolysis reaction with N, N-Dibutylpropylamine (DBP). Specifically, 200mg of mPEG-b-PLLMA-b-PBLA and 0.5mL of LDBP are dissolved in 15mL of DMF, the mixture is magnetically stirred at 35 ℃ for reaction for 12 hours, then the mixture is precipitated in ether, the precipitation is repeatedly carried out for three times, the centrifugation and the vacuum drying are carried out to obtain the polyethylene glycol-polymethacryloyl lysine-poly (asparaginyl-N, N-dibutylpropylamine) mPEG-b-PLLMA-b-PASp (DBP), and the product is a slightly yellow solid.

Finally, the cyclodextrin polypeptide conjugate CD-Peptide-SH prepared in example 1 and polymer mPEG-PLLMA-PAsp (DBP) are subjected to Michael-addition reaction to prepare the cyclodextrin polypeptide conjugate CD-Peptide-SH, and the specific operation is as follows: n is a radical of₂Under protection, 250mg of CD-Peptide-SH and 100mg of mPEG_5k-PLLMA-PASP (DBP) was dissolved in 10mL of anhydrous DMF, added to a 50mL Schlenk tube, and 100. mu.L of dimethylphenylphosphine was added, sealed and reacted at 40 ℃ for 72 h. After the reaction is finished, repeatedly precipitating in anhydrous ether for three times, centrifuging, and drying in vacuum to obtain a primary product. Dissolving the initial product in DMF, performing self-assembly under ultrasound to obtain nano micelle, placing the nano micelle in a dialysis bag (MWCO 14k Da) and putting the nano micelle in deionized water for dialysis to remove free small molecules, then performing rotary evaporation and concentration, and performing freeze-drying to obtain a polymer mPEG-PLLMA (CD-Peptide-SH) -PASp (DBP) and white solid powder. The nuclear magnetic shift is shown in fig. 4C: 5.0ppm (-COOCH)₂C₆H₅)，7.2ppm(-COOCH₂C₆H₅) The peak originally belonging to PBLA disappeared, while the characteristic peak of methyl group (overlapping with the methyl peak of leucine in CD-Peptide) appeared on N, N-Dibutylpropylenediamine (DBP) at 0.83ppm, and in addition, the obvious nuclear magnetic shift peak of cyclodextrin was seen in the nuclear magnetic map, while the double bond peak of PLLMA segment did not disappear completely. By nuclear magnetic analysis, it was calculated that approximately 45% of the double bonds had undergone an addition reaction with the cyclodextrin MMP enzyme-sensitive polypeptide conjugate, which may be related to a decrease in the click reactivity and efficiency in the polymer.

From the gel permeation chromatography GPC chart (FIG. 5), it can be seen that mPEG-PLLMA-PASP (DBP) and CD-Peptide-SH grafted block polymer mPEG-PLLMA (CD-Peptide-SH) -PASP (DBP) showed a clear separation, and both were unimodal and had a narrow peak shape distribution.

The results of the nuclear magnetic resonance hydrogen spectrum and the gel permeation chromatography show that the cyclodextrin polypeptide grafted block polymer mPEG-PLLMA (CD-Peptide-SH) -PASP (DBP) is successfully synthesized.

If other molecular weight raw materials are selected, the triblock polymer with other molecular weight blocks can be obtained according to the conditions of the embodiment.

EXAMPLE 3 preparation of empty Carrier nanoparticles

This example provides a method for preparing empty carrier nanoparticles (i.e., drug-loaded nanomicelles with tumor microenvironment response), as follows.

Dissolving a certain amount of polymer mPEG-PLLMA (CD-Peptide-SH) -PASp (DBP) in a proper amount of good solvent DMF, and dropwise adding the polymer solution into a large amount of selective solvent H under the condition of stirring or ultrasonic dispersion₂And (4) in O. Specifically, 20mg of the polymer was dissolved in 2mL of DMF, 20mL of deionized water was placed in a small beaker and placed in an ice-water bath, and the polymer solution was added dropwise to deionized water with ultrasonic oscillation. After all the drops were added, the micellar solution was placed in a dialysis bag and dialyzed against deionized water for 2 days to remove DMF. Dialyzing to remove organic solvent, concentrating by ultrafiltration or rotary evaporation, and collecting micelle solution with certain volume, lyophilizing, and weighing. The concentration of the prepared polymer micelle is 5-10 mg/mL.

Example 4 preparation of entrapped paclitaxel nanomicelle

First, 20mg of the polymer mPEG-PLLMA (CD-Peptide-SH) -PASP (DBP) and 2mg of PTX were dissolved in 2mLDMF, 20mL of deionized water was put into a small beaker, placed in an ice-water bath, and the polymer solution was added dropwise to the deionized water under ultrasonic oscillation. After all the drops are added, the micelle solution is filled into a dialysis bag and dialyzed by deionized water to remove DMF. Dialyzing to remove the organic solvent, and concentrating the micelle solution by ultrafiltration or rotary evaporation, wherein the micelle concentration is obtained by taking a certain volume of the micelle solution, freeze-drying and weighing. The concentration of the prepared polymer drug-loaded micelle is 6.3 mg/mL. The PTX loading was 5.1% as determined by high performance liquid chromatography HPLC.

Example 5 preparation of Sunitinib-entrapped nano-micelle

First, 20mg of polymer mPEG-PLLMA (CD-Peptide-SH) -PASP (DBP) is dissolved in 2mLDMF, 20mL of deionized water is filled in a small beaker and placed in an ice-water bath, and the polymer solution is dropwise added into the deionized water under ultrasonic oscillation. After all the addition was completed, 1mg of Sunitinib was then dissolved in 1ml of dmf and added to the above solution with rapid stirring. Stirring at room temperature for 2h in the dark. And then filling the micelle solution into a dialysis bag, dialyzing with deionized water to remove DMF, and then filtering and concentrating to obtain the micelle carrying the Sunitinib. The micelle-permeable concentration is obtained by taking a certain volume of micelle solution, freeze-drying and weighing. The concentration of the prepared polymer drug-loaded micelle is 5.5 mg/mL. The Sunitinib loading was 1.6% as determined by HPLC.

Example 6 preparation of nano-micelle simultaneously encapsulating PTX and Sunitinib

First, 20mg of the polymers mPEG-PLLMA (CD-Peptide-SH) -PASP (DBP) and 2mg PTX were dissolved in 2mLDMF, 20mL of deionized water was put in a small beaker and placed in an ice-water bath, and the polymer solution was added dropwise to the deionized water under ultrasonic oscillation. After all the addition was completed, 1mg of Sunitinib was then dissolved in 1mL of DMF and added to the above solution with rapid stirring. Stirring at room temperature for 2h in the dark. And filling the micelle solution into a dialysis bag, dialyzing with deionized water to remove DMF, and filtering and concentrating to obtain the micelle simultaneously encapsulating PTX and Sunitinib. Dialyzing to remove the organic solvent, and concentrating by ultrafiltration or rotary evaporation, wherein the micelle concentration is obtained by taking a certain volume of micelle solution, freeze-drying and weighing. The concentration of the prepared polymer micelle is 7.3 mg/mL. PTX and Sunitinib drug loading rates were determined by HPLC to be 5.34% and 1.12%, respectively.

Example 7 particle size and morphology characterization of nanoparticles

The particle Size (Size) and surface potential (Zeta potential) of the nanoparticles were determined by Dynamic Light Scattering (DLS) on a particle Size potentiometer of the type 90Plus/BI-MAS from Brookhaven. DLS used scattering angles of 90 and 15, temperature 25 ℃, and five measurements per sample were averaged.

The morphology of the nanoparticles was characterized by Transmission Electron Microscopy (TEM). The morphology of the nanoparticles was observed with a Hitachi model H-7650TEM, and the preparation process of the TEM sample was as follows: transferring 5 mu L of the prepared nanoparticle solution (1mg/mL) by using a liquid transfer gun, dripping the nanoparticle solution on a copper net supported by an amorphous carbon film, naturally drying at room temperature, transferring 5 mu L of 1 wt% uranium acetate solution, adding the solution on the spotted copper net, dyeing for half a minute, then sucking the solution by using filter paper, placing the sample in a dryer, drying at room temperature, and reserving the sample for TEM observation.

The polymer mPEG-PLLMA (CD-Peptide-SH) -PAsp (DBP) self-assembles to form uniform micelles with hydrated particle sizes of about 100nm, as shown in fig. 6A and 6C. When the polymer is loaded with the hydrophobic drugs PTX and Sunitinib, the particle size is slightly increased to about 160nm, and the particle size distribution is slightly widened, as shown in FIG. 6B and FIG. 6D, but is still uniform, which may be related to the change of the interaction force between the polymer molecular chains after the hydrophobic drugs are loaded by the cyclodextrin.

Example 8 drug Loading and drug Release behavior of nanoparticles

The drug loading of the nanoparticles was determined by reverse phase high performance liquid chromatography (RP-HPLC). First, 1mL of solution of Nanoparticles (NPs) loaded with Sunitinib or PTX was taken, lyophilized, weighed, dissolved in 40 μ L DMF (HPLC purity grade) and diluted with 2mL acetonitrile (HPLC purity grade), and the resulting solution was used for HPLC assay. The Sunitinib or PTX content in the solution was determined from a standard curve of the corresponding substances under the same conditions.

HPLC analysis of Sunitinib and PTX were both performed on a Waters 1525 HPLC by C18-

The column was separated, the column temperature was 40 ℃ and detection was performed with a Waters 2489 UV/Vis detector. HPLC separation conditions for Sunitinib: the mobile phase is acetonitrile/phosphoric acid-ammonium phosphate buffer (20mM, pH 3)65:35 volume ratio; the flow rate is 1.0 mL/min; the detection wavelength is 254 nm; the linear range of the standard curve is from 0.6 to 36. mu.g/mL. HPLC separation conditions for PTX: the mobile phase was acetonitrile/water (70:30v: v); the flow rate is 1.0 mL/min; the detection wavelength is 227 nm; the linear range of the standard curve is from 0.5 to 255. mu.g/mL.

Example 9 in vitro simulated drug Release assay

PTX release behaviour at pH 7.4 and pH 5.0 free PTX drug concentrations at different time points (0-48h) were determined by HPLC and the cumulative release was calculated and plotted against time to generate the corresponding curve. The specific operation is as follows: first, 2mL of nanoparticle solution of known concentration was filled into dialysis bags (MWCO 14kDa), placed in 10mL centrifuge tubes, and 4mL of phosphate buffer (20mM) at pH 7.4 and pH 5.0 were added to the tubes, respectively, and each sample was subjected to three parallel experiments. The sample was then placed in a 37 + -2 deg.C constant temperature shaker and 2mL of dialysis bag fluid was removed at intervals and supplemented with an equal amount of fresh release buffer and constant temperature shaking was continued until sampling was complete at all time points. As shown in FIG. 8A, it was found that less than 20% of the total PTX released over 48h at pH 7.4 was released, and that 50% of the total PTX released at 12h was released at pH 5.0. In addition, through a Transmission Electron Microscope (TEM), the appearance of the acid-treated nanoparticles is greatly changed, the shape of the nanoparticles is changed from spherical to irregular, and an aggregation phenomenon appears, as shown in FIG. 7A, which may be related to structural transformation of micelles caused by protonation of the micelle cores under acidic conditions.

The enzyme sensitive release behavior of the Sunitinib under the action of MMP-2 enzyme is characterized in that the concentration of the free drug Sunitinib at different time points (0-36h) is measured by HPLC, and the cumulative release amount is calculated and plotted against time to draw a corresponding curve. The specific operation is as follows: first, 2mL of nanoparticle solution of known concentration was filled in dialysis bag (MWCO 14k Da), placed in 10mL centrifuge tube, and 4mL of TCNB (composition 100mM Tris,5mM Calcium chloride,200mM NaCl, 0.1% Brij-35) buffer was added to each tube as a control group, while 4mL of three parallel experiments were added to the experimental group. Then, the samples were again placed in a 37 ± 2 ℃ constant temperature shaker and shaken, and the dialysis bag external solution TCNB buffer and MMP-2(10nM, with an or with MMP inhibitor) were taken at intervals, each set of samples was made up to 2mL and supplemented with an equal amount of fresh release buffer, and constant temperature shaking was continued until sampling was completed at all time points. As shown in FIG. 8B, we found that under the same conditions, when 10nM MMP-2 enzyme is added into the system, the cumulative release amount within 12h exceeds 60%, and after 36h, Sunitinib is basically completely released, showing better time and concentration dependent MMP-2 response release; when 10nM MMP-2 enzyme and 100nM MMP-2 small molecule inhibitor are added simultaneously, the release rate of the Sunitinib is obviously slowed down, but is slightly accelerated compared with the PBS group, which is probably that the addition of the MMP-2 inhibitor can compete with the Sunitinib to be combined with cyclodextrin, so that the release of the Sunitinib is accelerated. The morphology of the nanoparticles was observed by transmission electron microscopy, as shown in fig. 7B, the particle size of the drug-loaded nanoparticles was reduced after MMP-2 enzyme treatment, which was due to the removal of the cyclodextrin polypeptide structure in the middle layer from the nanoparticles under the action of MMP-2.

It will be appreciated by those of ordinary skill in the art that the examples provided herein are intended to assist the reader in understanding the principles of the invention and are to be construed as being without limitation to such specifically recited examples and embodiments. Those skilled in the art can make various other specific changes and combinations based on the teachings of the present invention without departing from the spirit of the invention, and these changes and combinations are within the scope of the invention.

Claims

1. A triblock polymer comprising a hydrophilic block polyethylene glycol, an intermediate poly (methacrylyl lysine-g- (cyclodextrin polypeptide)) and a hydrophobic block poly (aspartyl-N, N-dibutylpropylamine); the molecular weight of the polyethylene glycol is 2-10 kDa, the molecular weight of the poly (methacryloyl lysine-g- (cyclodextrin polypeptide)) is 2-12 kDa, and the molecular weight of the poly (asparaginyl-N, N-dibutylpropylamine) is 2-6 kDa.

2. The triblock polymer of claim 1, wherein the polyethylene glycol has a molecular weight of 5 kDa; the poly (methacrylyl lysine-g- (cyclodextrin polypeptide)) has a molecular weight of 10 kDa; the poly (asparaginyl-N, N-dibutylpropylamine) has a molecular weight of 5 kDa.

3. A process for preparing a triblock polymer according to any one of claims 1 to 2, comprising the steps of:

s1: synthesis by taking mPEG-OH as raw materialAminated mPEG-NH₂；

S2: with mPEG-NH₂Respectively initiating the ring-opening polymerization of a methacryloyl Lysine MA-Lysine-NCA monomer and an aspartic acid cyclic anhydride BLA-NCA monomer to synthesize a triblock polymer main chain mPEG-b-PLLMA-b-PBLA as raw materials;

s4: and (2) connecting the cyclodextrin polypeptide conjugate CD-Peptide-SH to the double bond of the PLLMA chain segment in the polymer mPEG-b-PLLMA-b-PASp (DBP) by utilizing the click reaction of thiol-double bond thiol-ene to obtain the triblock polymer.

4. The method of claim 3, wherein the cyclodextrin polypeptide conjugate CD-Peptide-SH of S4 is prepared by the following steps:

s401, β -cyclodextrin with p-toluenesulfonyl chloride to obtain CD-OTs;

s402: alkynylating CD-OTs to obtain alkynylated cyclodextrin CD-Yne;

5. A drug-loaded nano-micelle with tumor microenvironment response, which is characterized in that the drug-loaded nano-micelle is formed by self-assembly of the triblock polymer of any one of claims 1-2.

6. The use of the drug-loaded nanomicelle of claim 5 for loading a drug.

7. The use according to claim 6, wherein the drug is one or more of paclitaxel PTX and Sunitinib.

8. A pH and MMP dual-responsive nano-drug, which is obtained by encapsulating the Sunitinib and the PTX simultaneously by the drug-loaded nano-micelle of claim 5.

9. The pH and MMP dual-responsive nano-drug of claim 8, wherein the wrapping amount of PTX is 2.11-5.34% of the total mass of the micelle, and the wrapping amount of Sunitinib is 0.5-1.6% of the total mass of the micelle.

10. The method for preparing a pH and MMP dual-responsive nano-drug of any one of claims 8 to 9, comprising the steps of: