CN109985007A - Using borate ester as two block sensitive camptothecin polymeric object prodrugs of connection unit and preparation method thereof - Google Patents

Abstract

The invention discloses a two-block double-sensitive camptothecin polymer prodrug using phenylboronic acid ester as a connecting unit and a preparation method. The phenylboronic acid catechol ester (BC) is used as a connecting unit to synthesize polyethylene glycol ‑Polyglutamate dithiodiethanolcamptothecin diblock polymer (PEG‑BC‑PGlu‑ss‑CPT), and then construct its self-assembled prodrug nanomicelles, namely PEG‑BC@PGlu‑ss‑CPT. In the present invention, PEG-BC is used as a macromolecular initiator to carry out a ring-opening polymerization reaction on a reduction-sensitive camptothecin monomer to obtain the two-block double-sensitive camptothecin polymer prodrug PEG using phenylboronate as a connecting unit. ‑BC‑PGlu‑ss‑CPT. The prodrug micelle prepared by the invention can improve the solubility of camptothecin and increase the stability of the camptothecin lactone ring, so as to overcome the limitation of clinical treatment of camptothecin, and the prodrug micelle has good drug release property , acid-sensitive and reduction-sensitive dual sensitive properties.

Description

Two-block dual-sensitive camptothecin polymer prodrugs using phenylboronate as a linking unit and its preparation method

technical field

The invention belongs to the fields of biomedical technology, nano-medicine and new materials, and in particular relates to a method for synthesizing a two-block dual-sensitive camptothecin polymer prodrug using phenylboronic ester as a connecting unit and a self-assembling prodrug nanomicelle. Preparation method and application.

Background technique

Traditional chemotherapeutic drugs have achieved great success in the treatment of tumors, but there are many limitations that limit the further improvement of their clinical efficacy, such as severe side effects, poor selectivity, and tumor cells are prone to drug resistance. Camptothecin (CPT) is a tryptophan-terpene alkaloid anticancer drug, which has a significant effect on the treatment of various malignant tumors. It inhibits DNA by interacting with DNA topoisomerase I. replication, transcription and mitosis. The closed lactone ring form in its molecular structure is an effective form of its tumor-suppressive activity, but under physiological conditions, the 20-position hydroxyl group of the lactone ring of camptothecin can form an intramolecular hydrogen bond with the ortho-position ester carbonyl group, making it possible to form an intramolecular hydrogen bond. The lactone ring is hydrolyzed to open, resulting in a decrease in activity. In addition, the poor water solubility of camptothecin hinders its application in clinical treatment.

In the prior art, most of the camptothecin prodrugs are constructed by improving the solubility of camptothecin. On the basis of the linear cyclodextrin copolymer published in 2009, the polymer was modified by carboxylation, so that camptothecin was bonded together (Advance Drug Delivery Review, 2009, 61, 1193-1202), and the increase of camptothecin However, this polymer prodrug lacks targeting, has poor biodegradability, and does not have the function of specific intracellular release of drugs.

Nano-drug carriers provide an effective way to achieve targeted and rapid release of chemotherapeutic drugs in tumor tissues, reduce damage to normal tissues and organs, and avoid drug resistance caused by insufficient doses of effective drugs. The intelligent nano-drug carrier system is mainly based on the microenvironment difference between tumor tissue and normal human tissue. For example, tumor tissue has the characteristics of hypoxia, acidic pH, slightly higher temperature, a large number of growth factors and hydrolytic proteases. Using the high permeability and long retention effect (EPR effect) of solid tumors, nanocarrier drugs can be enriched in tumor tissue, rapidly disintegrated under the action of tumor microenvironment, and rapidly release antitumor drugs to "kill" tumor cells.

Phenylboronic acid and cis 1,2- or 1,3-dihydroxy compounds (such as sugars, catechol, etc.) can form boronate ester bonds. The boronic ester bond is relatively stable in basic and neutral environments, but cleaved in acidic environment, confirming that the boronic ester bond is very suitable as an acid-sensitive nano-drug carrier building unit. Prof. Pavel A.Levkin modified dextran with phenylboronic acid to obtain amphiphilic polymer B-Dex. The drug release rate of nanoparticles formed by self-assembly of B-Dex in an acidic medium of pH 5.0 is 5 times higher than that in a medium of pH 7.4, showing very good acid-sensitive drug release characteristics (Biomaterials, 2013, 34, 8504-8510). Professor Zhong Zhenlin's group found that phenylboronic acid covalently modified polyethyleneimine has a higher gene transfection efficiency for HepG2 cells. One of the reasons is that phenylboronic acid binds strongly to the oligosaccharide (Sialyl Lewis X) on the surface of the HepG2 cell membrane. Receptor-mediated endocytosis makes the gene carrier system easier to enter cells for transfection (Chemical Communication, 2010, 46, 5888-5890), confirming the potential of phenylboronic acid as a targeting group for drug carrier systems .

The traditional block polymer-constructed drug-loaded micelles usually use polyethylene glycol (PEG) as a hydrophilic segment, which can make full use of the high permeability and long retention effect (EPR effect) of solid tumors, so that drugs can be enriched in tumors. However, due to the existence of the PEG shell, this type of micelle is not conducive to cell endocytosis, and it is difficult for antitumor drugs to reach the target located in the cell in sufficient dose. Targeting groups such as galactose or antibody modification to achieve receptor-mediated endocytosis.

SUMMARY OF THE INVENTION

The object of the present invention is a kind of two-block dual-sensitive camptothecin polymer prodrug with phenylboronate as connecting unit proposed for the deficiencies of the prior art, and the drug-loaded glue constructed with the two-block polymer The bundle not only achieves long-term circulation in the body, but also can remove the PEG shell after accumulation in the tumor site to avoid PEG hindering endocytosis. The site-specific rapid release of the drug is achieved through a reduction response. Therefore, the construction of the micelle of the present invention realizes a new approach of "long-acting circulation, enhanced cell endocytosis and site-specific release". The two-block polymer prepared by the present invention can be used to construct nano micelles, and has good drug release, low cytotoxicity and good cell phagocytosis.

The concrete technical scheme that realizes the object of the present invention is:

A two-block double-sensitive camptothecin polymer prodrug with phenylboronate as a connecting unit is characterized by having the structure of formula (I):

In formula (I):

x=40-120

m=2-10.

Preferably, x=44 and m=5.

Wherein, the structural formula of PEG is shown in formula (i):

The structural formula of BC is shown in formula (ii):

The structural formula of PGlu-ss-CPT is shown in formula (iii):

The present invention also proposes a preparation method of the amphiphilic camptothecin macromolecular prodrug PEG-BC-PGluCPT using phenylboronate as a connecting unit, the method specifically comprises the following steps:

Step 1: Synthesis of macroinitiator PEG-BC

(a) In a solvent, 3,4-dihydroxyphenylacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, N, N-diisopropylethylamine and PEG-NH ₂ are subjected to amidation reaction to synthesize the dopa derivative PEG-3,4-DA of PEG-NH ₂ shown in formula (1);

(b) in a solvent, PEG-3,4-DA is dehydrated and condensed with 3-aminophenylboronic acid to obtain the phenylboronic acid ester derivative PEG-BC of PEG shown in formula (2);

The reaction process is shown in route (A):

route (A), x=40-120, m=2-10;

Step 2: Synthesis of Reduction-Sensitive Camptothecin Monomer (Glu-ss-CPT)-NCA

(c) in a solvent, camptothecin reacts with triphosgene, 4-dimethylaminopyridine and 2,2'-dithiodiethanol to obtain CPT-ss-OH as shown in formula (3);

(d) In solvent, CPT-ss-OH and Boc-L-glutamic acid-1-tert-butyl ester, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride, triethylamine, and esterification to obtain Boc-(Glu-ss-CPT)-OtBu as shown in formula (4);

(e) in a solvent, use TFA to remove the Boc protecting group in the Boc-(Glu-ss-CPT)-OtBu molecule to obtain (Glu-ss-CPT)-OH as shown in formula (5);

(f) In the solvent, with triphosgene as an initiator, (Glu-ss-CPT)-OH undergoes a cyclization reaction, and the α-amino acid structure in (Glu-ss-CPT)-OH forms an N-carboxy intracyclic anhydride, The reduction-sensitive camptothecin monomer (Glu-ss-CPT)-NCA shown in formula (6) is obtained; the reaction process is shown in route (B):

Step 3: Synthesis of Diblock Dual-Sensitive Camptothecin Polymer Prodrug PEG-BC-PGlu-ss-CPT

(g) In a solvent, using PEG-BC as a macroinitiator, the reduction-sensitive camptothecin monomer (Glu-ss-CPT)-NCA represented by formula (4) is subjected to ring-opening polymerization to obtain the following The two-block dual-sensitive camptothecin polymer prodrug PEG-BC-PGlu-ss-CPT represented by formula (I) using phenylboronate as a connecting unit.

The reaction process is shown in the route (C):

Wherein, x=40-120, m=2-10.

Preferably, x=44 and m=5.

Wherein, the structural formula of PEG is shown in formula (i):

Wherein, the structural formula of BC is shown in formula (ii):

Wherein, the structural formula of PGlu-ss-CPT is shown in formula (iii):

In (a) of step 1, the solvent is selected from dichloromethane or dimethylformamide; preferably, it is dichloromethane; more preferably, it is anhydrous dichloromethane.

In (a) of step 1, the 3,4-dihydroxyphenylacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole , the molar ratio of N,N-diisopropylethylamine and PEG-NH ₂ is (1.2-2): (1.2-3): (1.2-3): (2-4): 1; preferably, it is 1.5:1.6:1.6:3:1; the amidation reaction temperature is 20°C-37°C; preferably, room temperature is 25°C; the amidation reaction time is 8h-16h; preferably, 12h; It is preferably carried out under the protection of nitrogen in the dark.

In (b) of step 1, the solvent for the dehydration condensation reaction is selected from tetrahydrofuran or toluene; preferably, it is toluene.

In step 1 (b), the molar ratio of the PEG-3,4-DA to 3-aminophenylboronic acid is 1:(5-10); preferably, it is 1:10; the temperature of the dehydration condensation reaction It is 100-130°C; preferably, it is 120°C; the time of the dehydration condensation reaction is 3-8h; preferably, it is 5h; it is preferably carried out under nitrogen protection.

In (c) of step 2, the solvent is selected from dichloromethane or tetrahydrofuran; preferably, it is dichloromethane; more preferably, it is anhydrous dichloromethane.

In (c) of step 2, the molar ratio of camptothecin, triphosgene, 4-dimethylaminopyridine and 2,2'-dithiodiethanol is 1:(0.7-1):(5-6) : (1.5-5); preferably, 1:0.7:6:5; the reaction temperature is 20-37°C; preferably, 37°C; the reaction time is 3-8h; preferably, 5h ; preferably carried out under nitrogen protection.

In (d) of step 2, the solvent of the esterification reaction is selected from dichloromethane or dimethylformamide; preferably, it is dichloromethane.

In (d) of step 2, the Boc-L-glutamic acid-1-tert-butyl ester, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, 1-ethyl-3-(3- The molar ratio of dimethylaminopropyl)carbodiimide hydrochloride, triethylamine and CPT-ss-OH is (1.5-2):(0.25-1):(1-1.5):(1-1.5) : (1-1.5): 1; preferably, 2: 0.5: 1.2: 1.2: 1.2: 1; the temperature of the esterification reaction is 20-37°C; preferably, it is room temperature 25°C; the esterification The reaction time is 3-8h; preferably, it is 5h; it is preferably carried out under nitrogen protection.

In (d) of step 2, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, triethyl Amine is to activate the carboxylic acid in Boc-L-glutamic acid-1-tert-butyl ester, and then the carboxylic acid in the activated Boc-L-glutamic acid-1-tert-butyl ester and CPT-ss-OH The hydroxyl group in the molecule undergoes an esterification reaction.

In (e) of step 2, the solvent for the removal reaction is selected from dichloromethane or ethyl acetate; preferably, it is dichloromethane; more preferably, it is anhydrous dichloromethane.

In (e) of step 2, the molar ratio of TFA and Boc-(Glu-ss-CPT)-OtBu is (10-15): 1; preferably, it is 13: 1; the temperature of the removal reaction is 0-25°C; preferably, room temperature is 25°C; the removal reaction time is 1.5-4h; preferably, 3h; preferably under nitrogen protection; After OtBu is added to the solvent, preferably at 0°C, trifluoroacetic acid is slowly added dropwise to the reaction system.

In (f) of step 2, the initiator is selected from triethylamine, pyridine or N,N-diisopropylethylamine; the effect of the initiator is to promote triphosgene decomposition, and the excess light generated in the reaction Gas becomes salt.

In (f) of step 2, the solvent is selected from ethyl acetate or tetrahydrofuran; preferably, it is tetrahydrofuran; more preferably, it is anhydrous tetrahydrofuran.

In (f) of step 2, the molar ratio of triphosgene, triethylamine and (Glu-ss-CPT)-OH is (1/3-1):(1-2):1; preferably, it is: 2/3:1:1; the temperature of the cyclization reaction is 48-55°C; preferably, it is 50°C; the time of the cyclization reaction is 1-4h; preferably, it is 3h.

In (g) of step 3, the solvent for the ring-opening polymerization reaction is selected from tetrahydrofuran or dimethylformamide; preferably, it is tetrahydrofuran; more preferably, it is anhydrous tetrahydrofuran.

In (g) of step 3, the molar ratio of the PEG-BC and (Glu-ss-CPT)-NCA is 1:(3-10); preferably, it is 1:3.3; The temperature is 20-37°C; preferably, it is 25°C; the time of the ring-opening polymerization reaction is 24-72h; preferably, it is 48h; preferably, it is carried out under nitrogen protection.

The present invention also proposes a method for preparing the prodrug nanomicelle PEG-BC@PGlu-ss-CPT by using the two-block dual-sensitive camptothecin polymer prodrug with phenylboronate as a connecting unit, Described method is: take by weighing 2mg formula (I) PEG-BC-PGlu-ss-CPT, be dissolved in the mixed solution of analytically pure organic solvent and ultrapure water, analytical pure organic solvent and ultrapure water volume ratio is 1: 10 ; filter the obtained mixed solution, then put the filtrate into a dialysis bag for dialysis for 48h, and after freeze-drying, obtain the prodrug nanomicelle PEG-BC@PGlu-ss-CPT.

Wherein, the organic solvent is selected from tetrahydrofuran, dimethyl sulfoxide or acetone; preferably, it is tetrahydrofuran.

Wherein, in the mixed solution, the concentration of formula (I) PEG-BC-PGlu-ss-CPT is 0.1-0.5 mg/mL.

Wherein, in the formula (I) PEG-BC-PGlu-ss-CPT, x=44, m=5.

When the formula (I) PEG-BC-PGlu-ss-CPT with a high degree of polymerization (x) is selected, the aqueous solution is very easy to produce precipitation; and the polymer prodrug solution with m=5 is selected, when the concentration is equal to 1 mg/mL, the glue The micelle solution will have precipitation, but when the concentration is 0.1mg/mL-0.5mg/mL, the micelle solution is very stable. The polymer prodrug nanomicelle PEG-BC@PGlu-ss-CPT solution of ss-CPT can remain stable for a long time.

The boronic ester bond in the nanomicelle has acid-sensitive properties and is responsive to the tumor tissue microenvironment; the small molecule drug camptothecin in the micelle is chemically bonded to the carrier, and does not survive long-term circulation in vivo. will be released in advance.

The present invention also provides a prodrug nanomicelle PEG-BC@PGlu-ss-CPT prepared by the above method.

Wherein, the prodrug nanomicelles are both acid-sensitive and reduction-sensitive.

Among them, the prodrug nanomicelles are used to enhance the endocytic properties of tumor cells, enhance tumor cell inhibitory activity, realize long-acting circulation of drugs in vivo, realize stable release of drugs in cells, and can actively target diseased cells to enhance mediators. induce cell endocytosis.

Wherein, the tumor cells include liver cancer cells, pancreatic cancer cells, colon cancer cells, lung cancer cells, etc.; preferably, liver cancer cells.

The present invention utilizes the responsiveness of phenylboronic acid ester to the slightly acidic environment of the tumor, the phenylboronic acid has a very strong binding ability to the oligosaccharide on the surface of the cell membrane, and the 20-position hydroxyl group in the molecular structure of camptothecin can be modified effectively to improve camptothecin. The characteristics of water solubility and lactone ring stability, as well as the sensitivity of disulfide bonds to reduction in tumor cells, the use of phenylboronic acid catechol ester (BC) as the linking unit before the synthesis of diblock bi-sensitive camptothecin polymers drug (PEG-BC-PGlu-ss-CPT). The active and orderly response behavior and rapid drug release process of the prodrug nanomicelle PEG-BC@PGlu-ss-CPT to the tumor microenvironment were investigated to evaluate its antitumor activity. The active and ordered response behavior and rapid drug release process can be specifically described as follows: the prodrug micelles fully realize the EPR effect after long-term circulation and are enriched in the tumor tissue; In response, the PEG shell was broken and removed; phenylboronic acid was exposed, and phenylboronic acid combined with oligosaccharides on the cell membrane surface to achieve phenylboronic acid-mediated enhanced active endocytosis, and released camptothecin through a reduction response in the lysosome.

The beneficial effect of the present invention is that the present invention provides a preparation method of a two-block dual-sensitive camptothecin polymer prodrug PEG-BC-PGlu-ss-CPT, which uses phenylboronic acid catechol ester (BC) as a linker unit, synthesized polyethylene glycol-polyglutamic acid camptothecin two-block polymer (PEG-BC-PGlu-ss-CPT), and then constructed its self-assembled prodrug nanomicelles (PEG-BC@PGlu-ss -CPT). Aiming at poor water solubility of camptothecin, the invention synthesizes a kind of camptothecin as a hydrophobic end by modifying the 20-position hydroxyl group of camptothecin, which can effectively promote the assembly of two-block polymers into a polymer prodrug of micelles. Improve the solubility of camptothecin, increase the stability of camptothecin lactone ring, improve the efficacy and bioavailability, in order to overcome the limitations of clinical treatment of camptothecin. The nano-drug delivery system prepared by the PEG-BC-PGlu-ss-CPT of the present invention has good drug release, good responsiveness to the weakly acidic environment and reducing environment of tumor tissue, good cell phagocytosis and anti-tumor effect. Strong inhibitory activity against tumor cells.

Description of drawings

Fig. 1 is the ¹ H NMR spectrum of the prodrug PEG-BC-PGlu-ss-CPT of the present invention;

Fig. 2 is the structural representation of the prodrug nanomicelle PEG-BC@PGlu-ss-CPT of the present invention;

3 is a schematic structural diagram of the prodrug nanomicelle PEG-BC@PGlu-ss-CPT of the present invention when the phenylboronic acid ester bond is broken under acidic conditions, and the micelles remove the PEG outer layer and expose the phenylboronic acid end;

Figure 4 is a schematic diagram of PGlu-ss-CPT;

5 is a particle size distribution (DLS) image and a morphology (TEM) image of the prodrug nanomicelle PEG-BC@PGlu-ss-CPT of the present invention;

Figure 6 is a schematic diagram of the stability of the prodrug nanomicelle PEG-BC@PGlu-ss-CPT of the present invention in an aqueous environment at 4°C;

Fig. 7 is the ¹ H NMR spectrum of the acid degradation of the prodrug PEG-BC-PGlu-ss-CPT of the present invention in different pH media;

Figure 8 is a graph showing the particle size change of the prodrug nanomicelle PEG-BC@PGlu-ss-CPT in different pH buffer solutions;

Fig. 9 is the ¹ H NMR spectrum of the reductive degradation of the prodrug PEG-BC-PGlu-ss-CPT of the present invention over time under the condition of 10 mM dithiothreitol;

Figure 10 is a graph showing the cumulative drug release curve triggered by the reductive degradation of the prodrug nanomicelle PEG-BC@PGlu-ss-CPT of the present invention at different concentrations of dithiothreitol;

11 is a graph showing the cumulative drug release curve of the prodrug nanomicelle PEG-BC@PGlu-ss-CPT of the present invention under simulated tumor environment and simulated blood circulation conditions;

Figure 12 is a graph showing the fluorescence intensity statistics of phagocytosis of FITC-labeled prodrug nanomicelles PEG-BC@PGlu-ss-CPT by HepG2 and HL-7702 cells under different pH conditions.

Detailed ways

The present invention will be further described in detail with reference to the following specific embodiments and accompanying drawings. Except for the content specifically mentioned below, the process, conditions, experimental methods, etc. for implementing the present invention are all common knowledge and common knowledge in the field, and the present invention is not particularly limited.

Example 1

Synthesis of PEG-3,4-DA

Weigh 3,4-dihydroxyphenylacetic acid (0.126 g, 0.750 mmol) and dissolve it in 25 mL of anhydrous dichloromethane, ultrasonically promote its dispersion to form a suspension, and add 1-ethyl-(3-dimethylamino) in turn propyl)carbodiimide hydrochloride (0.154g, 0.800mmol), 1-hydroxybenzotriazole (0.108g, 0.800mmol), N,N-diisopropylethylamine (0.260mL, 1.500mmol) ), after 0.5h, PEG-NH ₂ (1.000g, 0.500mmol) was added under nitrogen protection, the reaction device was protected from light, and the reaction was carried out at room temperature overnight. After concentration, pure water was added to dissolve, and extracted with dichloromethane for several times. After the organic phase was dried with sodium, the filtrate was concentrated and the sample was mixed and purified by silica gel column. The mobile phase was a mixture of dichloromethane and methanol in a certain volume ratio (30:1). The purified product was concentrated and added to ice ether for precipitation, suction filtered to obtain a white precipitate, washed and filtered for several times and then dried to obtain 0.960 g of PEG-3,4-DA, with a yield of 85%.

The structure of the PEG-3,4-DA is shown in formula (1);

where x=44.

Example 2

Synthesis of PEG-BC

Weigh 3-aminophenylboronic acid (0.191g, 1.390mmol) and 200mL of re-distilled toluene into a 500mL single-neck flask, ultrasonically promote its dissolution, and add PEG-3,4-DA (0.300g, 0.139mmol) under nitrogen protection , react at 120°C for 5h, after concentrating the reaction solution, add 30mL of anhydrous tetrahydrofuran to dissolve, transfer the tetrahydrofuran solution to a dialysis bag with a molecular weight cut-off of 1000, dialyze the anhydrous tetrahydrofuran for 5h and then dropwise add it to 100mL of glacial ether, wash and filter several times After several times, 0.360 g of light yellow powder PEG-BC was obtained with a yield of 87%.

The structural formula of the PEG-BC is shown in formula (2);

where x=44.

Example 3

Synthesis of CPT-ss-OH

Weigh camptothecin (700 mg, 2 mmol), triphosgene (414 mg, 1.4 mmol) and 100 mL of anhydrous dichloromethane in a reaction flask, and after 0.5 h, under nitrogen protection, add 4-dimethylaminopyridine (1.464 g in turn) , 12 mmol) and 2,2'-dithiodiethanol (1.540 g, 10 mmol), after 5 h of reaction at room temperature, the reaction solution became clear. Extracted three times with saturated NaCl aqueous solution, dried the organic phase with anhydrous NaSO4, filtered, concentrated the filtrate, mixed the sample and purified by silica gel column, the mobile phase was that dichloromethane and methanol were mixed in a certain volume ratio (300:1), and the purified product was Light yellow powder CPT-ss-OH 0.95g, yield 90%.

The structural formula of the Boc-(Glu-ss-CPT)-OtBu is shown in formula (3);

Example 4

Synthesis of Boc-(Glu-ss-CPT)-OtBu

Weigh Boc-L-glutamic acid-1-tert-butyl ester (1.7416g, 5.74mmol), 4-dimethylaminopyridine (0.1754g, 1.44mmol), 1-hydroxybenzotriazole (0.4654g, 3.44g) mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.6604 g, 3.44 mmol), triethylamine (0.48 mL, 3.44 mmol), and 110 mL of anhydrous diethylamine Chloromethane was placed in the reaction flask, and after 0.5 h, CPT-ss-OH (1.5154 g, 2.87 mmol) was added under nitrogen protection, and the reaction solution became clear after 5 h at room temperature. Add the hydrochloric acid aqueous solution of pH 1 to terminate the reaction, extract three times with saturated NaCl aqueous solution, dry the organic phase with anhydrous NaSO , after filtration, the filtrate is concentrated and mixed with sample to be purified by silica gel column, and the mobile phase is methylene chloride and methanol at a certain volume ratio (300: 1) Mixing, the purified product is light yellow powder Boc-(Glu-ss-CPT)-OtBu 1.54g, the yield is 85%.

The structural formula of the Boc-(Glu-ss-CPT)-OtBu is shown in formula (4);

Example 5

Synthesis of (Glu-ss-CPT)-OH

Weigh Boc-(Glu-ss-CPT)-OtBu (0.641 g, 0.789 mmol) and 20 mL of anhydrous dichloromethane in a reaction flask, and slowly add trifluoroacetic acid dropwise to the reaction system under nitrogen protection and 0 °C, room temperature After reaction for 3 h, the concentrated reaction was added dropwise to 50 mL of glacial ether, suction filtered to obtain a yellow precipitate, washed and filtered for several times, and then vacuum dried to obtain light yellow powder (Glu-ss-CPT)-OH 0.363 g, yield 70%.

The structural formula of the (Glu-ss-CPT)-OH is shown in formula (5);

Example 6

Synthesis of (Glu-ss-CPT)-NCA

Weigh (Glu-ss-CPT)-OH (0.276g, 0.420mmol), triethylamine (0.058mL, 0.420mmol) was dissolved in 100mL anhydrous tetrahydrofuran, after 0.5h, add triphosgene (0.083g) dropwise to the reaction , 0.280 mmol) in anhydrous tetrahydrofuran solution, after the reaction system was reacted at 50 ° C for 3 h, the reaction system became clear, and the reaction ended. The remaining phosgene in the reaction system was treated with a nitrogen flow and a tail gas treatment device, and the concentrated reaction droplets were added to 50 mL of n-hexane, placed in a -20 °C refrigerator overnight, and suction filtered to obtain a yellow precipitate, which was washed several times, filtered and dried. A yellow powder (Glu-ss-CPT)-NCA 0.159 g was obtained with a yield of 75%.

The structural formula of the (Glu-ss-CPT)-NCA is shown in formula (6);

Example 7

Synthesis of PEG-BC-PGlu-ss-CPT

Weigh PEG-BC (0.500g, 0.222mmol), (Glu-ss-CPT)-NCA (0.909g, 1.332mmol) and 100mL of anhydrous tetrahydrofuran in a reaction flask, and ultrasonically promote their dispersion to form a suspension, nitrogen protection , reacted at room temperature for 48h, the concentrated reaction solution was transferred to a dialysis bag with a molecular weight cut-off of 2000, dialyzed against anhydrous tetrahydrofuran for 12h, the dialysate was concentrated and added dropwise to 100mL of ice ether, filtered and washed several times and dried to obtain the final product PEG -BC-PGlu-ss-CPT 1.190 g, 85% yield.

The structural formula of the final product PEG-BC-PGlu-ss-CPT is shown in the following formula (I); the ¹ H NMR spectrum is shown in FIG. 1 .

Wherein, x=44; m=5.

Example 8

The process of determining the optimal results of PEG-BC-PGlu-ss-CPT synthesis

Weigh PEG-BC (0.500g, 0.222mmol), (Glu-ss-CPT)-NCA (0.455g, 0.666mmol, or 1.516g, 2.220mmol) and 200mL of anhydrous tetrahydrofuran in a reaction flask, and ultrasonically promote their dispersion Form a suspension, protect with nitrogen, react at room temperature for 72 hours, transfer the concentrated reaction solution to a dialysis bag with a molecular weight cut-off of 2000, dialyze anhydrous tetrahydrofuran for 24 hours, concentrate the dialysate and add dropwise to 100 mL of ice ether, filter and wash for several times. After drying, the final products PEG-BC-PGlu-ss-CPT were 0.850 g and 1.028 g, respectively, and the yields were 88% and 51%, respectively.

The structural formula of the PEG-BC-PGlu-ss-CPT is shown in the following formula (I);

Wherein, x=44; m=2-10.

The product obtained by increasing the feeding ratio is not stable enough after self-assembly, which is related to the large steric hindrance of camptothecin and the extremely poor solvent property.

Example 9

Assembly and Characterization of Prodrug Nanomicelles PEG-BC@PGlu-ss-CPT

2 mg of PEG-BC-PGlu-ss-CPT of formula (I) (wherein, in formula (I), x=44, m=5) was dissolved in 1 mL of tetrahydrofuran, and added dropwise to 10 mL of ultrapure with a stirring speed of 500 r/min. The water was filtered with a 0.45 μm needle filter, put into a dialysis bag for dialysis for 48 hours (ultrapure water was changed every 6 hours), and freeze-dried to obtain the prodrug nanomicelle PEG-BC@PGlu-ss-CPT Lyophilized powder, the prodrug nanomicelle PEG-BC@PGlu-ss-CPT structure is shown in Figure 2, the PEG end forms a hydrophilic outer layer, and PGlu-ss-CPT forms a hydrophobic spherical inner core (as shown in Figure 4 ), phenylboronate is used as the linking unit between PEG and PGlu-ss-CPT. When the phenylboronic acid ester bond is broken under acidic conditions, the micelles remove the PEG outer layer, exposing the phenylboronic acid end, and the structure after removing the PEG outer layer is shown in Figure 3. TEM observed that it was spherical in shape, and its average particle size was about 160 nm as measured by dynamic light scattering.

The experimental results are shown in Figure 5. The nanoparticle size and distribution measured by DLS are in good agreement with the TEM results, and most of them are distributed around 160 nm, which is in line with the characteristics required for passive targeting of nanomicelles, that is, the nanoparticle size range in Nanomicelles of 5-500 nm can accumulate in tumor tissue through passive targeting.

Example 10

Stability of prodrug nanomicelles PEG-BC@PGlu-ss-CPT in aqueous environment at 4°C

When the formula (I) PEG-BC-PGlu-ss-CPT with a higher degree of polymerization (x) is selected, the aqueous solution is very easy to produce precipitation, but the drug loading is also relatively low when the degree of polymerization is low; and the polymerization of m=5 is selected. When the concentration is equal to 1 mg/mL, the micellar solution will precipitate, but when the concentration is 0.1 mg/mL and 0.5 mg/mL, the micellar solution is very stable and the drug loading can be as high as 32 %. The change of particle size of prodrug nanomicelles PEG-BC@PGlu-ss-CPT in aqueous phase was measured by DLS, which could reflect the storage stability of prodrug nanomicelles PEG-BC@PGlu-ss-CPT. From the 1st day to the 14th day, the change of the micelle particle size in the aqueous phase was monitored.

The experimental results are shown in Figure 6. From the 1st day to the 14th day, the particle size increased slightly and remained basically unchanged. The polymer prodrug nanomicelle PEG-BC@PGlu-ss-CPT solution of -ss-CPT (wherein, in formula (I), x=44, m=5) can remain stable for a long time.

Example 11

Acid degradation performance of prodrug nanomicelles PEG-BC@PGlu-ss-CPT

The acid degradation kinetics of the polymer prodrug PEG-BC-PGlu-ss-CPT was investigated by ¹ H NMR. Prepare Na ₂ HPO ₄ /NaH ₂ PO ₄ buffer with pH 7.4, 6.5, 6.0, 5.0 with D ₂ O, respectively, take 10 mg of the two-block polymer prodrug PEG-BC-PGlu-ss-CPT and dissolve it in 1 mL Add 100 μL of each pH buffer to DMSO-D ₆ and monitor changes by ¹ H NMR.

The experimental results are shown in Figure 7. As the acidity increases, the proportion of characteristic peaks of the hydrophobic part PGlu-ss-CPT part gradually decreases, suggesting that the acid degradation of the polymer prodrug PEG-BC-PGlu-ss-CPT is accelerated. The acid degradation half-life was 96.27h, 10.78h, 8.48h, and 5.7h under the conditions of pH 7.4, pH 6.5, pH 6.0, and pH 5.0, respectively. After the boronic ester bond is broken, the PGlu-ss-CPT part has poor solubility in DMSO-D6, while the PEG end is still soluble in DMSO-D6. decrease.

The particle size change of the prodrug nanomicelles PEG-BC@PGlu-ss-CPT in pH buffer was measured by DLS, which could reflect the degradation of the prodrug nanomicelles PEG-BC@PGlu-ss-CPT. 1 mg of prodrug nanomicelle PEG-BC@PGlu-ss-CPT lyophilized powder was dissolved in 10 mL of each pH buffer, and the particle size changes were monitored in pH 7.4, pH 6.5, pH 6.0, and pH 5.0 media.

The experimental results are shown in Figure 8. Under the condition of pH 7.4, the particle size of the nanomicelles remained basically unchanged, and the particle size was relatively stable. As the pH of the medium decreased, the particle size of the micelles increased significantly. At pH 6.5, At pH 6.0 and pH 5.0, the particle size of the prodrug nanomicelles PEG-BC@PGlu-ss-CPT increased to 586 nm, 683 nm, and 2380 nm, respectively. The reason is that the boronic ester bond is broken in an acidic environment, the hydrophilic and lipophilic equilibrium state of the nanomicelles is destroyed, and the PEG shell is slowly peeled off. The micelle structure after peeling off the PEG shell is shown in Figure 2. The micelles after peeling off the PEG shell coagulated, resulting in an increase in the DLS test results. This experiment demonstrated that the boronic ester bond in the prodrug nanomicelle PEG-BC@PGlu-ss-CPT possesses acid-sensitive properties.

Example 12

Reductive degradation performance of prodrug nanomicelles PEG-BC@PGlu-ss-CPT and drug release behavior in response to reductive stimuli

The reductive degradation kinetics of the polymer prodrug PEG-BC-PGlu-ss-CPT was investigated by ¹ H NMR. 10 mg of the polymer prodrug PEG-BC-PGlu-ss-CPT was dissolved in 1 mL of DMSO-D ₆ containing 10 mM dithiothreitol, and changes in ¹ H NMR were monitored.

The experimental results are shown in Figure 9. The polymer prodrug can be rapidly reduced and degraded into the CPT original drug when the concentration of dithiothreitol is 10 mM, and the degradation half-life is 0.95h.

The CPT release properties of prodrug nanomicelles PEG-BC@PGlu-ss-CPT in phosphate buffer at pH 7.4 with different dithiothreitol concentrations were determined by high performance liquid chromatography. The prodrug nanomicelles PEG-BC@PGlu-ss-CPT (0.1 mg/mL) were taken 10 mL and placed in phosphate buffer with dithiothreitol concentrations of 0.02, 1, 5, and 10 mM, respectively, and dialyzed for 48 h (pH 7.4). MWCO500), the CPT content of each group after dialysis was measured by high performance liquid chromatograph, and the drug release amount of each group was calculated by comparing with the standard curve of measured CPT.

The experimental results are shown in Figure 10. The drug release amount of the prodrug nanomicelles PEG-BC@PGlu-ss-CPT showed a certain dependence on the concentration of dithiothreitol. In the phosphate buffer medium of pH 7.4, the cumulative drug release amount was as high as 82.9±2.29% within 48h, and the drug release rate was fast in the first 12h, and the drug release rate was relatively slow in the following 24h. However, the cumulative drug release amount of the prodrug nanomicelle PEG-BC@PGlu-ss-CPT was only 8.5±1.73% under the condition of dithiothreitol concentration of 0.02 mM. Reduction-sensitive properties of disulfide bonds in BC@PGlu-ss-CPT.

Example 13

Drug release behavior of prodrug nanomicelles PEG-BC@PGlu-ss-CPT under simulated tumor environment and simulated blood circulation conditions

The drug release behavior of prodrug nanomicelles PEG-BC@PGlu-ss-CPT under simulated tumor environment and simulated blood circulation conditions was determined by high performance liquid chromatography. Prodrug nanomicelles PEG-BC@PGlu-ss-CPT (0.1 mg/mL) were placed in 10 mL of different media to simulate different physiological environments, namely, 0.02 mM dithiothreitol in pH 7.4 phosphoric acid Buffer, 0.02 mM dithiothreitol in pH 7.2 phosphate buffer, 0.02 mM dithiothreitol in pH 6.5 phosphate buffer, and 10 mM dithiothreitol in pH 5.0 phosphate buffer for 48 h (MWCO500), the CPT content of each group after dialysis was measured by high performance liquid chromatography, and the drug release amount of each group was calculated by comparing with the standard curve of measured CPT.

The experimental results are shown in Figure 11. In the pH 5.0 phosphate buffer medium with dithiothreitol concentration of 10 mM, the cumulative drug release amount reached 40% within 48 h, while in 0.02 mM dithiothreitol at different pH phosphates In the buffer medium, the cumulative drug release amount was less than 9%, suggesting that the prodrug nanomicelle PEG-BC@PGlu-ss-CPT could remain relatively stable under simulated in vivo circulating conditions, and could be more stable under simulated tumor intracellular conditions. Slow release of drugs, with the performance of drug fixed-point sustained release.

Example 14

Phagocytosis of prodrug nanomicelles PEG-BC@PGlu-ss-CPT

After pre-culturing HepG2 cells and HL7702 cells on a six-well plate (Greiner) for 24 hours (25×10 ⁴ cells/well), 2 mL of pH 7.4, 6.5, and 6.0 media were added respectively, and the CPT content was used as the standard. The CPT concentration of the group containing FITC-labeled PEG-BC@PGlu-ss-CPT was 0.6 μg/mL. After culturing for 4 h, the group was washed with PBS (pH 7.4) for several times, digested and collected by centrifugation, and transferred to a 96-well plate. The fluorescence intensity of each group of cells was measured by cytometry.

The experimental results are shown in Figure 12. The fluorescence intensity of HepG2 cells increased with the decrease of pH, and the fluorescence intensity of HL7702 cells remained basically unchanged, which was directly related to the SA content on the surface of the two cells. The content of SA on the surface of HepG2 cells is high. After the boronic ester bond of the prodrug nanomicelles PEG-BC@PGlu-ss-CPT is broken in an acidic environment, the exposed phenylboronic acid binds to the SA on the cell surface, enhancing the cellular Endocytosis, the fluorescence intensity of cells increased, while the SA content on the surface of HL7702 cells was low, and its fluorescence intensity was lower than that of HepG2 cells.

Example 15

_IC50 of prodrug nanomicelles PEG-BC@PGlu-ss-CPT inhibiting HepG2 under different culture conditions

HepG2 cells in the logarithmic growth phase were seeded on 96-well plates, with 9000 cells per well. After culturing in a constant temperature incubator for 12 h, 20 μL of medium with pH of 7.4, 6.5, and 6.0 were added respectively. Each group contained a series of concentration gradients. The final concentrations of PEG-BC@PGlu-ss-CPT and CPT were 0.039063, 0.078125, 0.15625, 0.3125, 0.625, 1.25 and 2.5 μg/mL based on the CPT content, respectively; 80 μL of medium was aspirated, and 10 μL of MTT was added. The solution (5mg/mL) was incubated for 4h, 50μL triple solution was added to dissolve the formazan crystals, and the absorbance was measured at a wavelength of 570nm with a microplate reader. Cell viability was calculated as follows:

Cell viability (%)=(OD experimental group-OD blank group/OD control group-OD blank group)×100%

In the present invention, the IC ₅₀ of the prodrug nanomicelle PEG-BC@PGlu-ss-CPT and the small molecule drug CPT under different culture conditions for HepG2 are shown in Table 1 below.

Table 1

At different pH, the IC ₅₀ of the prodrug nanomicelles PEG-BC@PGlu-ss-CPT is greater _than that of the free small molecule drug CPT, mainly because the intracellular release of CPT in the prodrug nanomicelles takes a certain time , while the free small molecule drug CPT can be directly taken up by cells to inhibit tumor cells; in addition, small molecule drugs enter cells through free diffusion to play their role, while nanomicelles enter cells through endocytosis and release drugs in lysosomes. The way the two enter the cell further affects their effect at the cellular level.

Combining Table 1 with Figure 12, it can be seen that the nanoprodrug delivery system involved in the present invention can achieve acid sensitivity, and can achieve receptor-mediated enhancement of cell endocytosis for cancer cells that overexpress SA on the cell surface, and has synergistic inhibition of tumor cells. activity.

The protection content of the present invention is not limited to the above embodiments. Variations and advantages that can occur to those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the appended claims are the scope of protection.

Claims (5)

1. a two-block double sensitive camptothecin polymer prodrug with phenylboronate as a linking unit, is characterized in that, has the structure of formula (I): In formula (I), x=40-120; m=2-10. 2. a synthetic method of the two-block double sensitive camptothecin polymer prodrug of claim 1, is characterized in that, the method comprises the following concrete steps: Step 1: Synthesis of macroinitiator PEG-BC (a) In a solvent, 3,4-dihydroxyphenylacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, N, N-diisopropylethylamine and PEG-NH ₂ carry out amidation reaction to synthesize the dopa derivative PEG-3,4-DA of PEG-NH ₂ shown in formula (1); (b) in a solvent, PEG-3,4-DA is dehydrated and condensed with 3-aminophenylboronic acid to obtain the phenylboronic acid ester derivative PEG-BC of PEG represented by formula (2); The reaction process is shown in route (A): route (A), x=40-120, m=2-10; Step 2: Synthesis of Reduction-Sensitive Camptothecin Monomer (Glu-ss-CPT)-NCA (c) in a solvent, camptothecin reacts with triphosgene, 4-dimethylaminopyridine and 2,2'-dithiodiethanol to obtain CPT-ss-OH shown in formula (3); (d) In solvent, CPT-ss-OH and Boc-L-glutamic acid-1-tert-butyl ester, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride, triethylamine, and esterification to obtain Boc-(Glu-ss-CPT)-OtBu represented by formula (4); (e) in a solvent, use TFA to remove the Boc protecting group in the Boc-(Glu-ss-CPT)-OtBu molecule to obtain (Glu-ss-CPT)-OH shown in formula (5); (f) In the solvent, with triphosgene as an initiator, (Glu-ss-CPT)-OH undergoes a cyclization reaction, and the α-amino acid structure in (Glu-ss-CPT)-OH forms an N-carboxy intracyclic anhydride, The reduction-sensitive camptothecin monomer (Glu-ss-CPT)-NCA represented by formula (6) is obtained; The reaction process is shown in route (B): Step 3: Synthesis of Diblock Dual-Sensitive Camptothecin Polymer Prodrug PEG-BC-PGlu-ss-CPT (g) In a solvent, using PEG-BC as a macroinitiator, the reduction-sensitive camptothecin monomer (Glu-ss-CPT)-NCA represented by formula (4) is subjected to ring-opening polymerization to obtain formula The two-block double-sensitive camptothecin polymer prodrug PEG-BC-PGlu-ss-CPT with phenylboronate as connecting unit shown in (1); The reaction process is shown in the route (C): Route (C), x=40-120, m=2-10; where: In (a) of the step 1, 3,4-dihydroxyphenylacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole , N,N-diisopropylethylamine and PEG-NH _The molar ratio is 1.2-2: 1.2-3: 1.2-3: 2-4: 1; the temperature of amidation reaction is 20-37 ℃; In (b) of the step 1, the molar ratio of PEG-3,4-DA and 3-aminophenylboronic acid is 1:5-10; the temperature of the dehydration condensation reaction is 100-130°C; In (c) of the step 2, the molar ratio of camptothecin, triphosgene, 4-dimethylaminopyridine and 2,2'-dithiodiethanol is 1:0.7-1:5-6:1.5-5 ; The reaction temperature is 20-37 ℃; In (d) of the step 2, Boc-L-glutamic acid-1-tert-butyl ester, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, 1-ethyl-3-(3- The molar ratio of dimethylaminopropyl)carbodiimide hydrochloride, triethylamine and CPT-ss-OH is 1.5-2:0.25-1:1-1.5:1-1.5:1-1.5:1; ester The temperature of the chemical reaction is 20-37 °C; In (e) of the step 2, the molar ratio of TFA and Boc-(Glu-ss-CPT)-OtBu is 10-15): 1; the temperature of the removal reaction is 0-25°C; In (f) of the step 2, the initiator is selected from triethylamine, pyridine or N,N-diisopropylethylamine; The molar ratio is 1/3-1:1-2:1; the temperature of the cyclization reaction is 48-55°C; In (g) of the step 3, the molar ratio of PEG-BC to (Glu-ss-CPT)-NCA is 1:3-10; the temperature of the ring-opening polymerization reaction is 20-37°C. 3. the preparation method of the two-block dual-sensitive camptothecin polymer prodrug nanomicelle of claim 1, is characterized in that, the method comprises the following concrete steps: Step 1: Dissolve the diblock dual-sensitive camptothecin polymer prodrug PEG-BC-PGlu-ss-CPT in a mixed solution of analytical pure organic solvent and ultrapure water; Step 2: filter the obtained mixed solution, and then put the filtrate into a dialysis bag for dialysis for 48 hours; freeze-dry the solution after dialysis to obtain the two-block dual-sensitive camptothecin polymer prodrug nanomicelle PEG -BC@PGlu-ss-CPT; Wherein: the organic solvent is tetrahydrofuran, dimethyl sulfoxide or acetone; the volume ratio of the analytically pure organic solvent to ultrapure water is 1:10; the concentration of the prodrug PEG-BC-PGlu-ss-CPT is 0.1-0.5 mg/mL; in the prodrug PEG-BC-PGlu-ss-CPT, x=44, m=5. 4. The prodrug nanomicelle PEG-BC@PGlu-ss-CPT prepared by the method of claim 3. 5 . The prodrug nanomicelle according to claim 3 , wherein the prodrug nanomicelle is an acid-sensitive and reduction-sensitive dual-sensitive type. 6 .