CN103479598B - The preparation method of polyethylene glycol ester triblock copolymer medicament-carried nano micelle - Google Patents

Abstract

The present invention provides the preparation method of a kind of polyethylene glycol polyester triblock copolymer medicament-carried nano micelle to be: is added drop-wise in polyethylene glycol polyester triblock copolymer by the mixture of small-molecule drug and organic solvent, obtains mixed solution；Dripping ultra-pure water while carrying out described mixed solution stirring for the first time, after continuing second time stirring, dialysis removes organic solvent and lyophilizing obtains medicament-carried nano micelle.The method prepares polyethylene glycol polyester triblock copolymer medicament-carried nano micelle, simple to operate, mild condition, the nano-micelle particle generated can present good monodisperse status, and the nano-micelle kernel prepared of the method with the small-molecule drug of coated water-soluble difference, and can be greatly improved envelop rate and the dissolubility of medicine, the polyethylene glycol polyester triblock copolymer medicament-carried nano micelle Stability Analysis of Structures of gained, particle diameter is little, and is prone to preserve.

Description

Preparation method of polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle

technical field

The invention relates to the field of macromolecules, in particular to a preparation method of polyethylene glycol-polyester triblock copolymer drug-loaded nano micelles.

Background technique

Polymer carrier drug is an emerging drug delivery technology with the development of pharmaceutical research, biomaterial science and clinical medicine. Low-molecular-weight drugs have the advantages of high curative effect and convenient use, but they also have great side effects. Generally, low-molecular-weight drugs enter the human body through oral administration or injection, with fast metabolism, short half-life, and lack of selectivity. A polymer carrier drug refers to a polymer that has no pharmacological effect and does not react with the drug itself as a carrier of the drug. A class of drugs obtained on the chain. Among them, the high-molecular compound acts as a delivery system for low-molecular drugs.

Using polymer materials as the carrier of small molecule drugs can increase the action time of drugs, improve the selectivity of drugs, reduce the toxicity of small molecule drugs, and position accurately. Micro- and nano-scale polymer carriers, such as nanomicelles, vesicles and nanoparticles, have been rapidly developed in the near future. Such polymer carriers can effectively disperse drug molecules into them, and utilize various response modes of the carrier. , to achieve drug delivery and controlled release.

Among them, nanomicelles can wrap drug molecules, and the stability of nanomicelles directly affects the performance of drug-loaded micelles. At present, the preparation methods of nanomicelle mainly include: self-assembly method, dialysis method, chemical combination method and electrostatic interaction method, etc., but these methods have disadvantages. The nanomicelles prepared by the self-assembly method only rely on non-covalent bonds such as Van der Waals force, hydrogen bond or electrostatic force to connect the material layers, so the mechanical stability of the nanomicelles is poor and the efficiency is low. The chemical combination method requires suitable functional groups to react, and the selection of polymer materials and small molecule drugs is relatively strict. Dialysis and electrostatic interaction methods are not suitable for large-scale production.

Therefore, the preparation method of drug-loaded micelles is also subject to certain limitations.

Contents of the invention

The technical problem solved by the present invention is to provide a preparation method of polyethylene glycol-polyester triblock copolymer drug-loaded nano-micelle, which is simple to operate and easy to industrialize, and the obtained polyethylene glycol-polyester triblock The copolymer drug-loaded nano-micelle has high encapsulation efficiency and stable structure.

The invention discloses a preparation method of polyethylene glycol-polyester triblock copolymer drug-loaded nano micelles, which comprises the following steps:

(A) Add the mixture of small molecule drug and organic solvent dropwise to polyethylene glycol-polyester triblock copolymer to obtain a mixed solution;

(B) adding ultrapure water dropwise to the mixed solution while stirring for the first time, and after continuing the second stirring, dialysis to remove the organic solvent and freeze-drying to obtain drug-loaded nanomicelles;

The polyethylene glycol-polyester triblock copolymer is shown in formula (I),

Among them, -R- is or

m is the degree of polymerization, 10≤m≤250; n is the degree of polymerization, 10≤n≤220.

Preferably, in the step (A), the small molecule drug is methotrexate, 5-fluorouracil, cyclophosphamide, daunorubicin, doxorubicin, epirubicin, pirarubicin , camptothecins or taxanes.

Preferably, in the step (A), the concentration of the small molecule drug in the organic solvent is 0.1-10 mg/mL.

Preferably, in the step (A), the mass ratio of the small molecule drug to the polyethylene glycol-polyester triblock copolymer is 0.01-1.

Preferably, the speed of the first stirring is 100-2000 rpm.

Preferably, the rate of adding the ultrapure water dropwise is 0.05-5 mL/min.

Preferably, the volume ratio of the amount of ultrapure water to the amount of organic solvent is 0.01-20.

Preferably, the organic solvent is tetrahydrofuran, 1,4-dioxane, dimethylsulfoxide or N,N-dimethylformamide.

Compared with the prior art, the invention adopts the nano-sedimentation method to prepare the drug-loaded nano micelles of the polyethylene glycol-polyester triblock copolymer. Add the mixture of the small molecule drug and the organic solvent dropwise into the polyethylene glycol-polyester triblock copolymer, stir to obtain a mixed solution, add ultrapure water dropwise to the mixed solution while stirring, and continue to the second Stir once, dialyze to remove the organic solvent and freeze-dry to obtain the drug-loaded nano-micelle. The preparation of polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelles by this method has simple operation and mild conditions, and the generated nanomicelle particles can present a good monodisperse state, and the nanomicelles prepared by this method The inner core can encapsulate small molecule drugs with poor water solubility, and greatly improve the encapsulation efficiency and solubility of the drug. The obtained drug-loaded nano-micelle has a stable structure, small particle size, and is easy to store.

Description of drawings

Fig. 1 is the nuclear magnetic resonance spectrum of the polyethylene glycol-polyester triblock copolymer that embodiment 9 obtains in chloroform;

Fig. 2 is the nuclear magnetic resonance spectrum of the polyethylene glycol-polyester triblock copolymer that embodiment 19 obtains in chloroform;

Fig. 3 is the particle size distribution figure of nano-micelles that embodiment 37～39, 62 and 84 obtain;

Fig. 4 is the particle size distribution figure of nano micelles prepared by embodiment 43～45;

Fig. 5 is the particle size distribution figure of the nano micelles of different concentrations prepared by 50～52;

Fig. 6 is the transmission electron micrograph of the nano micelles prepared by embodiment 62;

Fig. 7 is the graph of the impact of the nanomicelles obtained in Examples 47, 57 and 79 on the viability of MCF-7 cells;

Fig. 8 is the particle size distribution diagram of the polyethylene glycol-polyester triblock copolymer drug-loaded nano-micelle prepared in Examples 87-89;

Fig. 9 is a graph showing the particle size distribution of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelles prepared in Examples 87-89 within 3 weeks.

detailed description

In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with examples, but it should be understood that these descriptions are only to further illustrate the features and advantages of the present invention, rather than limiting the claims of the present invention.

The embodiment of the present invention discloses a preparation method of polyethylene glycol-polyester triblock copolymer drug-loaded nano-micelle, comprising the following steps:

(A) Add the mixture of small molecule drug and organic solvent dropwise to polyethylene glycol-polyester triblock copolymer to obtain a mixed solution;

(B) adding ultrapure water dropwise to the mixed solution while stirring for the first time, and after continuing the second stirring, dialysis to remove the organic solvent and freeze-drying to obtain drug-loaded nanomicelles;

The polyethylene glycol-polyester triblock copolymer is shown in formula (I),

Among them, -R- is or

m is the degree of polymerization, 10≤m≤250, preferably 40≤m≤200, more preferably 60≤m≤150; n is the degree of polymerization, 10≤n≤220, preferably 30≤n≤20, more preferably 60≤n≤150.

The present invention selects the nano-sedimentation method to prepare the polyethylene glycol-polyester triblock copolymer drug-loaded nano-micelle, which is simple to operate and has mild conditions. The inner core of the nano-micelle prepared by this method can wrap small-molecule drugs with poor water solubility, And the encapsulation efficiency and solubility of the drug are greatly improved, and the obtained drug-loaded nano-micelle has a stable structure, a small particle size, and is easy to store. The present invention preferably controls the particle size of the drug-loaded nanomicelles by changing the speed of adding ultrapure water, the amount of ultrapure water added, and the stirring speed during the preparation process, and adjusts the drug and polyethylene glycol-polyethylene glycol. The ratio of the ester triblock copolymer is selected to select the best drug-loading conditions. In the invention, firstly, the mixture of the small molecule drug and the organic solvent is added dropwise into the polyethylene glycol-polyester triblock copolymer to obtain a mixed solution. Since the finally formed micelles will encapsulate the small molecule drug inside, the small molecule drug is preferably methotrexate, 5-fluorouracil, cyclophosphamide, daunorubicin, doxorubicin, epirubicin , pirarubicin, camptothecins or taxanes, more preferably 10-hydroxycamptothecin, doxorubicin or docetaxel. The organic solvent is preferably tetrahydrofuran, 1,4-dioxane, dimethyl sulfoxide or N,N-dimethylformamide, more preferably tetrahydrofuran. The polyethylene glycol-polyester triblock copolymer is shown in formula (I),

Among them, -R- is or

m is the degree of polymerization, preferably 40≤m≤200, more preferably 60≤m≤150; n is the degree of polymerization, 10≤n≤220, preferably 30≤n≤20, more preferably 60≤n≤150.

The polyethylene glycol-polyester triblock copolymer is preferably obtained by polymerizing lactide or caprolactone with polyethylene glycol with a molecular weight of 1000-40000; the lactide is D-lactide or L - Lactide. Its preparation method is specifically:

Under anhydrous and oxygen-free conditions, add polyethylene glycol and ester monomers into an ampoule, add a certain amount of toluene after azeotropic water removal, and the volume of toluene (mL) is 10% of the weight of ester monomers (g). times, use a syringe to inject 0.1mol/L stannous octoate toluene solution, the volume of stannous octoate and the molar ratio of ester monomers is 1/1000, and put it in an oil bath at 120°C for 24 hours. After the reaction is completed, settle with a large amount of ether, the ratio of ether to toluene is 10/1, filter with a Buchner funnel, dissolve the product in chloroform, settle with ether, filter with a Buchner funnel, and dry the product in a vacuum atmosphere for 24 hours. Acquired polyethylene glycol-polyester triblock copolymer.

In the process of preparing the polyethylene glycol-polyester triblock copolymer, it is preferable to use an initiator for initiation, the initiator is preferably polyethylene glycol, and the molecular weight of the polyethylene glycol is preferably 4000. The number average molecular weight of the obtained polyester block is preferably 2,000 to 30,000.

In the present invention, the mixture of the small molecule drug and the organic solvent is a homogeneous solution, preferably obtained by stirring for 4 to 6 hours, and the concentration of the small molecule drug in the organic solvent is preferably 0.1 to 10 mg/mL, more preferably 0.4 ~ 8mg/mL. The mixture of the small molecule drug and the organic solvent is added dropwise in the polyethylene glycol-polyester triblock copolymer to obtain a mixed solution, and the mixture of the small molecule drug and the polyethylene glycol-polyester triblock copolymer The mass ratio is preferably 0.01 to 1, more preferably 0.05 to 0.5. Since the polyethylene glycol-polyester triblock copolymer can be selected from ester monomers of different configurations in the preparation process, it can also be selected to have different configurations of chains when preparing a mixed solution with the mixture. Segments of polyethylene glycol-polyester triblock copolymers are mixed, and the left-handed and right-handed segments can interact with each other, so that they have a stereocomplexation effect in the formation of micelles, thereby improving the encapsulation efficiency of drugs and Solubility. The mass ratio of polyethylene glycol-polyester triblock copolymers with different configuration segments is preferably 1:1. Preferably, after the mixture of the small molecule drug and the organic solvent is added dropwise into the polyethylene glycol-polyester triblock copolymer, stirring is performed; the stirring time is preferably 1 to 3 hours.

After the mixed solution is obtained, ultrapure water is added dropwise to the mixed solution while being stirred for the first time, and after the second stirring is continued, the organic solvent is removed by dialysis and freeze-dried to obtain drug-loaded nano micelles. The device for dropping the ultrapure water is preferably a syringe pump, and the speed of adding the ultrapure water is preferably 0.05-5 mL/min, more preferably 0.1-3 mL/min. The volume ratio of the amount of the ultrapure water to the amount of the organic solvent is preferably 0.01-20, more preferably 0.1-10. When the mixed solution is stirred for the first time, ultrapure water is added dropwise. The speed of the first stirring is preferably 100-2000 rpm, more preferably 800-1500 rpm. After the dropwise addition, the second stirring is continued, the organic solvent is removed by dialysis, and the drug-loaded nano-micelle is obtained by lyophilization. The time of the second stirring is preferably 8-12 hours. The speed of the second stirring is preferably the same as that of the first stirring. In order to improve the purity of the obtained drug-loaded nanomicelles, it is preferable to perform dialysis in ultrapure water after the second stirring, the dialysis time is preferably 20 to 30 hours, and the water is changed more than 5 times, and the dialysis is preferably dialysis with a MWCO of 3500 bag. In order to facilitate storage, the obtained polyethylene glycol-polyester triblock copolymer drug-loaded nano-micelle can also be freeze-dried.

The particle size of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelles measured by dynamic light scattering was continuously measured for 3 weeks, and the results showed that the particle size variation trend was basically the same. The polyethylene glycol-polyester triblock copolymer drug-loaded nano-micelle prepared by the method of the invention has a stable structure.

The preparation of polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelles by this method has simple operation and mild conditions, and the generated nanomicelle particles can present a good monodisperse state, and the nanomicelles prepared by this method The inner core can encapsulate small molecule drugs with poor water solubility, and greatly improve the encapsulation efficiency and solubility of the drug. The obtained drug-loaded nano-micelle has a stable structure, small particle size, and is easy to store.

In order to further understand the present invention, the preparation method of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle provided by the present invention will be described below in conjunction with the examples, and the protection scope of the present invention is not limited by the following examples.

Embodiment 1～6

Preparation of polyethylene glycol-poly(D-lactide) triblock copolymers initiated by polyethylene glycol with different number-average molecular weights

Weigh 1.67g, 6.67g, 13.33g, 16.67g, 33.33g, 66.57g of polyethylene glycol (PEG) with molecular weights of 1000, 4000, 8000, 10000, 20000, 40000 respectively into the reaction flask, and remove Water, add 12g of dextrolactide (DLA) in an anhydrous and oxygen-free environment, ventilate, the volume of toluene (mL) is 10 times the weight of the ester monomer (g) 120mL, the volume of stannous octoate and The molar ratio of ester monomers is 1/1000, inject 1 mL of 0.1 mol/L stannous octoate solution in toluene with a syringe, and put it in an oil bath at 120°C for 24 hours. After the reaction is complete, wait for the solution to cool down and settle with 1200mL of diethyl ether while stirring. The ratio of diethyl ether to toluene is 10/1. Filter the product through a Buchner funnel. Dissolve the product in chloroform and settle it with diethyl ether. Drying in a vacuum desiccator for 24 hours in a cold well

Polyethylene glycol-poly(D-lactide) triblock copolymer with number average molecular weight.

Table 1 The number-average molecular weight and reaction yield of the polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Examples 1-6

In Table 1, the number-average molecular weight _Mn is the number-average molecular weight of polyethylene glycol-poly(D-lactide) triblock copolymers initiated by polyethylene glycol with different number-average molecular weights, which are obtained by ¹ H NMR .

Examples 7-10

Preparation of polyethylene glycol-poly(D-lactide) triblock copolymers with different degrees of polymerization initiated by polyethylene glycol

Weigh 4g, 6g, 8g, and 12g of polyethylene glycol (PEG) with a molecular weight of 4000, respectively, and put them into the reaction bottle, remove water by azeotropy, add 12g of dextrolactide (DLA) in an anhydrous and oxygen-free environment, Ventilation, the amount of toluene volume (mL) is 10 times the weight (g) of the ester monomer 120mL, the volume amount of stannous octoate and the molar ratio of the ester monomer is 1/1000, inject 0.1mol/L octanoic acid with a syringe Put 1mL of stannous toluene solution in an oil bath at 120°C for 24h. After the reaction is complete, wait for the solution to cool down and settle with 1200mL of diethyl ether while stirring. The ratio of diethyl ether to toluene is 10/1. Filter the product through a Buchner funnel. Dissolve the product in chloroform and settle it with diethyl ether. Dry in a vacuum desiccator for 24 hours in a cold well to obtain polyethylene glycol-poly(D-lactide) triblock copolymers with different degrees of polymerization initiated by polyethylene glycol.

Table 2 Number average molecular weight and reaction yield of polyethylene glycol-poly(D-lactide) triblock copolymers prepared in Examples 7-10

In Table 2, the number-average molecular weight M _n is the number-average molecular weight of polyethylene glycol-poly(D-lactide) triblock copolymers with different degrees of polymerization initiated by polyethylene glycol, which was determined by ¹ H NMR.

See Figure 1 for the nuclear magnetic resonance spectrum of the polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Example 9. Figure 1 shows the polyethylene glycol-polyester triblock copolymer obtained in Example 9. NMR spectra of block copolymers in chloroform. In Figure 1, the assignment of each peak is: 1.6ppm (S, 3H, HO-CH(CH ₃ )OC(O)) 3.68ppm (S, 2H, CH ₂ CH ₂ O)), 5.2ppm (S, 3H ,HO-CH(CH ₃ )OC(O)). Figure 1 shows that the polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Example 9 has the structure of formula (I).

Examples 11-16

Preparation of polyethylene glycol-poly(L-lactide) triblock copolymers initiated by polyethylene glycol with different number-average molecular weights

Weigh 1.67g, 6.67g, 13.33g, 16.67g, 33.33g, 66.57g of polyethylene glycol (PEG) with molecular weights of 1000, 4000, 8000, 10000, 20000, 40000 respectively into the reaction flask, and remove Water, add L-lactide (LLA) 12g in an anhydrous and oxygen-free environment, ventilate, the volume of toluene (mL) is 10 times the weight (g) of the ester monomer, that is, 120mL, the volume of stannous octoate The molar ratio with the ester monomer is 1/1000, inject 1mL of 0.1mol/L stannous octoate toluene solution with a syringe, and put it in a 120°C oil bath to react for 24h. After the reaction is complete, wait for the solution to cool down and settle with 1200mL of diethyl ether while stirring. The ratio of diethyl ether to toluene is 10/1. Filter the product through a Buchner funnel. Dissolve the product in chloroform and settle it with diethyl ether. Dry in a vacuum desiccator for 24 hours in a cold well to obtain polyethylene glycol-poly(L-lactide) triblock copolymers initiated by polyethylene glycol with different number average molecular weights.

Table 3 Number average molecular weight and reaction yield of polyethylene glycol-poly(L-lactide) triblock copolymers prepared by polyethylene glycol initiated in Examples 11-16

In the above table, the number-average molecular weight M _n is the number-average molecular weight of polyethylene glycol-poly(L-lactide) triblock copolymers initiated by polyethylene glycol with different number-average molecular weights, which are obtained by ¹ H NMR .

Examples 17-20

Preparation of PEG-initiated PEG-poly(L-lactide) triblock copolymers with different number-average molecular weights.

Weigh 4g, 6g, 8g, and 12g of polyethylene glycol (PEG) with a molecular weight of 4000, respectively, and put them into the reaction flask, remove water by azeotropy, add 12g of L-lactide (LLA) in an anhydrous and oxygen-free environment, and replace Gas, the amount of toluene volume (mL) is 10 times the weight (g) of the ester monomer, that is, 120mL, the volume amount of stannous octoate and the molar ratio of the ester monomer is 1/1000, inject 0.1mol/L with a syringe 1 mL of toluene solution of stannous octoate was placed in an oil bath at 120°C for 24 h. After the reaction is complete, wait for the solution to cool down and settle with 1200mL of diethyl ether while stirring. The ratio of diethyl ether to toluene is 10/1. Filter the product through a Buchner funnel. Dissolve the product in chloroform and settle it with diethyl ether. Dry in a vacuum desiccator for 24 hours in a cold well to obtain polyethylene glycol-poly(L-lactide) triblock copolymers initiated by polyethylene glycol with different number average molecular weights.

Table 4 Number average molecular weight and reaction yield of polyethylene glycol-poly(L-lactide) triblock copolymers prepared in Examples 17-20

In Table 4, the number-average molecular weight _Mn is the number-average molecular weight of polyethylene glycol-poly(L-lactide) triblock copolymers initiated by polyethylene glycol with different number-average molecular weights, obtained by ¹ H NMR .

See Figure 2 for the nuclear magnetic resonance spectrum of the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Example 19, which shows the polyethylene glycol-polyester triblock copolymer obtained in Example 19 NMR spectrum of the compound in chloroform. In Figure 2, the assignment of each peak is: 1.6ppm (S, 3H, HO-CH(CH ₃ )OC(O)) 3.68ppm (S, 2H, CH ₂ CH ₂ O)), 5.2ppm (S, 3H ,HO-CH(CH ₃ )OC(O)). Figure 2 shows that the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Example 19 has the structure of formula (I).

Examples 21-26

Preparation of polyethylene glycol-initiated poly(ε-caprolactone) triblock copolymers with different number-average molecular weights

Weigh 1.67g, 6.67g, 13.33g, 16.67g, 33.33g, 66.57g of polyethylene glycol (PEG) with molecular weights of 1000, 4000, 8000, 10000, 20000, 40000 respectively into the reaction flask, and remove Water, add caprolactone 12g in an anhydrous and oxygen-free environment, ventilate, the volume of toluene (mL) is 10 times the weight of caprolactone monomer (g) 120mL, the volume of stannous octoate and caprolactone monomer The molar ratio is 1/1000, inject 1mL of 0.1mol/L stannous octoate toluene solution with a syringe, and put it in an oil bath at 120°C for 24h. After the reaction is complete, wait for the solution to cool down and settle with 1200mL of diethyl ether while stirring. The ratio of diethyl ether to toluene is 10/1. Filter the product through a Buchner funnel. Dissolve the product in chloroform and settle it with diethyl ether. Dry in a vacuum desiccator for 24 hours in a cold well to obtain polyethylene glycol-poly(ε-caprolactone) triblock copolymers with different number average molecular weights.

The number-average molecular weight and reaction yield of the polyethylene glycol-poly(ε-caprolactone) triblock copolymer prepared in Table 5 Examples 21-26

In Table 5, the number-average molecular weight _Mn is the number-average molecular weight of polyethylene glycol-poly(ε-caprolactone) triblock copolymers initiated by polyethylene glycol with different number-average molecular weights, which are obtained by ¹ H NMR .

Examples 27-30

Preparation of polyethylene glycol-initiated poly(ε-caprolactone) triblock copolymers with different number-average molecular weights

Weigh 4g, 6g, 8g, and 12g of polyethylene glycol (PEG) with a molecular weight of 4000, respectively, and put them into the reaction bottle, remove water by azeotropy, add 12g of caprolactone in an anhydrous and oxygen-free environment, ventilate, and the volume of toluene The amount (mL) is 10 times the weight of caprolactone monomer (g) 120mL, the volume dosage of stannous octoate and the molar ratio of caprolactone monomer is 1/1000, and the toluene of 0.1mol/L stannous octoate is injected with a syringe 1 mL of the solution was placed in an oil bath at 120°C for 24 hours. After the reaction is complete, wait for the solution to cool down and settle with 1200mL of diethyl ether while stirring. The ratio of diethyl ether to toluene is 10/1. Filter the product through a Buchner funnel. Dissolve the product in chloroform and settle it with diethyl ether. Dry in a vacuum desiccator for 24 hours in a cold well to obtain polyethylene glycol-poly(ε-caprolactone) triblock copolymers with different number average molecular weights.

Table 6 The polyethylene glycol-poly(ε-caprolactone) triblock copolymer prepared in Examples 27-30

In Table 6, the number-average molecular weight M _n is the number-average molecular weight of polyethylene glycol-poly(ε-caprolactone) triblock copolymers with different degrees of polymerization initiated by polyethylene glycol, measured by ¹ H NMR

Examples 31-33

Take 100 mg of the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Example 19 and dissolve them in tetrahydrofuran, 1,4-dioxane, dimethyl sulfoxide, N, N - In dimethylformamide, the concentration is 2.5mg/mL, stir for 3h, set the flow rate of the syringe pump to 0.1mL/min, the flow rate (mL) to 25mL, and set the stirring speed of the stirrer to 1000rpm. Stir the dissolved polyester block copolymer solution on a stirrer, and add Milli-Q dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q for 24 h with a dialysis bag with MWCO of 3500, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 34-36

Take 100 mg of the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Example 19, respectively, and prepare tetrahydrofuran solutions with concentrations of 2 mg/mL, 2.5 mg/mL, and 3 mg/mL, and stir for 3 h , Set the flow rate of the syringe pump to 0.1mL/min, the flow rate (mL) to 25mL, and set the stirring speed of the stirrer to 1000r/min. Stir the dissolved polyester block copolymer solution on a stirrer, and add Milli-Q dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 37-39

Take three parts of the polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Example 9, 100 mg each, dissolve in tetrahydrofuran solution at a concentration of 2.5 mg/mL, stir for 3 hours, and set the syringe pump The flow rate is 0.1mL/min, the flow rate (mL) is 25mL, and the stirring speed of the stirrer is set to 500rpm, 1000rpm, and 1500rpm respectively. Put the dissolved polyester lactic acid block copolymer solution on the stirrer to stir, and use the set syringe pump to drop Milli-Q into the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Fig. 3 is the nanomicelle size distribution figure that embodiment 37～39, 62 and 84 obtain, in Fig. 3, For the dynamic hydrodynamic radius distribution of the nanomicelle obtained in Example 37, For the dynamic hydrodynamic radius distribution of the nanomicelle obtained in Example 38, For the dynamic hydrodynamic radius distribution of the nanomicelle obtained in embodiment 39, For the dynamic hydrodynamic radius distribution of the nanomicelle obtained in embodiment 62, Dynamic hydrodynamic radius distribution of nanomicelles obtained for Example 84.

Examples 40-42

Take three parts of polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Example 19, 100 mg each, dissolve in tetrahydrofuran solution at a concentration of 2.5 mg/mL, stir for 3 hours, and set up a syringe pump The flow rate is 0.1mL/min, the flow rate (mL) is set to 20mL, 25mL, and 30mL, respectively, and the stirring speed of the stirrer is set to 1000rpm. Put the dissolved polylactic acid block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 43-45

Take three parts of polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Example 19, 100 mg each, dissolve in tetrahydrofuran solution at a concentration of 2.5 mg/mL, stir for 3 hours, and set up a syringe pump The flow rates were 0.1 mL/min, 0.3 mL/min, and 0.5 mL/min, respectively, the flow rate (mL) was set to 25 mL, and the stirring speed of the stirrer was set to 1000 rpm. Put the dissolved polylactic acid block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Fig. 4 is the particle size distribution figure of the obtained nanomicelle prepared by embodiment 43～45, in Fig. 4, For the dynamic hydrodynamic radius distribution of the nanomicelle obtained in Example 43, For the dynamic hydrodynamic radius distribution of the nanomicelle obtained in Example 44, Dynamic hydrodynamic radius distribution of nanomicelles obtained for Example 45.

Examples 46-49

Weigh 100 mg of the polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Examples 1 to 4, dissolve in THF solution at a concentration of 2.5 mg/mL, stir for 3 hours, and set the flow rate of the syringe pump The flow rate (mL) is set to 0.1mL/min, the flow rate (mL) is set to 25mL, and the stirring speed of the stirrer is set to 1000rpm. Put the dissolved polylactic acid block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 50-52

Weigh 100 mg of the polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Examples 7 to 9, dissolve in THF solution at a concentration of 2.5 mg/mL, stir for 3 hours, and set the flow rate of the syringe pump They are 0.1mL/min, 0.3mL/min, 0.5mL/min respectively, the flow rate (mL) is set to 25mL, and the stirring speed of the stirrer is set to 1000rpm. Put the dissolved polylactic acid block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Fig. 5 is the particle size distribution figure of the nano micelles of different concentrations that embodiment 50～52 prepares, in Fig. 5, The dynamic hydrodynamic radius distribution of the nanomicelle obtained for embodiment 50, For the dynamic hydrodynamic radius distribution of the nanomicelle obtained in Example 51, Dynamic hydrodynamic radius distribution of nanomicelles obtained for Example 52.

Examples 53-55

Take three parts of the polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Example 3, 100 mg each, dissolve in THF solution at a concentration of 2.5 mg/mL, stir for 3 hours, and set the syringe pump The flow rates were 0.1 mL/min, 0.3 mL/min, and 0.5 mL/min, respectively, the flow rate (mL) was set to 25 mL, and the stirring speed of the stirrer was set to 1000 rpm. Put the dissolved polylactic acid block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 56-59

Weigh 100 mg of the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Examples 11 to 14, dissolve in tetrahydrofuran solution at a concentration of 2.5 mg/mL, stir for 3 hours, and set the flow rate of the syringe pump 0.1mL/min, the flow rate (mL) is set to 25mL, and the stirring speed of the stirrer is set to 1000rpm. Put the dissolved polylactic acid block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 60-63

Weigh 100 mg of the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Examples 17-20, dissolve in tetrahydrofuran solution at a concentration of 2.5 mg/mL, stir for 3 hours, and set the flow rate of the syringe pump 0.1mL/min, the flow rate (mL) is set to 25mL, and the stirring speed of the stirrer is set to 1000rpm. Put the dissolved polylactic acid block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Fig. 6 is a transmission electron microscope image of the nanomicelle prepared in Example 62, and it can be known from Fig. 6 that the described method has prepared the nanomicelle.

Examples 64-66

Take three parts of the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Example 13, 100 mg each, dissolve in tetrahydrofuran solution with a concentration of 2.5 mg/mL, stir for 3 hours, and set the syringe pump The flow rates were 0.1mL/min, 0.3mL/min, and 0.5mL/min, the flow rate (mL) was set to 25mL, the flow rate (mL) was set to 25mL, and the stirring speed of the stirrer was set to 1000rpm. Put the dissolved polylactic acid block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 67-70

Weigh 100 mg of the polyethylene glycol-poly(ε-caprolactone) triblock copolymer prepared in Examples 21 to 24, dissolve in THF solution at a concentration of 2.5 mg/mL, stir for 3 hours, and set the flow rate of the syringe pump 0.1mL/min, the flow rate (mL) is set to 25mL, and the stirring speed of the stirrer is set to 1000rpm. Put the dissolved polycaprolactone block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 71-74

Weigh 100 mg of the polyethylene glycol-poly(ε-caprolactone) triblock copolymer prepared in Examples 27 to 30, dissolve them in tetrahydrofuran solution at a concentration of 2.5 mg/mL, stir for 3 hours, and set the flow rate of the syringe pump 0.1mL/min, the flow rate (mL) is set to 25mL, and the stirring speed of the stirrer is set to 1000rpm. Put the dissolved polycaprolactone block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 75-77

Take three parts of polyethylene glycol-poly(ε-caprolactone) triblock copolymer prepared in Example 23, 100 mg each, dissolve in tetrahydrofuran solution with a concentration of 2.5 mg/mL, stir for 3 hours, and set up a syringe pump The flow rates were 0.1mL/min, 0.3mL/min, and 0.5mL/min, the flow rate (mL) was set to 25mL, the flow rate (mL) was set to 25mL, and the stirring speed of the stirrer was set to 1000rpm. Put the dissolved polylactic acid block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 78-81

The polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Examples 1-4 and the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Examples 11-14 Mix 50 mg of each copolymer, dissolve in tetrahydrofuran solution at a concentration of 2.5 mg/mL, stir for 3 h, set the flow rate of the syringe pump to 0.1 mL/min, set the flow rate (mL) to 25 mL, and set the stirring speed of the stirrer to 1000 rpm. Put the dissolved polycaprolactone block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Examples 82-85

The polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Examples 7-10 and the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Examples 17-20 Mix 50 mg of each copolymer, dissolve in tetrahydrofuran solution at a concentration of 2.5 mg/mL, stir for 3 h, set the flow rate of the syringe pump to 0.1 mL/min, set the flow rate (mL) to 25 mL, and set the stirring speed of the stirrer to 1000 rpm. Put the dissolved polycaprolactone block copolymer solution on the stirrer to stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is constant to volume to obtain biologically active nanomicelles.

Example 86

The toxicity test of the nano micelles that embodiment 47,57,79 obtains in MCF-7 cell:

The nanomicelles and PEI obtained in Examples 47, 57, and 79 were prepared into cell culture media with concentrations of 0.01 μg/mL, 0.025 μg/mL, 0.05 μg/mL, and 0.10 μg/mL, respectively.

First, MCF-7 cells were planted in a 96-well culture plate, and the culture medium was removed after 24 hours of culture. The experimental group was added the cell culture medium of nanomicelles obtained in the above-mentioned various concentrations of Examples 47, 57, and 79, and the control group was added with the above-mentioned Various concentrations of PEI. MTT was added after culturing for 72 hours, the culture medium was aspirated after 4 hours, and DMSO was added to measure the OD value at 490nm wavelength of the control group and the experimental group on a microplate reader.

The experimental results are shown in Fig. 7, and Fig. 7 is a graph showing the effect of nanomicelles obtained in Examples 47, 57, and 79 on the survival rate of MCF-7 cells. In Fig. 7, The impact curve of the nanomicelle obtained in embodiment 47 of different concentrations on the cell viability, The impact curve of the nanomicelle obtained in embodiment 57 of different concentrations on cell viability, The influence curve of the nanomicelle obtained for the embodiment 79 of different concentrations on the cell viability; Effect curve of different concentrations of PEI on cell viability.

As shown in Figure 7, in all tested concentration ranges, the cell survival rate of the nanomicelles with the highest concentration of 0.1 mg L ^-1 was above 80% after 72 hours of culture. That is to say, nanomicelles have low cytotoxicity and can be safely used as biocompatible carriers for the delivery of biologically active substances.

Examples 87-89

Polyethylene glycol ether-polyester tri-block copolymers were respectively selected from 100 mg of polyethylene glycol-poly(D-lactide) tri-block copolymers prepared in Example 9, and polyethylene glycol-poly(D-lactide) tri-block copolymers prepared in Example 19. Poly(L-lactide) triblock copolymer 100mg, the polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Example 9 and the polyethylene glycol-poly(polyethylene glycol) prepared in Example 19 (L-lactide) triblock copolymer 50 mg each was mixed.

Dissolve 10-hydroxycamptothecin in tetrahydrofuran, the concentration of 10-hydroxycamptothecin is 0.4mg/mL, stir for 5h, after dissolving, add 10-hydroxycamptothecin-tetrahydrofuran solution dropwise to the weighed poly The mass ratio of 10-hydroxycamptothecin and polyethylene glycol-polyester triblock copolymer is configured in the ethylene glycol-polyester triblock copolymer as a solution that is 1:5 and continues to stir for 2h, and the flow rate of the syringe pump is set. 0.1mL/min, the flow rate (mL) is set to 25mL, and the stirring speed of the stirrer is set to 1000rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved with 10-hydroxycamptothecin on a stirrer and stir, and add Milli-Q dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain a polyethylene glycol-polyester triblock copolymer drug-loaded nano-micelle with biological activity.

After calculation, the encapsulation efficiency of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 87 is 100%, and the drug-loading efficiency is 13.1%; the polyethylene glycol-polyester triblock copolymer obtained in Example 88- The encapsulation efficiency of the polyester triblock copolymer drug-loaded nano-micelle was 88%, and the drug-loading efficiency was 11.7%; the polyethylene glycol-polyester tri-block copolymer drug-loaded nano-micelle obtained in Example 89 The encapsulation efficiency was 100%, and the drug loading efficiency was 13.2%.

Fig. 8 is a particle size distribution diagram of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle prepared in Examples 87-89, and the particle size distribution diagram is measured by dynamic light scattering (DLS). In Figure 8, For the dynamic hydrodynamic radius distribution of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 87, The dynamic hydrodynamic radius distribution of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 88, The dynamic hydrodynamic radius distribution of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 89.

Fig. 9 is the particle size distribution diagram of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelles prepared in Examples 87-89 within 3 weeks, a is the polyethylene glycol prepared in Examples 87-89. The particle size distribution of alcohol-polyester triblock copolymer drug-loaded nanomicelles in 1 week, b is the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelles prepared in Examples 87～89 at Particle size distribution during 1 week, c is the particle size distribution of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle prepared in Examples 87-89 during 1 week, For the dynamic hydrodynamic radius distribution of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 87, The dynamic hydrodynamic radius distribution of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 88, The dynamic hydrodynamic radius distribution of the polyethylene glycol-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 89.

It can be seen from Figure 9 that the polyethylene glycol-polyester triblock copolymer drug-loaded nano-micelle prepared by the present invention has good stability.

Examples 90-92

Polyethylene glycol-polyester tri-block copolymers were respectively selected from 100 mg of polyethylene glycol-poly(D-lactide) tri-block copolymers prepared in Example 3, and polyethylene glycol-polyester prepared in Example 13. (L-lactide) triblock copolymer 100 mg, the polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Example 3 and the polyethylene glycol-poly( L-lactide) triblock copolymer 50 mg each were mixed.

Dissolve 10-hydroxycamptothecin in tetrahydrofuran, the concentration of 10-hydroxycamptothecin is 0.4mg/mL, stir for 5h, after dissolving, add 10-hydroxycamptothecin-tetrahydrofuran solution dropwise to the weighed poly In the glycol ether-polyester triblock copolymer, configure a solution with a mass ratio of 10-hydroxycamptothecin to polyethylene glycol ether-polyester triblock copolymer of 1:5 and continue to stir for 2 hours, then set the syringe pump The flow rate was 0.1 mL/min, the flow rate (mL) was set to 25 mL, and the stirring speed of the stirrer was set to 1000 rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved with 10-hydroxycamptothecin on a stirrer and stir, and add Milli-Q dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 90 was 12.76%, and the drug-loading efficiency was 92.34%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 91 was 12.03%, and the drug-loading efficiency was 95.6%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 92 was 14.3%, and the drug-loading efficiency was 100%.

Examples 93-97

Weigh 100 mg of the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Example 13, respectively. Dissolve 10-hydroxycamptothecin in tetrahydrofuran, the concentration of 10-hydroxycamptothecin is 0.4mg/mL, stir for 5h, after dissolving, add 10-hydroxycamptothecin-tetrahydrofuran solution dropwise to the weighed poly In the glycol ether-polyester triblock copolymer, the mass ratios of 10-hydroxycamptothecin and polyethylene glycol-polyester triblock copolymer are 1:20, 1:10, and 3:20, respectively , The solution of 4:20 and 5:20 was continuously stirred for 2h, the flow rate of the syringe pump was set to 0.1mL/min, the flow rate (mL) was set to 25mL, and the stirring speed of the stirrer was set to 1000rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved with 10-hydroxycamptothecin on a stirrer and stir, and add Milli-Q dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 93 was 11.39%, and the drug-loading efficiency was 87.52%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 94 was 12.51%, and the drug-loading efficiency was 89.21%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 95 was 13.21%, and the drug-loading efficiency was 97.32%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 96 was 12.74%, and the drug-loading efficiency was 90.63%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 97 was 11.94%, and the drug-loading efficiency was 87.86%.

Examples 98-102

Weigh 100 mg of the polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Example 6, respectively. Dissolve 10-hydroxycamptothecin in tetrahydrofuran, the concentration of 10-hydroxycamptothecin is 0.4mg/mL, stir for 5h, after dissolving, add 10-hydroxycamptothecin-tetrahydrofuran solution dropwise to the weighed poly In the ethylene glycol-polyester tri-block copolymer, the mass ratios of 10-hydroxycamptothecin and polyethylene glycol-polyester tri-block copolymer are respectively 1:20, 1:10, 3:20, The solution of 4:20 and 5:20 was continuously stirred for 2 hours, the flow rate of the syringe pump was set to 0.1mL/min, the flow rate (mL) was set to 25mL, and the stirring speed of the stirrer was set to 1000rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved with 10-hydroxycamptothecin on a stirrer and stir, and add Milli-Q dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 98 was 12.17%, and the drug-loading efficiency was 86.24%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 99 was 12.84%, and the drug-loading efficiency was 91.63%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 100 was 13.42%, and the drug-loading efficiency was 98.87%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 101 was 12.76%, and the drug-loading efficiency was 91.22%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 102 was 12.16%, and the drug-loading efficiency was 87.26%.

Examples 103-107

The polyethylene glycol-poly(D-lactide) triblock copolymer prepared in Example 9 and the polyethylene glycol-poly(L-lactide) triblock copolymer prepared in Example 19 were each 50 mg By mixing, 5 parts of the same mixed polyglycol ether-polyester triblock copolymer were prepared. Dissolve 10-hydroxycamptothecin in tetrahydrofuran, the concentration of 10-hydroxycamptothecin is 0.4mg/mL, stir for 5h, after dissolving, add 10-hydroxycamptothecin-tetrahydrofuran solution dropwise to the weighed poly The mass ratios of 10-hydroxycamptothecin and polyethylene glycol ether-polyester triblock copolymer in the glycol ether-polyester triblock copolymer are 1:20, 1:10, and 3:20, respectively , The 4:20, 5:20 solution continued to stir for 2h, set the flow rate of the syringe pump to 0.1mL/min, the flow rate (mL) to 25mL, and set the stirring speed of the stirrer to 1000rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved with 10-hydroxycamptothecin on a stirrer and stir, and add Milli-Q dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 103 was 11.89%, and the drug-loading efficiency was 86.84%

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 104 was 12.51%, and the drug-loading efficiency was 92.52%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 105 was 13.42%, and the drug-loading efficiency was 98.62%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 106 was 12.35%, and the drug-loading efficiency was 94.23%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 107 was 11.72%, and the drug-loading efficiency was 88.24%.

Examples 108-110

Polyethylene glycol-polyester triblock copolymers were respectively selected from 100 mg of polyethylene glycol-poly(D-lactide) triblock copolymers prepared in Example 9, and 100 mg of polyethylene glycol-polyester triblock copolymers prepared in Example 19. (L-lactide) tri-block copolymer 100 mg, the polyethylene glycol-poly(D-lactide) tri-block copolymer prepared in Example 9 and the polyethylene glycol-poly( L-lactide) triblock copolymer 50 mg each were mixed.

Dissolve methotrexate in tetrahydrofuran, the concentration of methotrexate is 0.4mg/mL, stir for 5h, after dissolving, add methotrexate-tetrahydrofuran solution dropwise to the weighed polyethylene glycol ether-polyethylene glycol ether The ester triblock copolymer was configured so that the mass ratio of methotrexate to polyethylene glycol ether-polyester triblock copolymer was 1:5, and the solution was stirred continuously for 2 hours, and the flow rate of the syringe pump was set to 0.1mL/min. (mL) was set to 25 mL and the stirring speed of the stirrer was set to 1000 rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved in methotrexate on a stirrer and stir, and add Milli-Q dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 108 was 22.21%, and the drug-loading efficiency was 89.93%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 109 was 22.27%, and the drug-loading efficiency was 90.42%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 110 was 24.52%, and the drug-loading efficiency was 100%.

Examples 111-113

Polyethylene glycol-polyester triblock copolymers were respectively selected from 100 mg of polyethylene glycol-poly(D-lactide) triblock copolymers prepared in Example 9, and 100 mg of polyethylene glycol-polyester triblock copolymers prepared in Example 19. (L-lactide) tri-block copolymer 100 mg, the polyethylene glycol-poly(D-lactide) tri-block copolymer prepared in Example 9 and the polyethylene glycol-poly( L-lactide) triblock copolymer 50 mg each were mixed.

Dissolve cyclophosphamide in tetrahydrofuran, the concentration of cyclophosphamide is 0.4mg/mL, stir for 5h, after dissolving, add cyclophosphamide-tetrahydrofuran solution dropwise to the weighed polyethylene glycol ether-polyester ternary In the block copolymer, a solution with a mass ratio of cyclophosphamide and polyethylene glycol ether-polyester triblock copolymer of 1:5 was configured and stirred for 2 hours. Set the stirring speed of the stirrer to 1000 rpm at 25 mL. Put the cyclophosphamide-dissolved polyethylene glycol-polyester triblock copolymer solution on a stirrer and stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the drug-loaded polyethylene glycol ether-polyester triblock copolymer nano-micelle with biological activity.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 111 was 52.34%, and the drug-loading efficiency was 90.63%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 112 was 52.41%, and the drug-loading efficiency was 91.46%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 113 was 63.63%, and the drug-loading efficiency was 98.95%.

Examples 114-116

Polyethylene glycol-polyester triblock copolymers were respectively selected from 100 mg of polyethylene glycol-poly(D-lactide) triblock copolymers prepared in Example 9, and 100 mg of polyethylene glycol-polyester triblock copolymers prepared in Example 19. (L-lactide) tri-block copolymer 100 mg, the polyethylene glycol-poly(D-lactide) tri-block copolymer prepared in Example 9 and the polyethylene glycol-poly( L-lactide) triblock copolymer 50 mg each were mixed.

Dissolve 5-fluorouracil in tetrahydrofuran, the concentration of 5-fluorouracil is 0.4mg/mL, stir for 5h, after dissolving, add 5-fluorouracil-tetrahydrofuran solution dropwise to the weighed polyethylene glycol The ether-polyester tri-block copolymer is configured so that the mass ratio of 5-fluorouracil and polyethylene glycol ether-polyester tri-block copolymer is 1:5, and the solution is stirred for 2 hours, and the flow rate of the syringe pump is set to 0.1mL /min, flow rate (mL) is set to 25mL, and the stirring speed of the stirrer is set to 1000rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved in 5-fluorouracil on the stirrer and stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 114 was 43.34%, and the drug-loading efficiency was 84.52%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 115 was 45.63%, and the drug-loading efficiency was 84.63%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 116 was 51.37%, and the drug-loading efficiency was 95.47%.

Examples 117-119

Polyethylene glycol-polyester triblock copolymers were respectively selected from 100 mg of polyethylene glycol-poly(D-lactide) triblock copolymers prepared in Example 9, and 100 mg of polyethylene glycol-polyester triblock copolymers prepared in Example 19. (L-lactide) tri-block copolymer 100 mg, the polyethylene glycol-poly(D-lactide) tri-block copolymer prepared in Example 9 and the polyethylene glycol-poly( L-lactide) triblock copolymer 50 mg each were mixed.

Dissolve docetaxel in tetrahydrofuran, the concentration of docetaxel is 0.4mg/mL, stir for 5h, after dissolving, add the docetaxel-tetrahydrofuran solution dropwise to the weighed polyethylene glycol ether-polyester ternary The mass ratio of docetaxel and polyethylene glycol ether-polyester triblock copolymer is configured in the block copolymer to be respectively 1:20, 1:10, 3:20, 4:20, 5:20 and continue to stir 2h, set the flow rate of the syringe pump to 0.1mL/min, the flow rate (mL) to 25mL, and set the stirring speed of the stirrer to 1000rpm. The polyethylene glycol-polyester triblock copolymer solution in which docetaxel is dissolved is placed on a stirrer and stirred, and Milli-Q is added dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 117 was 14.37%, and the drug-loading efficiency was 84.58%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 118 was 14.47%, and the drug-loading efficiency was 86.54%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 119 was 16.74%, and the drug-loading efficiency was 96.35%.

Examples 120-122

Polyethylene glycol-polyester triblock copolymers were respectively selected from 100 mg of polyethylene glycol-poly(D-lactide) triblock copolymers prepared in Example 9, and 100 mg of polyethylene glycol-polyester triblock copolymers prepared in Example 19. (L-lactide) tri-block copolymer 100 mg, the polyethylene glycol-poly(D-lactide) tri-block copolymer prepared in Example 9 and the polyethylene glycol-poly( L-lactide) triblock copolymer 50 mg each were mixed.

Dissolve daunorubicin in tetrahydrofuran, the concentration of daunorubicin is 0.4mg/mL, stir for 5h, after dissolving, add daunorubicin-tetrahydrofuran solution dropwise to the weighed polyethylene glycol ether-polyethylene glycol ether The mass ratio of daunorubicin and polyethylene glycol ether-polyester triblock copolymer in the ester triblock copolymer is 1:20, 1:10, 3:20, 4:20, 5:20 respectively Continue to stir the solution for 2 h, set the flow rate of the syringe pump to 0.1 mL/min, set the flow rate (mL) to 25 mL, and set the stirring speed of the stirrer to 1000 rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved in daunorubicin on the stirrer, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 120 was 15.36%, and the drug-loading efficiency was 86.35%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 121 was 15.32%, and the drug-loading efficiency was 87.12%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 122 was 16.83%, and the drug-loading efficiency was 97.32%.

Examples 123-125

Polyethylene glycol-polyester triblock copolymers were respectively selected from 100 mg of polyethylene glycol-poly(D-lactide) triblock copolymers prepared in Example 9, and 100 mg of polyethylene glycol-polyester triblock copolymers prepared in Example 19. (L-lactide) tri-block copolymer 100 mg, the polyethylene glycol-poly(D-lactide) tri-block copolymer prepared in Example 9 and the polyethylene glycol-poly( L-lactide) triblock copolymer 50 mg each were mixed.

Dissolve doxorubicin in tetrahydrofuran, the concentration of doxorubicin is 0.4mg/mL, stir for 5h, after dissolving, add doxorubicin-tetrahydrofuran solution dropwise to the weighed polyethylene glycol ether-polyester In the block copolymer, the solution that the mass ratio of doxorubicin to polyethylene glycol ether-polyester triblock copolymer is respectively 1:20, 1:10, 3:20, 4:20, 5:20 is continued to stir 2h, set the flow rate of the syringe pump to 0.1mL/min, the flow rate (mL) to 25mL, and set the stirring speed of the stirrer to 1000rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved in doxorubicin on a stirrer and stir, and use the set syringe pump to add Milli-Q dropwise to the solution at a constant speed. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 123 was 15.23%, and the drug-loading efficiency was 86.34%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 124 was 15.47%, and the drug-loading efficiency was 86.25%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 125 was 17.82%, and the drug-loading efficiency was 96.35%.

Examples 126-128

Polyethylene glycol-polyester triblock copolymers were respectively selected from 100 mg of polyethylene glycol-poly(D-lactide) triblock copolymers prepared in Example 9, and 100 mg of polyethylene glycol-polyester triblock copolymers prepared in Example 19. (L-lactide) tri-block copolymer 100 mg, the polyethylene glycol-poly(D-lactide) tri-block copolymer prepared in Example 9 and the polyethylene glycol-poly( L-lactide) triblock copolymer 50 mg each were mixed.

Dissolve epirubicin in tetrahydrofuran, the concentration of epirubicin is 0.4mg/mL, stir for 5h, after dissolving, add epirubicin-tetrahydrofuran solution dropwise to the weighed polyethylene glycol ether-polyethylene glycol ether The mass ratio of epirubicin and polyethylene glycol ether-polyester triblock copolymer in the ester triblock copolymer is 1:20, 1:10, 3:20, 4:20, 5:20 respectively Continue to stir the solution for 2 h, set the flow rate of the syringe pump to 0.1 mL/min, set the flow rate (mL) to 25 mL, and set the stirring speed of the stirrer to 1000 rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved in epirubicin on a stirrer and stir, and add Milli-Q dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 126 was 15.78%, and the drug-loading efficiency was 86.34%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 127 was 15.62%, and the drug-loading efficiency was 85.96%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 128 was 17.35%, and the drug-loading efficiency was 97.42%.

Examples 129-131

Polyethylene glycol-polyester triblock copolymers were respectively selected from 100 mg of polyethylene glycol-poly(D-lactide) triblock copolymers prepared in Example 9, and 100 mg of polyethylene glycol-polyester triblock copolymers prepared in Example 19. (L-lactide) tri-block copolymer 100 mg, the polyethylene glycol-poly(D-lactide) tri-block copolymer prepared in Example 9 and the polyethylene glycol-poly( L-lactide) triblock copolymer 50 mg each were mixed.

Dissolve pirarubicin in tetrahydrofuran, the concentration of pirarubicin is 0.4mg/mL, stir for 5h, after dissolving, add the pirarubicin-tetrahydrofuran solution dropwise to the weighed polyethylene glycol ether-polyethylene glycol ether The mass ratio of pirarubicin to polyethylene glycol ether-polyester triblock copolymer in the ester triblock copolymer is 1:20, 1:10, 3:20, 4:20, 5:20 respectively Continue to stir the solution for 2 h, set the flow rate of the syringe pump to 0.1 mL/min, set the flow rate (mL) to 25 mL, and set the stirring speed of the stirrer to 1000 rpm. Put the polyethylene glycol-polyester triblock copolymer solution dissolved with pirarubicin on a stirrer and stir, and add Milli-Q dropwise to the solution at a constant speed with a set syringe pump. Continue to stir for 10 h after the dropwise addition, dialyze in Milli-Q with a dialysis bag (MWCO=3500) for 24 h, and change the water more than 5 times. After dialysis, the solution is freeze-dried to obtain the bioactive polyethylene glycol ether-polyester triblock copolymer drug-loaded nano-micelle.

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 129 was 15.31%, and the drug-loading efficiency was 86.34%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 130 was 15.53%, and the drug-loading efficiency was 87.53%;

The encapsulation efficiency of the polyethylene glycol ether-polyester triblock copolymer drug-loaded nanomicelle obtained in Example 131 was 17.83%, and the drug-loading efficiency was 97.36%.

The descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (7)

1. a preparation method for polyethylene glycol ester triblock copolymer medicament-carried nano micelle, comprises the following steps:

(A) mixture of small-molecule drug and organic solvent is added drop-wise in polyethylene glycol ester triblock copolymer, is mixed Close solution；

Described small-molecule drug is methotrexate, 5-fluorouracil, cyclophosphamide, daunorubicin, amycin, epirubicin, pyrrole Soft than star, camptothecin or Ramulus et folium taxi cuspidatae class；

(B) dripping ultra-pure water while carrying out described mixed solution stirring for the first time, after continuing second time stirring, dialysis removes Organic solvent lyophilizing obtain medicament-carried nano micelle；

Shown in described polyethylene glycol ester triblock copolymer such as formula (I),

Wherein ,-R-isOr

M is the degree of polymerization, 10≤m≤250；N is the degree of polymerization, 10≤n≤220.

Preparation method the most according to claim 1, it is characterised in that in described step (A), described small-molecule drug is having Concentration in machine solvent is 0.1～10mg/mL.

Preparation method the most according to claim 1, it is characterised in that in described step (A), described small-molecule drug is with poly- The mass ratio of ethylene glycol-polyester three-block copolymer is 0.01～1.

Preparation method the most according to claim 1, it is characterised in that described first time stirring speed be 100～ 2000rpm。

Preparation method the most according to claim 1, it is characterised in that the speed of described dropping ultra-pure water is 0.05～5mL/ min。

Preparation method the most according to claim 1, it is characterised in that the consumption of described ultra-pure water and consumption of organic solvent Volume ratio is 0.01～20.

Preparation method the most according to claim 1, it is characterised in that described organic solvent is oxolane, Isosorbide-5-Nitrae-dioxy Six rings, dimethyl sulfoxide or N,N-dimethylformamide.