A polymeric vesicle, its preparation method and application in a drug delivery carrier

Abstract

The present invention discloses a polymeric vesicle, a preparation method thereof and an application in a drug delivery carrier. The amphiphilic block polymer vesicle in the present invention is prepared by dissolving an amphiphilic block polymer in methoxypolyethylene glycol with a molecular weight of 200 to 600 and then dropping water thereto. The polymer vesicle has a nanoscale vesicle structure in which a cell membrane-like bilayer formed by hydrophobic interaction of the amphiphilic polymer wraps a hydrophilic cavity, and can load hydrophilic drugs and hydrophobic drugs. The preparation of the polymer vesicle and the drug loading process in the present invention are relatively simple, avoiding the use of toxic and harmful solvents. In addition, the polymer vesicle in the present invention has a high encapsulation efficiency and drug loading capacity, has a high drug release amount in a slightly acidic tumor environment, increases the concentration of the drug reaching the tumor, enhances the inhibitory effect of the drug on the tumor, and improves the therapeutic effect of the drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polymeric vesicle, a preparation method thereof, and an application thereof in a drug delivery carrier. Background Art

[0002] Glioblastoma is a tumor originating from glial cells in neuroepithelial tissue of the brain, growing infiltratively. It is the most common primary intracranial malignant tumor, with a high fatality rate and recurrence rate, and a poor prognosis. The standardized treatment plan is surgical resection combined with postoperative radiotherapy and chemotherapy to kill or inhibit tumor cells and prolong the survival period of patients. Currently, commonly used chemotherapeutic drugs include temozolomide, carmustine, irinotecan, etoposide, cisplatin, carboplatin and other chemotherapeutic drugs. In addition, other drug treatments such as molecular targeting and biological immunotherapy are also constantly developing. However, chemotherapeutic drugs have the disadvantages of poor targeting and selectivity, so they will kill a large number of normal cells and have great side effects. At the same time, chemotherapeutic drugs are difficult to cross the blood-brain barrier to reach an effective drug concentration in the intracranial lesion area, having the disadvantage of low bioavailability.

[0003] Paclitaxel (PTX) is a natural anti-cancer active ingredient extracted from the trunk and bark of Taxus plants. It can effectively inhibit the division of active tumor cells and kill tumor cells. At present, paclitaxel has been widely used in the treatment of various cancers such as breast cancer, ovarian cancer, head and neck cancer and lung cancer. However, it has the disadvantages of poor water solubility and poor stability. Therefore, its wider anti-tumor application is limited. Clinically, paclitaxel injection is mostly used, with polyoxyethylene castor oil and ethanol as solvents to increase the solubility of paclitaxel and improve its stability. However, histamine produced by the degradation of polyoxyethylene castor oil in the body can cause fatal allergic reactions.

[0004] Nanoscale particles are promising drug delivery systems for the treatment of glioblastoma because they not only have the characteristics of long blood circulation, strong tumor penetration and enrichment ability, controllable and targeted drug delivery due to their small volume, but also can reduce the systemic toxicity of anti-cancer drugs. Therefore, an effective way to improve the therapeutic effect of PTX is to find a suitable drug delivery carrier. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this purpose, the present invention provides an amphiphilic block polymer vesicle, a preparation method thereof, and an application thereof in a drug delivery carrier. The amphiphilic block polymer vesicle is prepared by dissolving an amphiphilic block polymer in methoxypolyethylene glycol with a molecular weight of 200 to 600 and then dropping water thereto. The preparation of the polymer vesicle and the loading of the drug in the present invention are relatively simple, avoiding the use of toxic and harmful organic solvents. In addition, the polymer vesicle in the present invention has a high encapsulation efficiency and drug loading capacity, and has a high drug release amount in a slightly acidic tumor environment, which can increase the concentration of the drug reaching the tumor, enhance the inhibitory effect of the drug on the tumor, and improve the therapeutic effect of the drug.

[0006] In a first aspect of the present invention, there is provided a method for preparing an amphiphilic block polymer vesicle, the method comprising the following steps: dissolving an amphiphilic block polymer in methoxypolyethylene glycol with a molecular weight of 200 to 600 and then dropping water thereto to obtain an amphiphilic block polymer vesicle.

[0007] According to the content of the first aspect of the present invention, in some embodiments of the present invention, 0.05 to 0.15 mg of the amphiphilic block polymer is added per microliter of methoxypolyethylene glycol with a molecular weight of 200 to 600.

[0008] In some preferred embodiments of the present invention, the average rate of dropping the water is 100 μL / min to 1000 μL / min.

[0009] In some more preferred embodiments of the present invention, the water is dropped in three portions.

[0010] In some preferred embodiments of the present invention, the amphiphilic block polymer is prepared by reacting methoxypolyethylene glycol with a molecular weight of 1500 to 2500 and a lactone monomer in an organic solvent.

[0011] In some preferred embodiments of the present invention, the lactone monomer includes ε-caprolactone.

[0012] In some preferred embodiments of the present invention, the mass ratio of the methoxypolyethylene glycol with a molecular weight of 1500 to 2500 to the lactone monomer is (4 to 6):(7 to 9).

[0013] In some preferred embodiments of the present invention, the organic solvents used in the preparation process of the amphiphilic block polymer include toluene, cold hexane, and tetrahydrofuran.

[0014] In some preferred embodiments of the present invention, a catalyst is further added in the preparation process of the amphiphilic block polymer.

[0015] In some more preferred embodiments of the present invention, the catalyst comprises methanesulfonic acid, stannous octoate, and stannous isooctoate.

[0016] In some preferred embodiments of the present invention, purification is further included in the preparation process of the amphiphilic block polymer.

[0017] In some more preferred embodiments of the present invention, the purification method is to dissolve the amphiphilic block polymer in one of tetrahydrofuran, chloroform, and dichloromethane and then perform precipitation.

[0018] In some more preferred embodiments of the present invention, precipitation is carried out with one of cold hexane, ether, and cold methanol.

[0019] In the second aspect of the present invention, there is provided a polymer vesicle prepared by the preparation method described in the first aspect of the present invention.

[0020] According to the content of the second aspect of the present invention, in some embodiments of the present invention, the particle size of the polymer vesicle is 100 nm to 200 nm.

[0021] In some preferred embodiments of the present invention, the Zeta potential of the polymer vesicle is -7 to -10 mV.

[0022] In the third aspect of the present invention, there is provided an application of the polymer vesicle described in the second aspect of the present invention in a drug delivery carrier.

[0023] According to the content of the third aspect of the present invention, in some embodiments of the present invention, the drug is an anti-tumor drug.

[0024] In some preferred embodiments of the present invention, the tumor includes any one of glioma, lung cancer, breast cancer, ovarian cancer, gastric cancer, and pancreatic cancer.

[0025] In some more preferred embodiments of the present invention, the anti-tumor drug includes any one of doxorubicin hydrochloride, gemcitabine, temozolomide, camptothecin, doxorubicin, erlotinib, and paclitaxel.

[0026] In some preferred embodiments of the present invention, the drug loading method is to add the drug and the amphiphilic block polymer together to methoxypolyethylene glycol with a molecular weight of 200 to 600 during the preparation process of the polymer vesicle.

[0027] In some preferred embodiments of the present invention, the mass ratio of the drug to the amphiphilic block polymer is 1:(15 - 25).

[0028] In some preferred embodiments of the present invention, the drug loading amount of the drug is 2 - 3%.

[0029] In some preferred embodiments of the present invention, the particle size of the polymeric vesicles is 100 nm to 200 nm.

[0030] In some preferred embodiments of the present invention, the Zeta potential of the polymeric vesicles is -7 to -10 mV.

[0031] In some preferred embodiments of the present invention, the polymeric vesicles have a bilayer membrane structure.

[0032] In some preferred embodiments of the present invention, the thickness of the bilayer membrane is 15 to 25 nm.

[0033] The polymeric vesicles in the present invention have a nanoscale vesicle structure in which an amphiphilic polymer forms a cell membrane-like bilayer to wrap a hydrophilic cavity through hydrophobic interactions. They can load hydrophilic drugs and hydrophobic drugs. When the drug is hydrophobic, the drug is embedded in the hydrophobic bilayer membrane of the polymeric vesicles during the formation process of the polymeric vesicles. When the drug is hydrophilic, the drug is embedded in the hydrophilic cavity of the polymeric vesicles during the formation process of the polymeric vesicles.

[0034] The beneficial effects of the present invention are as follows:

[0035] (1) The preparation of the polymeric vesicles and the drug loading process in the present invention are relatively simple, avoiding the use of toxic and harmful organic solvents. In addition, the polymeric vesicles in the present invention have a high encapsulation efficiency and drug loading capacity, and have a high drug release amount in a slightly acidic tumor environment, which can increase the concentration of the drug reaching the tumor, enhance the inhibitory effect of the drug on the tumor, and improve the therapeutic effect of the drug.

[0036] (2) The polymeric vesicles are formed by the direct hydration method in the present invention. The polymeric vesicles in the present invention have a nanoscale vesicle structure in which an amphiphilic polymer forms a cell membrane-like bilayer to wrap a hydrophilic cavity through hydrophobic interactions. They can load hydrophilic drugs and hydrophobic drugs. When the drug is hydrophobic, the drug is embedded in the hydrophobic bilayer membrane of the polymeric vesicles during the formation process of the polymeric vesicles. When the drug is hydrophilic, the drug is embedded in the hydrophilic cavity of the polymeric vesicles during the formation process of the polymeric vesicles. Description of the Drawings

[0037] Figure 1 It is the reaction process of the amphiphilic block polymer in the example;

[0038] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of the amphiphilic block polymer prepared in the example;

[0039] Figure 3 It is the gel permeation chromatography elution curve of the amphiphilic block polymer prepared in the example;

[0040] Figure 4 Particle size diagrams of the polymer vesicles and the polymer vesicles loaded with paclitaxel prepared in the examples;

[0041] Figure 5 Particle size diagram of the polymer vesicles loaded with paclitaxel prepared in the comparative example;

[0042] Figure 6 Transmission electron microscopy images of the polymer vesicles and the polymer vesicles loaded with paclitaxel prepared in the examples;

[0043] Figure 7 Quantitative analysis diagram of the transmission electron microscopy images of the polymer vesicles and the polymer vesicles loaded with paclitaxel prepared in the examples;

[0044] Figure 8 In vitro drug release curves of free paclitaxel, paclitaxel liposomes for clinical use, and polymer vesicles loaded with paclitaxel in an environment with a pH value of 7.4;

[0045] Figure 9 In vitro drug release curves of the polymer vesicles loaded with paclitaxel in environments with a pH value of 7.4 and a pH value of 5;

[0046] Figure 10 Statistical chart of the survival rate of mouse glioblastoma cells in the blank control group and the experimental group;

[0047] Figure 11 Relative luminescence intensity diagram of mouse glioblastoma cells in the experimental group and the blank control group;

[0048] Figure 12 Flow cytometry apoptosis diagram of mouse glioblastoma cells in the blank control group;

[0049] Figure 13 Flow cytometry apoptosis diagram of mouse glioblastoma cells in the free paclitaxel group;

[0050] Figure 14 Flow cytometry apoptosis diagram of mouse glioblastoma cells in the paclitaxel liposomes for clinical use group;

[0051] Figure 15 Flow cytometry apoptosis diagram of mouse glioblastoma cells in the polymer vesicles loaded with paclitaxel group;

[0052] Figure 16 Fluorescence diagrams at different time points after treating mouse glioblastoma cells with free coumarin-6 and polymer vesicles loaded with coumarin-6;

[0053] Figure 17 Fluorescence detection diagram of reactive oxygen species in cells in the experimental group and the blank control group;

[0054] Figure 18 are the fluorescence detection and quantitative analysis results of reactive oxygen species in the experimental group and the blank control group;

[0055] Figure 19 are the fluorescence images of free paclitaxel, paclitaxel-loaded polymeric vesicles, and clinical paclitaxel liposomes affecting the cytoskeleton;

[0056] Figure 20 are the electrophoresis band diagrams of B-cell lymphoma-2 gene, cell proliferation marker Ki-67, and P-glycoprotein in the experimental group and the blank control group;

[0057] Figure 21 Quantitative analysis results of the electrophoresis band diagrams of B-cell lymphoma-2 gene, cell proliferation marker Ki-67, and P-glycoprotein in the experimental group and the blank control group;

[0058] Figure 22 are the growth conditions of mouse glioma in the blank control group (PBS) and the experimental group after administration. Specific implementation manners

[0059] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

[0060] The test materials and reagents used in the following examples can be obtained from commercial channels without special instructions.

[0061] Example

[0062] (1) Amphiphilic block polymer PEG 2k -b-PCL 4.3k (PEG 2000 -b-PCL 4300 ) Preparation: Dissolve 5 g of dry methoxypolyethylene glycol (MeO-PEG 44 -OH) with a molecular weight of 2000 and 8 g of ε-caprolactone (ε-CL, 99%, Sigma-Aldrich) in 500 mL of toluene, equilibrate at 30 °C for 10 minutes, then add 170 μL of methanesulfonic acid (MSA), and stir at 30 °C for 2.5 hours. After cooling to 25 °C, add A21 macroporous resin to adsorb the anions dissociated by MSA, and filter to remove A 21 macroporous resin, and the obtained product was precipitated in 300 mL of cold hexane to obtain a crude product of amphiphilic block polymer PEG 2k -b-PCL 4.3k The obtained crude product was dissolved in 100 mL of tetrahydrofuran solvent and precipitated twice again with 300 mL of cold hexane to obtain the final amphiphilic block polymer PEG 2k -b-PCL 4.3k The reaction process was as Figure 1 shown

[0063] (2) Preparation of polymer vesicles: 10 mg of PEG 2k -b-PCL 4.3k prepared in step (1) was placed in a 1.5 mL centrifuge tube, 100 μL of methoxypolyethylene glycol (MeO-PEG 11 -OH) with a molecular weight of 550 was added, and it was stirred at 60 °C for 20 min to dissolve completely. The stirring speed was 300 rpm. After cooling to 50 °C, 100 μL of ultrapure water was added dropwise at an average rate of 500 μL / min and stirred for 30 min. Then, 200 μL of ultrapure water was added dropwise at an average rate of 500 μL / min and stirred for 30 min. Finally, 700 μL of ultrapure water was added dropwise at an average rate of 500 μL / min and stirred for 30 min. The prepared polymer vesicles were denoted as PS

[0064] (3) Preparation of polymer vesicles loaded with paclitaxel (PTX): 0.5 mg of PTX and 10 mg of PEG 2k -b-PCL 4.3k prepared in step (1) were placed in a 1.5 mL centrifuge tube, 100 μL of methoxypolyethylene glycol (MeO-PEG 11 -OH) with a molecular weight of 550 was added, and it was stirred at 60 °C for 20 min to dissolve completely. The stirring speed was 300 rpm. After cooling to 50 °C, 100 μL of ultrapure water was added dropwise at an average rate of 500 μL / min and stirred for 30 min. Then, 200 μL of ultrapure water was added dropwise at an average rate of 500 μL / min and stirred for 30 min. Finally, 700 μL of ultrapure water was added dropwise at an average rate of 500 μL / min and stirred for 30 min. The obtained solution was centrifuged at 3500 rpm for 20 min using a 100 kDa ultrafiltration centrifuge tube to remove the free PTX that was not loaded into the polymer vesicles. The prepared polymer vesicles loaded with PTX were denoted as PTX@PS

[0065] Comparative example

[0066] In the comparative example, PEG 2k -b-PCL 4.3kThe preparation method is the same as that of the example. The specific preparation steps of the polymer vesicles loaded with PTX in the comparative example are as follows: Weigh 10 mg of PEG 2k -b-PCL 4.3k and 0.5 mg of PTX into a 10 mL centrifuge tube, add 6 mL of chloroform to dissolve them completely, transfer to a round-bottomed flask, and use the rotary evaporation under reduced pressure method to make a uniform film. Then add 10 mL of double-distilled water, and place it in a probe sonicator to sonicate for 20 min at a power of 200 W, and thus obtain the polymer vesicle dispersion loaded with PTX in the comparative example. Centrifuge for 20 min at a speed of 3500 rpm using a 100 kDa ultracentrifugation tube to remove the free drug PTX that has not entered the polymer vesicles. The prepared polymer vesicles loaded with PTX are denoted as D-PTX@PS.

[0067] I. Basic Characterization

[0068] (1) Nuclear magnetic resonance analysis:

[0069] The 1H nuclear magnetic resonance spectrum of the amphiphilic block copolymer prepared in the example is as shown in Figure 2 . The nuclear magnetic resonance result is NMR(400 MHz, CDCl3): δ 4.01 (t, J = 6.6 Hz, 2H), 3.59 (s, 4H), 2.26 (t, J = 7.5 Hz, 2H), 1.59 (ddd, J = 9.8, 7.3, 3.0 Hz, 4H), 1.44–1.26 (m, 2H), indicating that the amphiphilic block copolymer PEG 2k -b-PCL 4.3k is prepared in the example of the present invention.

[0070] (2) Gel permeation chromatography (GPC) analysis:

[0071] The gel permeation chromatography determination results of the amphiphilic block copolymer are as shown in Figure 3 . It can be seen from Figure 3 that the elution peak in the polymer elution curve is a single peak and the distribution is relatively narrow, indicating that the polymer is a single block copolymer and there is no unreacted PCL and PEG, and the purity is relatively high. Further analysis and calculation from the figure show that the weight-average molecular weight Mw of the polymer is 5577, close to the theoretical molecular weight of 6300, and the polydispersity index PDI is 1.3696, between 1 and 2, indicating that the prepared PEG 2k -b-PCL 4.3k block copolymer has a relatively narrow molecular weight distribution and is relatively uniform.

[0072] (3) Analysis of particle size and Zeta potential:

[0073] The polymer vesicles (PS) and PTX-loaded polymer vesicles (PTX@PS) prepared in the embodiment of the present invention and the PTX-loaded polymer vesicles (D-PTX@PS) prepared in the comparative example were diluted 10 times with ultrapure water and the particle sizes and zeta potentials of PS, PTX@PS and D-PTX@PS were measured on a Malvern nanometer Zetasizer Nano S90. The test results of the particle sizes of PS and PTX@PS are shown in Figure 2. Figure 4 As shown, the test results of the particle size of D-PTX@PS in the comparative example are as follows Figure 5 As shown in Table 1, the average particle size and Zeta potential of PS, PTX@PS and D-PTX@PS are statistically shown.

[0074] Table 1 Average particle size and Zeta potential of PS, PTX@PS and D-PTX@PS

[0075]

[0076] As can be seen from Table 1, the average particle size of the PS in the embodiment of the present invention is (118.3 ± 0.97) nm, the average particle size of the PTX@PS prepared in the embodiment is (136.5 ± 0.44) nm, and the average particle size of the D-PTX@PS prepared in the comparative example is (625.9 ± 48.51), indicating that the particle size of the drug-loaded polymer vesicles prepared using the chloroform organic solvent in the comparative example is larger; in addition, the dispersion index (PDI) of the PS and the PTX@PS in the embodiment is between 0.10 and 0.17, while the PDI of the D-PTX@PS in the comparative example is about 0.918, indicating that the particle size distribution of the PS and PTX@PS prepared in the embodiment of the present invention is relatively uniform, while the particle size distribution of the D-PTX@PS prepared in the comparative example is uneven. And the smaller the particle size of the prepared polymer vesicles, the more conducive it is to escape the recognition and clearance of the reticuloendothelial system (RES), which can prolong its circulation time in the body, and is conducive to the uptake and endocytosis of the drug by tumor cells. The higher the absolute value of the Zeta potential, the more stable the prepared nanosystem. As can be seen from Table 1, the absolute value of the Zeta potential of the D-PTX@PS prepared in Comparative Example 1 is lower than that of the drug-loaded polymer vesicles prepared by the direct hydration method in the embodiment, indicating that the stability of the D-PTX@PS prepared in Comparative Example 1 is poor.

[0077] (4) Morphology analysis

[0078] The morphology of PS and PTX@PS was analyzed using a transmission electron microscope. 10 μL of the prepared PS solution or PTX@PS solution was dropped onto a copper grid, air-dried, and then stained with 2.5% phosphotungstic acid. The morphology of PS and PTX@PS was observed using a transmission electron microscope. Figure 6are the transmission electron micrographs of PS and PTX@PS. As can be seen from Figure 6 , PS has a nanoscale vesicle structure with a cell membrane-like bilayer wrapping a hydrophilic cavity. Among them, the cavity in PS is hydrophilic and can be used to load hydrophilic drugs, and the bilayer membrane is hydrophobic and can be used to load hydrophobic drugs. After loading PTX, the thickness between the bilayer membranes of the polymeric vesicle PS increases, indicating that PTX is loaded between the bilayer membranes of PS, and the distribution of the drug PTX is also observed between the bilayer membranes of PTX@PS. Quantitative analysis of the obtained transmission electron micrographs of PS and PTX@PS gives the sizes of the cavities and the thicknesses between the bilayer membranes of PS and PTX@PS. The results are as Figure 7 shown, where Figure 7 a is the thickness between the bilayer membranes of PS and PTX@PS, Figure 7 b is the size of the cavity of PS and PTX@PS. As can also be seen from Figure 7 , after loading PTX, the cavity size of the polymeric vesicle PS decreases and the thickness between the bilayer membranes increases, indicating that PTX is loaded between the bilayer membranes of PS.

[0079] II. Determination of Encapsulation Efficiency and Drug Loading

[0080] The absorbance values of PTX at different concentrations (5 - 30 μg / mL) at 227 nm were measured respectively, and the standard concentration curve of the absorbance values of PTX was plotted. 20 μL of PTX@PS and 20 μL of D-PTX@PS were dissolved in 180 μL of methanol respectively. After dissolution, their absorbance at 227 nm was detected by an ultraviolet spectrophotometer. According to the measured absorbance values and the plotted standard concentration curve of PTX, the concentrations of PTX in PTX@PS and D-PTX@PS were obtained, and the encapsulation efficiency and drug loading of PTX@PS and D-PTX@PS were calculated.

[0081]

[0082]

[0083] According to the above formula, the drug loading of PTX@PS in the examples was calculated to be (2.08 ± 0.14)%, and the encapsulation efficiency was (39.93 ± 2.43)%. The drug loading of D-PTX@PS in the comparative examples was (1.95 ± 0.31)%, and the encapsulation efficiency was (28.82 ± 4.63)%. It shows that the drug-loaded polymeric vesicles prepared by the direct hydration method in the examples of the present invention have higher drug loading and encapsulation efficiency.

[0084] III. In Vitro Drug Release Behavior

[0085] 500 μL of PTX@PS was put into a dialysis bag (molecular weight cut-off: 3500 Da), sealed with a clip, and placed in 40 mL of phosphate buffer solution release medium with a pH of 7.4 to simulate the drug release environment in body fluids. It was placed in a shaker at 37 °C and 100 rpm. Starting from when the shaker began to work, at different time points (0, 0.5, 2.5, 4.0, 6.0, 9.0, 12.0, 24.0, 56.0, 100.0, 150.0, 200.0, 250.0, 300.0, 350.0, 400.0, 450.0 h), 10 mL of dialysis fluid was taken out, and 10 mL of phosphate buffer solution was added to the dialysis bag. The concentration of the drug PTX in the taken-out dialysis fluid was measured by an ultraviolet spectrophotometer, and the drug release curve of PTX@PS was plotted. Free PTX and PTX@LS were obtained with the same method to get their drug release curves. Among them, PTX@LS is paclitaxel liposome, which is a microvesicle formed by encapsulating PTX in a lipid bilayer. It is a clinical drug purchased from Nanjing Luye Pharmaceutical Co., Ltd. The content of PTX in PTX@LS is 3 mg / mL. Figure 8 Figure 2 is the in vitro drug release curves of free PTX, PTX@LS, and PTX@PS in an environment with a pH of 7.4. Among them, Figure 8 a is the in vitro drug release curve of free PTX, Figure 8 b is the in vitro drug release curves of PTX@LS and PTX@PS. It can be seen from Figure 8 Figure 2 that free PTX was completely released within 60 min, while PTX@LS and PTX@PS enabled PTX to be released in a controllable and slow continuous manner. Moreover, compared with PTX@LS, the drug release time of PTX@PS was significantly prolonged, and the cumulative drug release amount was also higher than that of PTX@LS. Within the first 100 hours, the cumulative drug release percentage of PTX@PS was (54.65 ± 2.15)%, while the cumulative drug release percentage of PTX@LS was (42.61 ± 5.69)%. The higher cumulative drug release percentage and longer drug release time are helpful for PTX to inhibit the growth of tumor cells for a long time. In addition, it can be seen from the slope of the drug release curves in Figure 8 Figure 2 that within the first 80 h, the drug release rate of PTX@PS was faster than that of PTX@LS, which is helpful for PTX to inhibit the growth of tumor cells in a short time. After that, within 80 - 100 h, the drug release rate of PTX@PS was slower than that of PTX@LS, which provided a guarantee for PTX to continuously play an anti-tumor role.

[0086] In addition, the drug release curve of PTX@PS in an environment with a pH of 5 was detected by the same method. The environment with a pH of 5 simulated the acidic microenvironment of glioma. Figure 9In vitro drug release curves of PTX@PS at pH 7.4 and pH 5. As can be seen from Figure 9 , within the first 150 h, the drug release rate of PTX@PS under the condition of pH 7.4 was significantly faster than that under the condition of pH 5. However, in the final acidic environment of pH 5, the cumulative drug release percentage of PTX@PS was higher and the release time was longer. The cumulative drug release percentages of PTX@PS under the conditions of final pH 5 and pH 7.4 were (79.79±5.89)% and (63.39±1.46)% respectively. The drug release characteristics of PTX in PTX@PS in body fluid (pH 7.4) and the acidic microenvironment of glioma (pH 5) can prolong the concentration and time of PTX aggregation at the tumor site, thereby more effectively inhibiting tumor growth and better killing tumors.

[0087] IV. Anti-mouse glioblastoma cell effect

[0088] 1) Inhibitory effect on mouse glioblastoma cells

[0089] The inhibitory effects of free PTX and PTX@PS on mouse glioblastoma cells (GL261 cells) were studied. GL261 cells were inoculated into 96-well plates at a density of 5×10 3 cells / well and cultured in an incubator at 37°C and 5% CO2 for 24 hours. Then, according to the concentration of PTX, the experiment was divided into ten groups. PTX@PS was diluted into different concentrations (calculated by the concentration of PTX) using DMEM medium. The concentrations of PTX were 0, 0.05, 0.2, 0.5, 0.8, 1, 5, 10, 20, 50 μg / mL respectively. Each group had 8 replicate wells. Among them, the group with a PTX concentration of 0 was used as the blank control group, and the other concentrations were used as experimental groups. Different concentrations of PTX@PS were added to the corresponding wells respectively. After culturing in an incubator at 37°C and 5% CO2 for 24 hours, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT solution) with a concentration of 5 mg / mL was added to each well. After continuing to culture for 4 h, the medium was removed, and 200 μL of dimethyl sulfoxide (DMSO) was added to each well. It was oscillated in a micro oscillator for 5 min to fully dissolve the crystals. The absorbance value (i.e., OD value) of each well at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, the results were recorded, and the cell survival rate was calculated to characterize the effect of PTX@PS on the survival rate of GL261 cells. The effect of free PTX on the survival rate of GL261 cells was measured using the same procedure. Taking the group as the abscissa and the cell survival rate as the ordinate, a graph was plotted, and the effects of PTX and PTX@PS on the proliferation of GL261 cells were analyzed according to the cell survival rate. The results are as Figure 10 shown. Figure 10Statistical graph of the survival rate of GL261 cells in the blank control group and the experimental group. It can be seen from Figure 10 that under the same PTX concentration, the inhibitory effects of PTX@PS and PTX on GL261 cells are similar, indicating that encapsulating the drug PTX in polymer vesicles does not affect the inhibitory effect of the drug PTX on GL261 cells.

[0090] The calculation formula for the cell survival rate is:

[0091] where the blank OD is the OD value in the well with the same operation procedure as the test well but without cells.

[0092] 2) Luciferase detection experiment

[0093] GL261 cells expressing luciferase (GL261.luc+) were seeded in a 96-well black culture plate at a seeding density of 5×10 3 cells / well and cultured at 37°C and 5% CO2 for 24 hours. According to the concentration of PTX, different series of gradient concentrations of free PTX, PTX@PS, and PTX@LP were prepared using complete medium (DMEM medium containing 10% fetal bovine serum by volume). Among them, the series of gradient concentrations were 0, 5, 20, and 50 μg / mL respectively. Among them, the group with a concentration of 0 was used as the blank control group, and the groups with the remaining concentrations were used as the experimental groups. 200 μL of the above-prepared solutions were added to the corresponding wells respectively, and the cells were cultured for another 24 h. After removing the medium, 200 μL of the working solution of D-luciferin (150 μg / mL) diluted with DMEM medium was added to each well, and the cells were cultured for another 15 min and then the luminescence was detected using a microplate reader. Among them, the intensity of luminescence is positively correlated with cell viability. A graph was plotted with the grouping as the abscissa and the relative luminescence intensity as the ordinate. Among them, the calculation formula for the relative luminescence intensity is:

[0094] where the blank CD is the CD value in the well with the same operation procedure as the test well but without cells.

[0095] The inhibitory effect on GL261 cells was characterized by the calculated relative luminescence intensity, and the results are as Figure 11 shown. Figure 11 Relative luminescence intensity of GL261 cells in the experimental group and the blank control group. It can be seen from Figure 11It can be seen that at the same PTX concentration, the inhibitory effect of PTX@PS on GL261 cells is better than that of PTX@LS, and the inhibitory effect of PTX@PS on GL261 cells is comparable to that of free PTX. This indicates that PTX@PS in the embodiments of the present invention has a better inhibitory effect on GL261 cells in vitro than PTX@LS used clinically. This may be because the drug release rate of PTX@PS is faster than that of PTX@LS in a short time, and the drug release is more complete. The above results show that compared with liposome carriers, polymer vesicles can enable PTX to fully exert its inhibitory effect on tumors and have better application potential.

[0096] 3) Apoptosis assay

[0097] GL261 cells were seeded in 6-well plates at a density of 1×10 5 cells / well and cultured at 37 °C and 5% CO2 for 24 hours. Then, 2 mL of complete medium containing PTX@LP, PTX@PS, and free PTX (at a concentration of 5 μg / mL in terms of PTX) was added as the experimental group, and only 2 mL of complete medium was added to the blank control group. The cells were cultured for another 24 hours. An AnnexinV-FITC / PI apoptosis detection kit was used to detect the apoptosis of tumor cells. First, after the culture was completed, the cells were digested with trypsin, and the digested cells were centrifuged at 1000 rpm for 3 min. The cells were washed twice with phosphate buffer solution and resuspended with 400 μL of binding buffer (from the Annexin V-FITC apoptosis detection kit). The resuspended cells were incubated with 5 μL of annexin V-FITC (from the Annexin V-FITC apoptosis detection kit) on ice for 15 min, and then 10 μL of PI (from the Annexin V-FITC apoptosis detection kit) was added and incubated for another 5 min. The samples were analyzed by flow cytometry, and the data collected were analyzed using FlowJo software. The cells were classified into intact cells V-FITC(-) / PI(-) (Q4), early apoptotic cells V-FITC(+) / PI(-) (Q3), late apoptotic cells V-FITC(+) / PI(+) (Q2), and necrotic cells V-FITC(-) / PI(+) (Q1). The results are as Figures 12 - 15 shown. It can be seen from Figures 12 - 15 that the proportion of apoptotic cells, the number of early apoptotic cells, and the number of late apoptotic cells in the cells cultured with DMEM medium containing PTX@PS are higher than those in the cells cultured with DMEM medium containing PTX@LP. The above results indicate that at the same PTX concentration, PTX@PS can better promote the apoptosis of GL261 cells.

[0098] 4) Cell uptake experiment

[0099] Coumarin-6 was encapsulated for labeling PS, and the uptake behavior of the drug by tumor cells was observed through fluorescence. Take a clean cell slide and soak it in ethanol with a concentration of 95% for 5 minutes, dry and sterilize it under an alcohol lamp, and place it in a 12-well plate at room temperature. Inoculate GL261 cells on the slide in the 12-well plate, and the inoculation density is 1.0×10 5 cells / well. Add 1 mL of complete medium (DMEM medium containing 10% fetal bovine serum by volume percentage) to each well and culture it for 24 h under the conditions of 37 °C and 5% CO2. Subsequently, add free coumarin-6 with a coumarin-6 concentration of 300 ng / mL and coumarin-6-loaded PS (coumarin-6@PS), and the culture times are 0.5 h, 2 h, and 4 h respectively. After reaching the specified culture time, discard the medium, wash the cells 3 times with phosphate buffer solution, then add DMEM medium containing 60 nM lysosomal red fluorescent probe (Lyso-Tracker Red), and continue to incubate at 37 °C for 30 min, and wash the cells 3 times with phosphate buffer solution. Transfer the slide to a glass slide, and observe the uptake of coumarin-6@PS and free coumarin-6 by cells through a laser scanning super-resolution microscope.

[0100] Most drug-loaded nanoparticles need to be taken up and phagocytosed by cells and then enter lysosomes. However, the action target of PTX is tubulin in the cytoplasm, so it is very meaningful to achieve lysosomal escape of PTX@PS. In the examples of the present invention, Lyso-Tracker Red (red) was used to localize lysosomes, and coumarin-6 (green) was used to localize PTX@PS. The results are as Figure 16 shown. As can be seen from Figure 16 , compared with free coumarin-6, there is a large overlap between coumarin-6 and lysosomes in coumarin-6@PS (yellow fluorescence in the figure, which is the overlap of green fluorescence and red fluorescence). This may be because PTX@PS mainly enters cells through the lysosomal pathway by endocytosis, while free PTX enters cells by passive diffusion. In addition, it can be seen from the figure that as time increases, the intensity of green fluorescence in the cytoplasm gradually increases, indicating that as the culture time increases, PTX@PS not only accumulates in lysosomes, but gradually distributes in the cytoplasm, indicating that a large amount of PTX@PS escapes from lysosomes and is released into the cytoplasm, so that PTX@PS is finally localized in the cytoplasm. At the same time, lysosomes may serve as effective targets for PTX@PS to exert its antitumor effect. For example, the polymer vesicles in PTX@PS may cause tumor cell death by swelling lysosomes. The above results show that the construction of polymer vesicles can enhance the ability of drugs to be taken up by cells and exhibit good lysosomal escape ability, which is beneficial to improving the drug delivery efficiency to tumors.

[0101] 5) Reactive oxygen species detection experiment

[0102] GL261 cells were seeded in 24-well plates at a seeding density of 5×10 4 cells / well and cultured for 24 h at 37 °C under 5% CO2. After adding 500 μL of complete medium containing PTX@LP, PTX@PS, and free PTX (at a concentration of 5 μg / mL in terms of PTX), the cells were cultured for another 24 h as the experimental group. In the blank control group, only 500 μL of complete medium was added. Reactive oxygen species in the cells were detected according to the operation instructions in the reactive oxygen species detection kit, which was purchased from Beyotime Biotechnology. The fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA) (from the above-mentioned reactive oxygen species detection kit) was diluted with serum-free DMEM medium at a volume ratio of 1:1000, and the probe loading time was 30 min. The fluorescence of the cells was photographed with a laser scanning super-resolution microscope.

[0103] Intracellular mitochondria produce oxidants, namely reactive oxygen species (ROS), including hydrogen peroxide (H2O2), oxygen free radicals (·O 2- ) and hydroxyl radicals (·OH). When the cellular redox balance is disrupted, the level of ROS increases significantly. After exceeding a certain threshold, ROS can act as an upstream signal regulator to trigger oxidative stress, and ultimately induce apoptosis through multiple pathways, such as damaging cell membrane lipids, proteins, and nucleic acids, and can also regulate the expression of cell-related genes, such as Bcl-xL and B-cell lymphoma-2 gene (Bcl-2), resulting in enhanced cytotoxicity of anticancer drugs, thereby inducing apoptosis of cancer cells. The fluorescence detection and quantitative analysis of cellular ROS in the experimental group and the blank control group are shown in Figure 17 and Figure 18 . Figure 17 Figure shows the fluorescence detection of cellular reactive oxygen species in the experimental group and the blank control group. Figure 18 Figure shows the quantitative analysis results of the fluorescence detection of cellular reactive oxygen species in the experimental group and the blank control group. As can be seen from Figure 17 and 18 , the DCF fluorescence intensity of GL261 cells treated with PTX@PS was (24.37±7.13)%, which was significantly higher than that of the free PTX group (P < 0.05) and the PTX@LS group (P < 0.01), indicating that PTX@PS can effectively promote the production of ROS in GL261 cells, thereby inducing apoptosis and necrosis of cells and enhancing the antitumor effect of PTX. Thus, it can be seen that redox imbalance is one of the important mechanisms for PTX@PS to induce apoptosis in tumor cells.

[0104] 6) α-tubulin fluorescence staining experiment

[0105] GL261 cells were seeded into an 8-well chamber (Nunc TM Lab-Tek TM II, Thermo Fisher Scientific, USA) at a seeding density of 5×10 4 cells per well. After culturing for 24 hours at 37°C and 5% CO2, 400 μL of complete medium containing PTX@LP, PTX@PS, and free PTX (at a concentration of 5 μg / mL in terms of PTX) was added and the cells were further cultured for 24 h as the experimental group. In the blank control group, only 400 μL of complete medium was added. After culturing for another 24 h, the medium was removed. 400 μL of 4% paraformaldehyde solution was added to each well and the cells were fixed on ice for 30 min, stabilized at 25°C for 10 min. Then, 0.1% Triton X-100 pre-warmed at 37°C was added to each well and incubated for 15 min. The cells were washed 3 times with phosphate buffer solution. 1% bovine serum albumin (BAS) (BAS was diluted with PBST, and PBST is phosphate buffer solution containing 0.1% (v / v) Tween 20) was added and the cells were blocked at 25°C for 15 min. Then, 200 μL of primary antibody (α-tubulin antibody at 1 μg / mL) was added and incubated at 37°C for 2 h. The cells were washed 3 times with phosphate buffer solution. Then, 200 μL of corresponding secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG (H+L)) and phalloidin-fluorescein (both diluted with antibody diluent at a volume ratio of 1:1000) were added and co-incubated at 37°C for 1 h. Then, 200 μL of 4',6-diamidino-2-phenylindole (DAPI) was added and incubated at 37°C for 30 min. Subsequently, the cells were washed 3 times with phosphate buffer solution and immediately observed using a laser scanning confocal microscope (FV3000, Olympus, JPN), and the images were analyzed using Image J software.

[0106] PTX binds to tubulin, stabilizes microtubules by inhibiting microtubule depolymerization, impairs the formation of the mitotic spindle, arrests tumor cells at the mitotic division phase, and finally induces apoptosis of tumor cells. The effects of PTX@LS, PTX@PS, and free PTX on the nucleus, α-tubblin, and β-actin were studied by fluorescence co-localization of the nucleus and α-tubulin. The results are as Figure 19 shown. Figure 19Green represents the distribution of α-tubblin, red represents the distribution of β-actin, and blue represents the distribution of the nucleus. It can be seen from the figure that the nuclei in the blank control group are all in a complete form, while in the experimental group, the chromatin condenses and is randomly dispersed. The nuclei in the experimental group show varying degrees of fragmented and densely stained masses, indicating that all experimental groups exhibit chromatin condensation in the nuclei during the M arrest phase, and the microtubulin and microfilament proteins are more dense and significantly concentrated at the cell edge, suggesting that PTX can significantly promote the stability of microtubules and microfilaments. For the localization of the nucleus, it can be seen that the distribution of the nuclei in the cells treated with PTX@PS is more compact, and the cells cannot undergo mitosis, so the cells will initiate programmed cell death, that is, apoptosis, further indicating that PTX@PS can maintain the pharmacological activity of PTX-induced apoptosis.

[0107] In addition, the cell microtubules in the cells treated with PTX@PS are more dense and complete, indicating that its effect on enhancing microtubule stability is significantly better than that of free PTX and the PTX@LS group, further demonstrating that the PTX@PS system can effectively enhance the pharmacological activity of PTX, and the polymer vesicle system may be a promising delivery platform for paclitaxel in anti-tumor therapy.

[0108] 7) Western blot

[0109] Protein blot analysis was performed on GL261 cells, and the Western blot was used to detect the effects of the use of PTX@PS on the expression of the proliferation marker protein ki-67, the apoptosis marker protein Bcl-2, and the drug resistance marker protein P-gp, in order to explore the anti-tumor mechanism of PTX@PS.

[0110] GL261 cells were seeded in six-well plates at a seeding density of 2×10 5Cells per well were cultured for 24 h at 37 °C under 5% CO2. After adding 2 mL of complete medium containing PTX@LS, PTX@PS, and free PTX (at a concentration of 5 μg / mL in terms of PTX), the culture was continued for 24 h as the experimental group. In the blank control group, only 2 mL of complete medium was added. After the culture ended, the above cells were collected, and the total proteins of GL261 cells in the experimental group and the blank control group were extracted and prepared by adding 5×SDS (purchased from Beyotime Biotechnology) loading buffer. The prepared samples were subjected to vertical polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis), where the initial voltage of electrophoresis was 60 V, the separation voltage was 100 V, and electrophoresis was stopped when the bromophenol blue migrated to 1 cm from the bottom of the polyacrylamide gel electrophoresis gel (PAGE gel). Then, wet transfer was performed (transfer conditions: 250 mA, 90 min). After transfer, the membrane was blocked with a 5% BSA solution at 25 °C for 30 min, and 3 mL of diluted primary antibodies (Anti-Bcl antibody, Anti-MDR1 antibody, Anti-Ki67 antibody, and Anti-β-Actin antibody, all antibodies were diluted with antibody diluent at a volume ratio of 1:1000) were added and incubated at 4 °C for 24 h. After incubation, the primary antibodies were recovered, and the membrane was washed 3 times with 1X TBST (purchased from Beyotime Biotechnology), 5 min each time. Then, it was blocked with 5% BSA for 30 min, 3 mL of the corresponding secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG (H+L) or horseradish peroxidase-labeled goat anti-rabbit IgG (H+L)) was added and incubated at 25 °C for 1 h, and the membrane was washed 3 times with 1X TBST. An enhanced chemiluminescence reagent (ECL) developer was used for development on a chemiluminescence imager, and quantitative analysis of the expression of related proteins was performed using Image J and Graphpad Prism.

[0111] Bcl family proteins control the occurrence of apoptosis by regulating the mitochondrial pathway. Bcl-2 is a gene that inhibits apoptosis. The stronger the pro-apoptotic effect of a drug on cells, the lower the expression level of this protein. The cell proliferation marker Ki-67 is a measure of the proliferative potential of cancers (including gliomas). The higher its positive rate, the faster the tumor proliferates. PTX resistance is one of the main obstacles leading to cancer chemotherapy failure. Multidrug resistance (MDR) is mainly mediated by membrane transport proteins caused by ATP-binding cassette transporters (including P-glycoprotein (P-gp), multidrug resistance protein, and breast cancer resistance protein). These proteins are usually overexpressed in drug-resistant tumors and pump drugs out of the cell membrane by hydrolyzing ATP, thereby reducing the intracellular drug concentration below its lethal threshold, decreasing the cytotoxicity of chemotherapeutic drugs and ultimately developing MDR. Among them, P-gp is the most common drug efflux pump. PTX has been confirmed to be a substrate of P-gp. Studies have found that cells resistant to PTX show cross-resistance to various other hydrophobic drugs, and the level of P-gp is significantly increased.

[0112] The electrophoretic band diagrams of Bcl-2, cell proliferation marker Ki-67, P-gp, and β-actin in the experimental group and the blank control group are as Figure 20 shown. The quantitative analysis results of the electrophoretic band diagrams of Bcl-2, cell proliferation marker Ki-67, and P-gp in the experimental group and the blank control group are as Figure 21 shown, where Figure 21 a is the quantitative analysis result of Bcl-2 in the experimental group and the blank control group, Figure 21 b is the quantitative analysis result of cell proliferation marker Ki-67 in the experimental group and the blank control group, Figure 21 c is the quantitative analysis result of P-gp in the experimental group and the blank control group. From Figure 20 and Figure 21 it can be seen that compared with free PTX, the expression level of Bcl-2 in the PTX@PS and PTX@LS groups was significantly decreased (P < 0.05), indicating that the effect of PTX in inducing apoptosis of tumor cells in the PTX@PS and PTX@LS groups was more obvious, which may be related to the ability of liposomes and polymer vesicles to enhance the effective delivery of PTX. In addition, compared with the blank control group, the expression level of Ki-67 in the PTX@PS group and the PTX@LS group was significantly decreased (P < 0.05), indicating that the liposome and polymer vesicle delivery systems played an obvious role in promoting the activation of the PTX-inhibited tumor cell proliferation pathway. And compared with the blank control group, both the PTX@LS group and the free PTX group significantly overexpressed P-gp (P < 0.05), while the increase in the level of cell P-gp protein in the PTX@PS group was smaller, indicating that the polymer vesicle system could significantly reduce the multidrug resistance caused by PTX treatment, and the effect was better than that of the liposome system.

[0113] 8) Inhibiting tumor growth in an orthotopic glioma model in mice

[0114] Inject a phosphate buffer solution containing 1×10 6 GL261 cells into the intracranial cavity of C57BL / 6 mice to establish an orthotopic glioma model. Ten days after model establishment, randomly divide the C57BL / 6 mice into an experimental group and a blank control group. The experimental group is further divided into a free PTX group, a PTX@LS group, and a PTX@PS group, with 5 mice in each group. Dilute free PTX, PTX@LS, and PTX@PS with a phosphate buffer solution, and the diluted volume is 6 μL. Among them, the mass of PTX is 20 μg. Inject the above diluted solutions into the tumor implantation sites of the mice in each group respectively. The mice in the blank control group are injected with an equal volume of phosphate buffer solution. Twenty days after administration, sacrifice the mice in the experimental group and the blank control group, remove the brain, and evaluate the tumor size and shape, prognosis, and blood vessel tissue formation pattern. The results are as Figure 22 shown. As can be seen from Figure 22 , histological analysis in the blank control group (PBS) shows that the tumor develops in the area of the residual cavity. The average volume of the tumor is 73.39±12.62 mm 3 (Volume = 0.5×minimum diameter×square of the maximum diameter), and the tumor grows irregularly with unclear edges and shows an obvious infiltrating state, invading adjacent lymphatic vessels, blood vessels, or infiltrating along the perineural space. In contrast, PTX in the experimental group (including the free PTX group, the PTX@LS group, and the PTX@PS group) significantly inhibits tumor growth by more than 97%. The tumor images in the experimental group show a small tumor mass with clear edges and few signs of local infiltration. In addition, the PTX@PS group has the most obvious tumor-killing effect, and in the PTX@PS group, visible tumors are not detected in 50% of the treated mice. The results prove that PTX@PS has excellent anti-tumor efficacy against gliomas in the brain in vivo.

[0115] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of polymeric vesicles in paclitaxel delivery carriers, characterized in that: The polymeric vesicles are prepared by a preparation method including the following steps: dissolving an amphiphilic block polymer in methoxypolyethylene glycol with a molecular weight of 200 - 600, and then adding water dropwise thereto to prepare amphiphilic block polymer vesicles, wherein 0.05 - 0.15 mg of the amphiphilic block polymer is added per microliter of methoxypolyethylene glycol with a molecular weight of 200 - 600; Wherein, the amphiphilic block polymer is prepared by reacting methoxypolyethylene glycol with a molecular weight of 1500 - 2500 and a lactone monomer in an organic solvent; wherein, the lactone monomer includes ε-caprolactone; The polymeric vesicles have a bilayer membrane structure; The average rate of water dropwise addition is 100 μL / min - 1000 μL / min; The Zeta potential of the polymeric vesicles is -7 to -10 mV.

2. The application according to claim 1, characterized in that, The particle size of the polymeric vesicles is 100 nm - 200 nm.

3. The application according to claim 1, wherein The drug loading amount of paclitaxel is 2 - 3%.

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