CN109679087B - A boronate functionalized pluronic polymer, preparation method and application in preparation of drug delivery system - Google Patents

Abstract

The invention discloses a boronate functionalized Pluronic polymer, the structure of which is shown in formula I:

The synthetic route of the polymer represented by formula I is as follows:

The preparation method of the polymer represented by the formula I includes the preparation of the carboxyl-modified pluronic shown in the formula I-2 and the preparation of the polymer of the formula I. At the same time, the present invention applies the boronate ester-functionalized pluronic polymer prepared above. In the preparation of a drug delivery system, it is used to provide a drug delivery system that can significantly treat tumors, which is embodied in the formation of the boronate-functionalized pluronic polymer and doxorubicin prepared by the technical solution disclosed in the present invention. The drug-loaded micelles show significant anti-tumor effect and have the application prospect of being prepared as a drug delivery system.

Description

A boronate functionalized pluronic polymer, preparation method and preparation method thereof Applications in Material Delivery Systems

technical field

The invention relates to the technical field of preparation of a pluronic polymer carrier, in particular to a boronate functionalized pluronic polymer, a preparation method and an application in the preparation of a drug delivery system.

Background technique

Multidrug resistance of tumor cells has been one of the major obstacles to successful chemotherapy in clinical practice. When cancer cells are induced by long-term anticancer drugs, the intracellular drug efflux protein will be significantly increased, which will lead to a lower concentration of drugs in the cells. In addition, with the occurrence of tumors, some microenvironments within the tumor, such as hypoxia, acid sequestration, and up-regulated enzymes, will also lead to weaker therapeutic effects of drugs. In particular, the highly expressed GSH in tumor cells can down-regulate the oxidative damage induced by chemotherapeutic drugs and reduce the anti-tumor efficacy.

With the development of nanotechnology, some functional nanocarriers have been widely designed and developed in recent years to overcome the multidrug resistance of tumors.

Among them, Pluronic, as an amphiphilic block copolymer, has been reported to effectively reverse the multidrug resistance of tumor cells. Its mechanism of action is mainly to induce mitochondrial depolarization, down-regulate ATP and interfere with the fluidity of cell membranes, thereby inhibiting the function of ATP-dependent drug efflux pumps. However, its reversal drug resistance performance is related to its hydrophilic-hydrophobic ratio (HLB). Therefore, it is necessary to further modify Pluronic to achieve the performance of both.

Recent research reports have shown that quinones (curcumin, phenylboronate) can undergo an addition reaction with intracellular GSH, thereby downregulating intracellular GSH and improving the oxidative damage effect of drugs. Based on this concept, we propose a combined strategy that simultaneously inhibits the drug efflux pump and downregulates GSH. In the present invention, we use hydrophobic phenylboronic ester molecules to degraft pluronics. The introduction of boronate esters can not only synergistically reverse tumor multidrug resistance with pluronics, but also improve the stability of the carrier and mediate the responsive release of drugs to stimuli.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a boronate functionalized pluronic polymer, a preparation method and an application in the preparation of a drug delivery system.

The present invention solves the above-mentioned technical problems through the following technical solutions:

A boronate functionalized Pluronic polymer, the structure is shown in formula I:

The synthetic route of the above-mentioned boronate functionalized Pluronic polymer shown in formula I is as follows:

The preparation method of the boronate functionalized Pluronic polymer represented by the above formula I comprises the following steps:

S1. Preparation of carboxyl-modified pluronic represented by formula I-2:

Pluronic P123 shown in formula I-1, adipic anhydride and anhydrous triethylamine were successively added to the reactor, and 20 mL of organic chlorine solvent was added. After 12 hours of reaction at room temperature, the reaction was completed, and the solvent was evaporated to remove the product. After dissolving in ethanol, adding it to a dialysis bag for 24 hours of dialysis, and then freeze-drying to obtain the carboxyl-modified pluronic shown in formula I-2;

S2. Preparation of boronate functionalized Pluronic polymer represented by formula I:

The carboxyl-modified pluronic shown in the formula I-2, the 4-(hydroxymethyl)benzeneboronic acid pinacol ester shown in the formula I-3, DCC, and DMAP prepared in step S1 were sequentially added to the reactor. 20 mL of organic chlorine solvent, under nitrogen protection, react at room temperature for 24 hours, the reaction solution is filtered, concentrated, and separated by column chromatography to obtain the boronate ester-functionalized Pluronic polymer represented by formula I.

Preferably, the Pluronic P123, adipic anhydride and anhydrous triethylamine shown in I-1 in the step S1 are sequentially added to the reactor according to the molar ratio of 1:3:3.

Preferably, in the step S2, the carboxyl-modified pluronic shown in formula I-2, 4-(hydroxymethyl) phenylboronic acid pinacol ester shown in formula I-3, DCC, and DMAP are in a molar ratio of 1:2.5: 2.2: The addition amount of 0.5 is sequentially added to the reactor.

Preferably, in the steps S1 and S2, the organic chlorine solvent is dichloromethane.

Preferably, the dialysis bag in the step S1 is a dialysis bag with a molecular weight cut-off of 3500 Da.

The invention also discloses the application of the above boronate functionalized pluronic polymer in the preparation of a drug delivery system, the drug delivery system comprising boronate functionalized pluronic polymer, antitumor drugs and acceptable pharmaceutical preparations of accessories.

Preferably, the antitumor drug is any one of doxorubicin, paclitaxel and camptothecin.

Preferably, the antitumor drug is doxorubicin.

Compared with the prior art, the present invention has the following advantages:

The invention discloses a boronate functionalized pluronic polymer. The polymer uses pluronic P123 as the parent nucleus and undergoes an acylation reaction to obtain carboxyl-modified pluronic. The carboxyl-modified pluronic can inhibit the drug efflux pump to reverse drug resistance.

A phenylboronate group was introduced into the carboxyl-modified Pluronic nucleus to obtain a boronate-functionalized Pluronic polymer. The boronate group could downregulate GSH to reverse drug resistance. Molecular fragments, according to the drug conjugation theory, obtain boronate-functionalized Pluronic polymers with reversal of drug resistance.

The boronate-functionalized Pluronic polymer disclosed in the present invention improves the stability of the micelle carrier due to the grafting of hydrophobic groups on the parent chain.

Description of drawings

Fig. 1 is the ¹ H NMR of carboxyl-modified pluronic in Example 1 of the present invention;

2 is the ¹ H NMR of the boronate functionalized Pluronic polymer in Example 1 of the present invention;

Fig. 3a is the particle size detection diagram of Pluronic P123 blank micelles in the embodiment of the present invention 2;

Fig. 3b is the topography detection diagram of Pluronic P123 blank micelle in Example 2 of the present invention;

Fig. 3c is the particle size detection diagram of boronate functionalized Pluronic polymer blank micelles in Example 2 of the present invention;

Fig. 3d is the morphology detection diagram of the boronate functionalized Pluronic polymer blank micelle in Example 2 of the present invention;

Figure 4a is a graph showing the results of the detection of the drug loading of Pluronic P123 drug-loaded micelles and boronate-functionalized Pluronic polymer drug-loaded micelles in Example 3 of the present invention;

4b is a graph showing the results of the detection of the encapsulation efficiency of Pluronic P123 drug-loaded micelles and boronate-functionalized Pluronic polymer drug-loaded micelles in Example 3 of the present invention;

Fig. 5 is the release result diagram of drug-loaded micelle particles releasing doxorubicin in Example 4 of the present invention;

Figure 6a is an electron microscope image of human breast cancer cells observed by laser confocal microscope in Example 5 of the present invention;

Figure 6b is an electron microscope image of human breast cancer doxorubicin-resistant cells observed by laser confocal microscope in Example 5 of the present invention;

Figure 7a is a graph showing the intensity detection of mitochondrial red light and depolarized mitochondrial green light in Example 6 of the present invention;

Figure 7b is a graph of the statistical result of the ratio of red to green light in Example 6 of the present invention;

8 is a graph showing the results of changes in intracellular ATP content after the nanomedicine micelle acts on human breast cancer cells and human breast cancer doxorubicin-resistant cells in Example 7 of the present invention;

9 is a graph showing changes in intracellular glutathione content after the nanomedicine micelles act on human breast cancer cells and human breast cancer doxorubicin-resistant cells in Example 8 of the present invention;

Figure 10a shows the effects of free 4-(hydroxymethyl) phenylboronic acid pinacol ester, Pluronic P123 blank micelles, and boronate-functionalized Pluronic polymer blank micelles on human breast cancer cells in Example 9 of the present invention. (MCF-7) cytotoxicity test result chart;

Figure 10b is a graph showing the cytotoxicity detection results of free doxorubicin, pluronic P123 drug-loaded micelles, and boronate-functionalized pluronic polymer drug-loaded micelles on human breast cancer cells in Example 9 of the present invention;

Figure 10c shows that in Example 9 of the present invention, free 4-(hydroxymethyl) phenylboronic acid pinacol ester, Pluronic 123 blank micelles, and boronate-functionalized Pluronic polymer blank micelles were resistant to human breast cancer. Result of doxorubicin cytotoxicity test;

Figure 10d shows the cytotoxicity detection of free doxorubicin, pluronic P123 drug-loaded micelles, and boronate-functionalized pluronic polymer drug-loaded micelles on human breast cancer doxorubicin-resistant cells in Example 9 of the present invention result graph;

In the figure, P123 micelles represent Pluronic P123 blank micelles; PHA micelles represent boronate functionalized Pluronic polymer blank micelles;

P123 drug-loaded micelle means Pluronic P123 drug-loaded micelle;

PHA drug-loaded micelles represent boronate functionalized Pluronic polymer drug-loaded micelles;

Phenylboronate means 4-(hydroxymethyl)phenylboronic acid pinacol ester.

Detailed ways

The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and provides a detailed implementation manner and a specific operation process, but the protection scope of the present invention is not limited to the following implementation. example.

Example 1

Preparation of boronate functionalized Pluronic polymers:

The synthetic route of boronate functionalized Pluronic polymers is shown below:

The preparation method of boronate functionalized Pluronic polymer comprises the following steps:

S1. Preparation of carboxyl-modified pluronic represented by formula I-2:

Pluronic P123 (10 g, 1.7 mmol, ), adipic anhydride (0.7 g, 5.5 mmol), and anhydrous triethylamine (0.55 g, 5.4 mmol), as shown in formula I-1, were added to a 100 mL circular In the bottom reaction flask, 20 mL of dichloromethane was added as a solvent. The reaction was carried out for 12 h, the dichloromethane was removed by rotary evaporation, the product was dissolved in ethanol, and dialyzed with a dialysis bag with a molecular weight cut-off of 3500 Da. Freeze-drying to obtain 8.7 g of the carboxyl-modified Pluronic product shown in formula I-2, with a yield of 85.04%;

S2. Preparation of boronate functionalized Pluronic polymer represented by formula I:

The carboxyl-modified pluronic (5 g, 0.8 mmol) represented by the formula I-2 prepared in step S1, 4-(hydroxymethyl)phenylboronic acid pinacol ester (5 g, 2.1 mmol) represented by the formula I-3 were successively prepared in step S1. ), DCC (dicyclohexylcarbodiimide) (0.38g, 1.8mmol), DMAP (4-dimethylaminopyridine) (0.05g, 0.41mmol) were added to the reactor, and then 10mL of anhydrous dichloromethane was added , pass nitrogen protection, and react at room temperature for 24h. Then filter with sand core funnel and rotary evaporation to obtain crude product. The crude product was subjected to gel column chromatography, the eluent was (methanol:dichloromethane=1:2), the eluate was collected, and the boronic ester-functionalized Pluronic polymer 3.8 represented by formula I was obtained by rotary evaporation and drying. g, 69.1% yield.

The structural characterization of the carboxyl-modified pluronic shown in formula I-2 is shown in Figure 1, and the structural characterization of the boronate-functionalized pluronic polymer shown in formula I is shown in Figure 2.

Example 2

Preparation of micelles and their particle size and morphology:

1. Preparation of Pluronic P123 blank micelles:

Weigh 30 mg of Pluronic P123 shown in formula I-1 into a 25 mL eggplant-shaped bottle, add 1 mL of dichloromethane, rotate at 50 °C for 30 min, and then put it in a 60 °C oven to dry overnight; then put the round bottom bottle into a 50 Hydrate and heat in a ℃ water bath for 15 min, add 10 mL of deionized water (pH 7.4) at 50 ℃ to the bottle, quickly stir and vortex, and filter the solution with a 0.22 μm filter to obtain a milky white or transparent micellar emulsion, which is Pulan Nick P123 blank micelles.

Take 1 mL of micellar emulsion for particle size detection using a nanoparticle size analyzer (DLS), and a transmission electron microscope for morphology detection. The results are shown in Figure 3a and Figure 3b.

It can be seen from Figure 3a and Figure 3b that the particle size of the Pluronic P123 blank micelles is about 50 nm, showing a regular morphology.

2. Preparation of boronate functionalized Pluronic polymer blank micelles:

Weigh 30 mg of boronate-functionalized Pluronic polymer represented by formula I into a 25 mL eggplant-shaped bottle, add 1 mL of dichloromethane, rotate at 50 °C for 30 min, and then put it in a 60 °C oven to dry overnight; Put the round-bottom bottle into a 50°C water bath for hydration and heating for 15min, add 10mL of 50°C deionized water (pH 7.4) to the bottle, quickly stir and vortex, and filter the solution with a 0.22μm filter to obtain milky white or transparent The micellar emulsion is a boronate functionalized Pluronic polymer blank micelle.

Take 1 mL of micellar emulsion for particle size detection using a nanoparticle size analyzer (DLS), and a transmission electron microscope for morphology detection. The results are shown in Figure 3c and Figure 3d.

It can be seen from Figure 3c and Figure 3d that the hydrated diameter of the boronate functionalized Pluronic polymer blank micelles is less than 100 nm, showing a spherical outline and uniform dispersion.

Example 3

Encapsulation efficiency, drug loading efficiency and particle size of drug-loaded micelle particles:

1. Preparation of Pluronic P123 drug-loaded micelles:

Weigh 30 mg of Pluronic P123 and doxorubicin to co-dissolve in 1 mL of anhydrous dichloromethane, steam at 50 °C for 30 min, and then put it in a 60 °C oven to dry overnight; then put the round-bottom bottle into a 50 °C water bath. Heat for 15 min, add 10 mL of deionized water (pH 7.4) at 50°C to the bottle, stir and vortex quickly, filter the solution with a 0.22 μm filter to obtain a red emulsion or a transparent solution, which is Pluronic P123 drug-loaded gel bundle.

2. Preparation of boronate functionalized Pluronic polymer drug-loaded micelles:

Weigh 30 mg of boronate-functionalized pluronic polymer and doxorubicin to dissolve in 1 mL of anhydrous dichloromethane, steam at 50 °C for 30 min, and then put it in a 60 °C oven to dry overnight; Put it into a 50°C water bath for hydration and heating for 15min, add 10mL of 50°C deionized water (pH 7.4) to the bottle, quickly stir and vortex, and filter the solution with a 0.22μm filter to obtain a red emulsion or a transparent solution, namely Pluronic polymer drug-loaded micelles functionalized with boronate esters.

The Pluronic 123 drug-loaded micellar particles prepared above and the boronate-functionalized Pluronic polymer drug-loaded micellar particles were measured for their absorbance at a wavelength of 481 nm by a microplate reader, thereby determining the drug loading and packaging of the micelles. The results of encapsulation efficiency and drug loading of micelles are shown in Figure 4a, and the results of encapsulation efficiency detection are shown in Figure 4b.

It can be seen from Figure 4a and Figure 4b that due to the hydrophobic modification of the boronate-functionalized pluronic polymer, the boronate-functionalized pluronic polymer drug-loaded micelles have better drug-loaded micelles than the pluronic P123 drug-loaded micelles. High encapsulation efficiency and drug loading efficiency. At the same time, it can be found that the particle size of the two drug-loaded micelles increases with the increase of the dosage, and the dispersibility becomes relatively poor. Taking into account comprehensively, the dosage of doxorubicin was 5 mg for follow-up experiments.

Wherein, drug loading (%) = amount of doxorubicin in micelles/total amount of drug-loaded micelles × 100%

Encapsulation efficiency (%) = the amount of doxorubicin in the micelle / the total amount of doxorubicin added × 100%

Example 4

In vitro drug preparation of drug-loaded micellar particles:

The Pluronic P123 drug-loaded micelles prepared in Example 3 and the boronate-functionalized Pluronic polymer drug-loaded micelles were prepared into drug-loaded micelles with a doxorubicin concentration of 500 μg/ml, respectively, and 1 mL was measured. The above-mentioned drug-loaded micelles were placed in a dialysis bag with a molecular weight cut-off of 8kD-14kD, and the dialysis bag was fastened with cotton thread, put into a 50mL EP tube, and then added with or without 10mM H ₂ O in the EP tube ₂ of 5 mL of phosphate buffer, with 3 replicates.

Put into a shaker, shake at 37°C and 100rpm, take out the old buffer at 0.5, 1, 2, 4...12, 24, 36, 48h, and add 5mL of new buffer , and then detect the concentration of doxorubicin in the buffer, and then calculate the release amount of doxorubicin, and the release results are shown in Figure 5.

It can be seen from Figure 5 that the drug-loaded micelles of Pluronic P123 have poor stability, and the drug is released quickly regardless of the presence or absence of H ₂ O ₂ . The boronate functionalized Pluronic polymer drug-loaded micelles are relatively stable, without H ₂ O ₂ stimulation, the cumulative release is less than 35% in 48h, but under H ₂ O ₂ stimulation, most of the drugs will be released within 24h.

Example 5

Qualitative cellular uptake of drug-loaded micellar particles:

Measure 0.2 mL each of the pluronic P123 drug-loaded micelles prepared in Example 3, the boronate-functionalized pluronic polymer drug-loaded micelles, and 0.2 mL of free doxorubicin, and set aside.

Human breast cancer cells (MCF-7) and human breast cancer doxorubicin-resistant cells (MCF-7/ADR) were added to two cell culture dishes respectively, and the final concentration of doxorubicin in the control cell culture dish solution system was 4 μg /mL, cultured for 24h, after allowing the cells to adhere, the old medium was aspirated, and 1.8mL of fresh medium, as well as the above free doxorubicin and boronate functionalized pluronic polymerization were added to the above cell culture dishes in turn. Drug-loaded micelles and Pluronic P123-loaded micelles.

After culturing for two hours, the old medium was aspirated, and 2 mL of fresh medium was added to continue the culture for 4 hours. Finally, the medium was aspirated, washed twice with PBS, fixed with 4% paraformaldehyde solution (5min), washed twice with PBS, stained with DAPI nuclei (5min), washed twice with PBS, and then washed with laser Focusing microscope observation, the detection results of human breast cancer cells (MCF-7) are shown in Figure 6a, and the detection results of human breast cancer cells (MCF-7/ADR) are shown in Figure 6b.

From Figure 6a and Figure 6b, it can be seen that in MCF-7 cells, several drug preparations could be internalized by cells; in MCF-7/ADR, only a small amount of free doxorubicin was taken up by cells, while Pluronic P123 was loaded with drugs. Micelles can increase the retention of drugs in cells due to their function of inhibiting drug efflux pumps, and it can be found that the boronate functionalized Pluronic polymer-loaded micelle group has the highest intracellular drug enrichment due to its function of inhibiting drug efflux pumps. Combined effect of inhibition of drug efflux pump and down-regulation of intracellular glutathione.

Example 6

The free 4-(hydroxymethyl)benzeneboronic acid pinacol ester, the Pluronic P123 blank micelle prepared in Example 2, and the boronate-functionalized Pluronic polymer blank micelle were each weighed to 0.2 mL, and set aside.

Human breast cancer cells (MCF-7) and human breast cancer doxorubicin-resistant cells (MCF-7/ADR) were added to two cell culture dishes, respectively, and cultured for 24 h to allow the cells to adhere. Then, the old medium was aspirated, 1.8 mL of fresh medium was added, and the above free 4-(hydroxymethyl)phenylboronic acid pinacol ester, Pluronic P123 blank micelle, and boronate-functionalized Pluronic were polymerized in turn. The blank micelles were added to two cell culture dishes, and after co-cultivation for 4 h, the old medium was aspirated, 2 mL of new medium and 0.1 mL of diluted mitochondrial probe JC-1 working solution were added, and incubated in the incubator for 15 min. Washed twice with PBS, the intensity of red light of healthy mitochondria and green light of depolarized mitochondria was observed by fluorescence microscope, and the ratio of red and green light was counted. The green light ratio statistics are shown in Fig. 7b.

It can be seen from Figure 7a and Figure 7b that the boronate functionalized Pluronic polymer blank micelles can significantly induce mitochondrial damage in both cells, the Pluronic P123 blank micelles and free 4-(hydroxymethyl)benzene. The induction effect of pinacol borate was relatively poor.

Example 7

Changes in ATP content after nanomedicine micelles act on cells:

Cell culture and sample incubation were the same as in Example 6. After co-cultivation for 4 h, the old medium was aspirated, PBS was added to wash the cells twice, and 0.2 mL of lysis buffer was added to lyse the cells. After lysis, centrifuge at 12000g at 4°C for 5 min, and take the supernatant for subsequent determination. Add 0.1 mL of ATP detection working solution to the detection well, then add 20 μL of sample or standard to the detection well, and quickly mix with a gun (micropipette), after at least 2s interval, use the chemiluminescence luminometer of the microplate reader to measure the RLU value, and the results are shown in Figure 8.

It can be seen from Figure 8 that in the two cells, the boronate functionalized Pluronic polymer blank micelles can significantly down-regulate intracellular ATP, followed by the Pluronic P123 blank micelles, free 4-(hydroxymethyl)benzene Pinacol borate is basically ineffective.

Example 8

Changes in intracellular glutathione (GSH) content after nanomedicine micelles act on cells:

Cell culture and sample incubation were the same as in Example 6. After co-cultivation for 4 h, the old medium was aspirated, PBS was added to wash the cells twice, and 0.2 mL of lysis buffer was added to lyse the cells. After lysis, centrifuge at 3500g at 4°C for 10min. Take the supernatant and mix it with GSH working solution, let stand for 5 min, and measure the absorbance value of each well at a wavelength of 405 nm. The results are shown in Figure 9.

It can be seen from Figure 9 that the decrease of intracellular GSH is mainly related to phenylboronic ester, and is weakly related to pluronic P123, and the combined effect of boronate functionalized pluronic polymer can significantly down-regulate intracellular GSH.

Example 9

Cytotoxicity assay:

Human breast cancer cells (MCF-7) or human breast cancer doxorubicin-resistant cells (MCF-7/ADR) were added to a 96-well plate, about 5,000 cells per well. After culturing for 24 hours, after removing the medium, Add 180 μL of fresh medium, 20 μL of free 4-(hydroxymethyl)phenylboronic acid pinacol ester, Pluronic P123 blank micelles, and boronate-functionalized Pluronic polymer blank micelles (carrier concentrations from 3.125- 100 μg/mL) and free doxorubicin, pluronic P123 drug-loaded micelles or boronate functionalized pluronic polymer drug-loaded micelles (doxorubicin concentration from 0.5-10 μg/mL).

After two hours of co-cultivation, the old medium was aspirated, 200 μL of fresh medium was added, and the culture was continued for 24 h. After that, the medium was removed, and 180 μL of fresh medium and 20 μL of MTT (5 mg/mL) were added after co-cultivation for 4 h. Finally, the medium was removed, 150 μL of DMSO was added, and after shaking for 10 min, the absorbance of crystal violet produced by living cells was detected at a wavelength of 570 nm, and the cell survival rate was calculated;

Among them, the cytotoxicity of free 4-(hydroxymethyl) phenylboronic acid pinacol ester, pluronic P123 blank micelles, and boronate functionalized pluronic polymer blank micelles on human breast cancer cells (MCF-7) The detection results are shown in Figure 10a;

It can be seen from Figure 10a that both blank micelles exhibited relatively good cytocompatibility.

Figure 10b shows the cytotoxicity test results of free doxorubicin pluronic 123 drug-loaded micelles and boronate-functionalized pluronic polymer drug-loaded micelles on human breast cancer cells (MCF-7).

It can be seen from Figure 10a that the cytotoxicity of the above two drug-loaded micelles increases with the increase of drug concentration, and the boronate functionalized Pluronic polymer blank micelles have the strongest cell killing ability.

Free 4-(hydroxymethyl)phenylboronic acid pinacol ester, pluronic P123 blank micelles, and boronate-functionalized pluronic polymer blank micelles are effective in human breast cancer adriamycin-resistant cells (MCF-7/ADR). ) cytotoxicity assay results are shown in Figure 10c.

It can be seen from Figure 10c that the above two blank micelles have weak killing ability to cells and have good biological safety.

The cytotoxicity test results of free doxorubicin, pluronic P123 drug-loaded micelles, and boronate-functionalized pluronic polymer drug-loaded micelles on human breast cancer doxorubicin-resistant cells (MCF-7/ADR) are shown in the figure 10d.

It can be seen from Figure 10d that in drug-resistant cells, the ability of free doxorubicin to kill cells was significantly inhibited, while pluronic P123 drug-loaded micelles and boronate-functionalized pluronic polymer drug-loaded micelles could reverse this effect. Inhibitory effect, that is, reversal of drug resistance. At the same time, it can be found that the reversal ability of the boronate functionalized Pluronic polymer drug-loaded micelles is the strongest.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (9)

1. a boronate functionalized Pluronic polymer, characterized in that, the structure is as shown in formula I:

2. a method for preparing the boronate functionalized Pluronic polymer as claimed in claim 1, is characterized in that, the synthetic route of the boronate functionalized Pluronic polymer shown in the formula I is as follows:

3. the method for preparing boronate functionalized pluronic polymer according to claim 2, is characterized in that, the preparation method of boronate functionalized pluronic polymer shown in the formula I comprises the following steps: S1. Preparation of carboxyl-modified pluronic represented by formula I-2: Pluronic P123 shown in formula I-1, adipic anhydride and anhydrous triethylamine were successively added to the reactor, and 20 mL of organic chlorine solvent was added. After 12 hours of reaction at room temperature, the reaction was completed, and the solvent was evaporated to remove the product. After dissolving in ethanol, adding it to a dialysis bag for 24 hours of dialysis, and then freeze-drying to obtain the carboxyl-modified pluronic shown in formula I-2; S2. Preparation of boronate functionalized Pluronic polymer represented by formula I: The carboxyl-modified pluronic shown in the formula I-2, the 4-(hydroxymethyl)benzeneboronic acid pinacol ester shown in the formula I-3, DCC, and DMAP prepared in step S1 were sequentially added to the reactor. 20 mL of organic chlorine solvent, under nitrogen protection, react at room temperature for 24 hours, the reaction solution is filtered, concentrated, and separated by column chromatography to obtain the boronate ester-functionalized Pluronic polymer represented by formula I. 4. The method for preparing boronate-functionalized Pluronic polymers according to claim 3, wherein the Pluronic P123, adipic anhydride, anhydrous triethyl shown in I-1 in the step S1 The amines were sequentially added to the reactor according to the molar ratio of 1:3:3. 5. The method for preparing boronate-functionalized Pluronic polymers according to claim 3, characterized in that in the step S2, the carboxyl-modified Pluronic shown in formula I-2, the carboxyl-modified Pluronic shown in formula I-3, 4 -(Hydroxymethyl) phenylboronic acid pinacol ester, DCC, and DMAP were added to the reactor in order according to the molar ratio of 1:2.5:2.2:0.5. 6. The method for preparing boronate-functionalized Pluronic polymers according to claim 3, wherein the organic chlorine solvent in the steps S1 and S2 is dichloromethane. 7 . The method for preparing boronate functionalized Pluronic polymers according to claim 3 , wherein the dialysis bag in the step S1 is a dialysis bag with a molecular weight cut-off of 3500 Da. 8 . 8. a kind of application of adopting boronate functionalized pluronic polymer as claimed in claim 1 in the preparation of drug delivery system, it is characterized in that, described drug delivery system comprises boronate functionalized pluronic polymer Drugs, antitumor drugs and acceptable excipients in pharmaceutical preparations. 9 . The application of the boronate functionalized pluronic polymer according to claim 8 in the preparation of a drug delivery system, wherein the antitumor drug is doxorubicin. 10 .

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