CN109679087B - Borate functionalized pluronic polymer, preparation method and application in preparation of drug delivery system - Google Patents
Borate functionalized pluronic polymer, preparation method and application in preparation of drug delivery system Download PDFInfo
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- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
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- C08G2650/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
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- C08G2650/04—End-capping
Abstract
The invention discloses a boric acid ester functionalized pluronic polymer, which has a structure shown as a formula I:
the synthesis route of the polymer shown in the formula I is as follows:
the preparation method of the polymer shown in the formula I comprises the preparation of carboxyl modified pluronic shown in a formula I-2 and the preparation of the polymer shown in the formula I, and meanwhile, the prepared boronic acid ester functionalized pluronic polymer is applied to the preparation of a drug delivery system to provide the drug delivery system capable of remarkably treating tumors.
Description
Technical Field
The invention relates to the technical field of preparation of a Pluronic polymer carrier, in particular to a Pluronic polymer functionalized by boric acid ester, a preparation method and application in preparation of a drug delivery system.
Background
Multidrug resistance in tumor cells has been one of the major obstacles that clinically hamper successful chemotherapy. Cancer cells are induced by long-term anticancer drugs, and intracellular drug efflux proteins are remarkably improved, which can result in lower enrichment of the drugs in the cells. In addition, as the tumor occurs, some microenvironments inside the tumor, such as hypoxia, acid sequestration and up-regulation of enzymes, can cause the therapeutic effect of the drug to be weak. In particular, high-expression GSH in tumor cells can lower oxidative damage induced by chemotherapy drugs, and reduce the anti-tumor curative effect.
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, is reported to be able to effectively reverse the multidrug resistance of tumor cells. Its action mechanism mainly induces depolarization of mitochondria, down-regulates ATP and interferes with fluidity of cell membranes, thereby inhibiting the function of ATP-dependent drug efflux pumps. However, the property of reversing drug resistance is related to the hydrophile-hydrophobicity ratio (HLB), the strong hydrophilicity has no reversing effect, and the strong hydrophobicity is easy to cause carrier instability and bring certain toxic and side effects. Therefore, it is necessary to further modify pluronic to achieve both of the properties.
Recent research reports show that quinone substances (curcumin and phenylboronate) can perform addition reaction with intracellular GSH, so that intracellular GSH is reduced, and the oxidative damage effect of the medicine is improved. Based on this concept, we propose a combination strategy, i.e. simultaneous inhibition of drug efflux pumps and down-regulation of GSH. In the present invention, we used hydrophobic phenylboronate molecules to graft pluronic. The introduction of the borate can not only generate the effect of synergistically reversing the multidrug resistance of the tumor with pluronic, but also improve the stability of the carrier and mediate the stimulation and response release of the drug.
Disclosure of Invention
The invention aims to solve the technical problem of providing a borate functionalized pluronic polymer, a preparation method and application in preparation of a drug delivery system.
The invention solves the technical problems through the following technical scheme:
a borate functionalized pluronic polymer has a structure shown as formula I:
the synthesis route of the borate functionalized pluronic polymer shown as the formula I is as follows:
the preparation method of the borate functionalized pluronic polymer shown as the formula I comprises the following steps:
s1 preparation of carboxy-modified Pluronic of formula I-2:
sequentially adding pluronic P123 shown as a formula I-1, adipic anhydride and anhydrous triethylamine into a reactor, adding 20mL of organic chlorine solvent, reacting at room temperature for 12 hours, then finishing the reaction, removing the solvent by rotary evaporation, dissolving the product with ethanol, adding the product into a dialysis bag, dialyzing for 24 hours, and freeze-drying to obtain the carboxyl modified pluronic shown as a formula I-2;
preparation of S2, boronic ester functionalized pluronic Polymer of formula I:
and (2) sequentially adding the carboxyl-modified pluronic shown as the formula I-2, the 4- (hydroxymethyl) phenylboronic acid pinacol ester shown as the formula I-3, DCC and DMAP which are prepared in the step S1 into a reactor, adding 20mL of organic chlorine solvent, introducing nitrogen for protection, reacting at normal temperature for 24 hours, and filtering, concentrating and separating by column chromatography to obtain the functional pluronic polymer shown as the formula I.
Preferably, the pluronic P123, adipic anhydride and anhydrous triethylamine shown in I-1 in the step S1 are mixed according to a molar ratio of 1: 3: 3, and sequentially adding the components into the reactor.
Preferably, in the step S2, the carboxyl-modified Pluronic represented by the formula I-2, the 4- (hydroxymethyl) phenylboronic acid pinacol ester represented by the formula I-3, DCC and DMAP are mixed according to a molar ratio of 1: 2.5: 2.2: 0.5, added into the reactor in sequence.
Preferably, the organic chlorine solvent in step S1 and step S2 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 application of the borate functionalized pluronic polymer in preparing a drug delivery system, wherein the drug delivery system comprises the borate functionalized pluronic polymer, an anti-tumor drug and an acceptable auxiliary material on a pharmaceutical preparation.
Preferably, the anti-tumor drug is any one of adriamycin, paclitaxel and camptothecin.
Preferably, the anti-tumor drug is doxorubicin.
Compared with the prior art, the invention has the following advantages:
the invention discloses a boronic acid ester functionalized pluronic polymer, which is prepared by taking pluronic P123 as a mother nucleus through acylation reaction, wherein the pluronic modified by carboxyl can inhibit the action of a drug efflux pump so as to reverse drug resistance.
Introducing phenylboronate into a carboxyl-modified pluronic molecule mother nucleus to obtain a boronic acid ester functionalized pluronic polymer, wherein the boronic acid ester group can down-regulate GSH (glutathione) to reverse drug resistance, and obtaining the drug molecule fragment with the reverse drug resistance from the drug molecule fragment with the reverse drug resistance according to a drug combination theory.
The invention discloses the borate functionalized pluronic polymer, which has the advantage that the stability of a micelle carrier is improved due to the fact that the hydrophobic group is grafted on the parent chain of the polymer.
Drawings
FIG. 1 is a diagram of a carboxyl group-modified Pluronic1H NMR；
FIG. 2 is a representation of a boronate functionalized pluronic polymer according to example 1 of the present invention1H NMR；
FIG. 3a is a particle size measurement of a Pluronic P123 blank micelle according to example 2 of the present invention;
FIG. 3b is a topographical view of a Pluronic P123 blank micelle in example 2 of the present invention;
FIG. 3c is a particle size detection graph of boronate functionalized Pluronic polymer blank micelles according to example 2 of the present invention;
FIG. 3d is a topographical view of a boronic ester functionalized Pluronic polymer blank micelle according to example 2 of the present invention;
fig. 4a is a graph of the drug-loading detection results of the pluronic P123 drug-loaded micelle and the borate functionalized pluronic polymer drug-loaded micelle in example 3 of the present invention;
FIG. 4b is a graph showing the encapsulation efficiency test results of the Pluronic P123 drug-loaded micelles and the Pluronic polymer drug-loaded micelles functionalized by borate in example 3 of the present invention;
FIG. 5 is a graph showing the release of doxorubicin from the micelle particles loaded with drugs in example 4 of the present invention;
FIG. 6a is the electron microscope of the laser confocal microscope for observing human breast cancer cells in example 5 of the present invention;
FIG. 6b is an electron microscope of human breast cancer adriamycin-resistant cells observed by a laser confocal microscope in example 5 of the present invention;
FIG. 7a is a graph of mitochondrial red light and depolarized mitochondrial green light intensity detection in example 6 of the invention;
FIG. 7b is a graph of the red/green ratio statistics in embodiment 6 of the present invention;
FIG. 8 is a graph showing the results of changes in intracellular ATP levels after the effect of the nanomedicine micelles on human breast cancer cells and human breast cancer adriamycin-resistant cells in example 7 of the present invention;
FIG. 9 is a graph showing the change in glutathione content in human breast cancer cells and human breast cancer adriamycin-resistant cells after the nanomedicine micelles of example 8 of the present invention act on the cells;
FIG. 10a is a graph showing the cytotoxicity test results of free 4- (hydroxymethyl) phenylboronic acid pinacol ester, Pluronic P123 blank micelle and borate-functionalized Pluronic polymer blank micelle on human breast cancer cells (MCF-7) in example 9 of the present invention;
FIG. 10b is a graph showing the cytotoxicity test results of free doxorubicin, Pluronic P123 drug-loaded micelles, and Pluronic polymer drug-loaded micelles functionalized with borate on human breast cancer cells in example 9 of the present invention;
FIG. 10c is a graph showing the results of cytotoxicity test of free 4- (hydroxymethyl) phenylboronic acid pinacol ester, Pluronic 123 blank micelle, and boronic ester functionalized Pluronic polymer blank micelle against human breast cancer doxorubicin-resistant cells in example 9 of the present invention;
FIG. 10d is a graph showing the results of the cytotoxicity assays 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;
in the figure, P123 micelles represent pluronic P123 blank micelles; PHA micelles represent boronate functionalized pluronic polymer blank micelles;
p123 drug-loaded micelles represent pluronic P123 drug-loaded micelles;
PHA drug-loaded micelles represent boronic acid ester functionalized pluronic polymer drug-loaded micelles;
phenylboronate represents the pinacol ester of 4- (hydroxymethyl) phenylboronic acid.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
Preparation of boronic ester functionalized pluronic polymers:
the synthetic route for the boronic ester functionalized pluronic polymer is shown below:
the preparation method of the borate functionalized pluronic polymer comprises the following steps:
s1 preparation of carboxy-modified Pluronic of formula I-2:
pluronic P123(10g, 1.7 mmol) shown as formula I-1, adipic anhydride (0.7g, 5.5mmol) and anhydrous triethylamine (0.55g, 5.4mmol) were added into a 100mL round bottom reaction flask, and then 20mL dichloromethane was added as a solvent. Reacting for 12h, removing dichloromethane by rotary evaporation, dissolving the product with ethanol, dialyzing with dialysis bag with molecular weight cutoff of 3500Da, wherein the dialysate is 80% ethanol and deionized water respectively, and dialyzing for 24h respectively. Freeze drying to obtain 8.7g of carboxyl modified Pluronic product shown as formula I-2 with 85.04% yield; .
Preparation of S2, boronic ester functionalized pluronic Polymer of formula I:
the carboxyl-modified pluronic (5g, 0.8mmol) represented by the formula I-2 prepared in the step S1, the 4- (hydroxymethyl) phenylboronic acid pinacol ester (5g, 2.1mmol) represented by the formula I-3, DCC (dicyclohexylcarbodiimide) (0.38g, 1.8mmol) and DMAP (4-dimethylaminopyridine) (0.05g, 0.41mmol) were sequentially added into a reactor, 10mL of anhydrous dichloromethane was added, nitrogen protection was performed, and the reaction was performed for 24 hours at normal temperature. Then filtering by a sand core funnel, and carrying out rotary evaporation to obtain a crude product. And (3) carrying out gel column chromatography on the crude product, wherein an eluent is (methanol: dichloromethane: 1: 2), collecting eluent, and drying by rotary evaporation to obtain 3.8g of the boronic ester functionalized pluronic polymer shown as the formula I, wherein the yield is 69.1%.
The structural characterization of carboxyl modified pluronic of formula i-2 is shown in figure 1 and the structural characterization of boronic ester functionalized pluronic polymers of formula i is shown in figure 2.
Example 2
Preparation of micelle and particle size and morphology thereof:
firstly, preparing a pluronic P123 blank micelle:
weighing 30mg of pluronic P123 shown as formula I-1, placing the pluronic P123 into a 25mL eggplant-shaped bottle, adding 1mL of dichloromethane, carrying out rotary evaporation at 50 ℃ for 30min, and then placing the bottle into a 60 ℃ oven for drying overnight; and then putting the round-bottom bottle into a 50 ℃ water bath kettle for hydration and heating for 15min, adding 10mL of 50 ℃ deionized water (pH 7.4) into the bottle, quickly stirring and swirling, and filtering the solution by using a 0.22 mu m filter head to obtain milky white or transparent micelle emulsion, namely the Pluronic P123 blank micelle.
The particle size of 1mL of micelle emulsion was measured using a nanometer particle sizer (DLS), and the morphology was measured using a transmission electron microscope, the results being shown in fig. 3a and 3 b.
As can be seen from fig. 3a and 3 b: the particle size of the Plannik P123 blank micelle is about 50nm, and the blank micelle has a regular shape.
Secondly, preparing the blank micelle of the borate functionalized pluronic polymer:
weighing 30mg of the borate functionalized pluronic polymer shown as the formula I, placing the weighed polymer into a 25mL eggplant-shaped bottle, adding 1mL of dichloromethane, carrying out rotary evaporation at 50 ℃ for 30min, and then placing the bottle into a 60 ℃ oven for drying overnight; and then putting the round-bottom bottle into a 50 ℃ water bath kettle for hydration and heating for 15min, adding 10mL of 50 ℃ deionized water (pH 7.4) into the bottle, quickly stirring and swirling, and filtering the solution by using a 0.22 mu m filter head to obtain milky white or transparent micelle emulsion, namely the boric acid ester functionalized Pluronic polymer blank micelle.
The particle size of 1mL of micelle emulsion was measured using a nanometer particle sizer (DLS) and the morphology was measured using a transmission electron microscope, the results are shown in fig. 3c and fig. 3 d.
As can be seen from fig. 3c and 3 d: the hydration diameter of the borate functionalized pluronic polymer blank micelle is less than 100nm, the blank micelle presents a spherical contour and is uniformly dispersed.
Example 3
The encapsulation efficiency, the drug loading efficiency and the particle size of the drug-loaded micelle particle are as follows:
firstly, preparing a pluronic P123 drug-loaded micelle:
weighing 30mg of pluronic P123 and adriamycin to be dissolved in 1mL of anhydrous dichloromethane, carrying out rotary evaporation at 50 ℃ for 30min, and then putting the mixture into a 60 ℃ oven to be dried overnight; and then putting the round-bottomed bottle into a 50 ℃ water bath kettle for hydration heating for 15min, adding 10mL of 50 ℃ deionized water (pH 7.4) into the bottle, quickly stirring and swirling, and filtering the solution by using a 0.22-micron filter head to obtain red emulsion or transparent solution, namely the Plannik P123 drug-loaded micelle.
Secondly, preparing the pramipexole polymer drug-loaded micelle functionalized by boric acid ester:
weighing 30mg of boric acid ester functionalized pluronic polymer and adriamycin to be dissolved in 1mL of anhydrous dichloromethane, carrying out rotary evaporation at 50 ℃ for 30min, and then putting the obtained product into a 60 ℃ oven to be dried overnight; and then putting the round-bottom bottle into a 50 ℃ water bath kettle for hydration heating for 15min, adding 10mL of 50 ℃ deionized water (pH 7.4) into the bottle, quickly stirring and swirling, and filtering the solution by using a 0.22 mu m filter head to obtain red emulsion or transparent solution, namely the borate functionalized Pluronic polymer drug-loaded micelle.
The absorbance of the prepared pluronic 123 drug-loaded micelle particles and the pluronic polymer drug-loaded micelle particles functionalized by boric acid ester is measured at the wavelength of 481nm by an enzyme-labeling instrument, so that the drug-loaded capacity and the encapsulation rate of the micelle are determined, wherein the detection result of the drug-loaded capacity of the micelle is shown in a figure 4a, and the detection result of the encapsulation rate is shown in a figure 4 b.
As can be seen from fig. 4a and 4 b: due to the hydrophobic modification of the borate functionalized pluronic polymer, the borate functionalized pluronic polymer drug-loaded micelle has higher encapsulation efficiency and drug-loaded efficiency than the pluronic P123 drug-loaded micelle. Meanwhile, the particle sizes of the two drug-loaded micelles are increased along with the increase of the dosage, and the dispersibility is relatively poor. Comprehensively considering, the subsequent experiment is carried out by selecting the dosage of adriamycin of 5 mg.
Wherein the drug loading (%) is the amount of adriamycin in the micelle/the total amount of the drug-loaded micelle multiplied by 100%
Encapsulation ratio (%). The amount of doxorubicin in the micelle/the amount of doxorubicin added in total. times.100%
Example 4
In-vitro drug preparation of drug-loaded micelle particles:
the pluronic P123 drug-loaded micelles and the borate functionalized pluronic polymer drug-loaded micelles prepared in the example 3 are respectively prepared into drug-loaded micelles with the doxorubicin concentration of 500 mug/mL, 1mL of the drug-loaded micelles are respectively measured, the dialysis bags are tightly tied by cotton threads in dialysis bags with the molecular weight cut-off of 8kD-14kD and are placed into a 50mL EP tube, and then 10mM or not H is added into the EP tube2O25mL of phosphate buffer solution, and 3 replicates were used.
The buffer was shaken in a shaker at 37 ℃ and 100rpm, the old buffer was removed at 0.5, 1, 2, 36, 48h, and 5mL of fresh buffer was added, then the doxorubicin concentration in the buffer was measured, and the released amount of doxorubicin was calculated, and the release results are shown in fig. 5.
As can be seen from fig. 5: the stability of the Plannik P123 drug-loaded micelle is poor regardless of H2O2The drug is released quickly in the presence or absence. The borate functionalized pluronic polymer drug-loaded micelle is relatively stable and free of H2O2Stimulation, 48H cumulative release less than 35%, but at H2O2Most of the drug will be released within 24h under stimulation.
Example 5
Qualitative uptake of drug-loaded micelle granulocytes:
the pluronic P123 drug-loaded micelles prepared in example 3, the borate functionalized pluronic polymer drug-loaded micelles each 0.2mL, and free doxorubicin 0.2mL were measured for use.
Respectively adding human breast cancer cells (MCF-7) and human breast cancer adriamycin-resistant cells (MCF-7/ADR) into two cell culture dishes, controlling the final concentration of adriamycin in a cell culture dish solution system to be 4 mu g/mL, culturing for 24h, sucking out an old culture medium after allowing cells to adhere to the wall, and sequentially and respectively adding 1.8mL of fresh culture medium, the free adriamycin, the borate functionalized Planciny polymer micelle carrying the medicine and the Planciny P123 medicine carrying micelle into the cell culture dishes.
After two hours of incubation, the old medium was aspirated and 2mL of fresh medium was added and incubation continued for 4 h. Finally, the culture medium is aspirated, the cells are washed twice with PBS (5min) and fixed with 4% paraformaldehyde solution, the cells are washed twice with PBS, the nuclei are stained with a DAPI staining reagent (5min), the cells are washed twice with PBS again, and then the cells are observed by a laser confocal microscope, so that the detection result of the human breast cancer cells (MCF-7) is shown in figure 6a, and the detection result of the human breast cancer adriamycin-resistant cells (MCF-7/ADR) is shown in figure 6 b.
As can be seen from fig. 6a and 6 b: in MCF-7 cells, several pharmaceutical agents are internalized by the cell; in MCF-7/ADR, only a small amount of free adriamycin is taken up by cells, and the Pluronic P123 drug-loaded micelle can increase the retention of the drug in the cells due to the function of inhibiting a drug efflux pump, and meanwhile, the Pluronic polymer drug-loaded micelle group functionalized by the borate ester has the highest intracellular drug enrichment due to the combined action of inhibiting the drug efflux pump and reducing intracellular glutathione.
Example 6
0.2mL of each of the free 4- (hydroxymethyl) phenylboronic acid pinacol ester, the pluronic P123 blank micelle prepared in example 2, and the borate functionalized pluronic polymer blank micelle was measured for use.
Human breast cancer cells (MCF-7) and human breast cancer adriamycin-resistant cells (MCF-7/ADR) are respectively added into two cell culture dishes and cultured for 24h, and the cells are allowed to adhere to the wall. Then, the old culture medium is aspirated, 1.8mL of fresh culture medium is added, the free 4- (hydroxymethyl) phenylboronic acid pinacol ester, the Pluronic P123 blank micelle and the Pluronic polymer blank micelle functionalized by borate are sequentially added into two cell culture dishes, after 4 hours of co-culture, the old culture medium is aspirated, 2mL of new culture medium and 0.1mL of diluted working solution of mitochondrial probe JC-1 are added, and the culture box is incubated for 15 minutes. And (3) cleaning twice by using PBS, observing the green light intensity of the healthy mitochondria and the depolarized mitochondria by using a fluorescence microscope, and counting the red-green light ratio, wherein the detection result of the red light intensity of the healthy mitochondria and the green light intensity of the depolarized mitochondria is shown in a figure 7a, and the counting result of the red-green light ratio is shown in a figure 7 b.
As can be seen from fig. 7a and 7 b: in both cells, the boronate functionalized pluronic polymer blank micelle was able to significantly induce mitochondrial damage, and the induction of the pluronic P123 blank micelle and free 4- (hydroxymethyl) phenylboronic acid pinacol ester was relatively poor.
Example 7
The ATP content of the nano-drug micelle after acting on cells changes:
cell culture and sample incubation were as in example 6. After co-culturing for 4h, the old medium was aspirated, the cells were washed twice with PBS, and then lysed with 0.2mL of lysis buffer. After lysis at 4 ℃ 12000g, centrifugation was carried out for 5min and the supernatant was taken for subsequent measurement. Adding 0.1ml of detection working solution to the detection well, adding 20. mu.L of sample or standard substance into the detection well, mixing well with a gun (micropipette), and measuring the RLU value with a chemiluminescence luminometer of an microplate reader after at least 2s interval, the result is shown in FIG. 8.
As can be seen from fig. 8: in two cells, the blank micelle of the boronate functionalized pluronic polymer can obviously reduce intracellular ATP, and then the blank micelle of the pluronic P123 has no effect basically when the free 4- (hydroxymethyl) phenylboronic acid pinacol ester is used.
Example 8
After the nano-drug micelle acts on a cell, the content of Glutathione (GSH) in the cell changes:
cell culture and sample incubation were as in example 6. After co-culturing for 4h, the old medium was aspirated, the cells were washed twice with PBS, and then lysed with 0.2mL of lysis buffer. After lysis, 3500g at 4 ℃ were centrifuged for 10 min. Mixing the supernatant with GSH working solution, standing for 5min, and measuring absorbance value of each well at 405nm wavelength. The results are shown in FIG. 9.
As can be seen from fig. 9: intracellular GSH reduction is mainly related to phenylboronate, and is weakly related to pluronic P123, while the combined action of the boronate functionalized pluronic polymers can significantly down-regulate intracellular GSH.
Example 9
And (3) detecting cytotoxicity:
human breast cancer cells (MCF-7) or human breast cancer doxorubicin-resistant cells (MCF-7/ADR) were added to a 96-well plate at about 5,000 cells per well, and after 24h of incubation, after removal of the medium, 180. mu.L of fresh medium, 20. mu.L of free 4- (hydroxymethyl) phenylboronic acid pinacol ester, Pluronic P123 blank micelles and borate functionalized Pluronic polymer blank micelles (carrier concentration from 3.125-100. mu.g/mL) and free doxorubicin, Pluronic P123 drug-loaded micelles or borate functionalized Pluronic polymer drug-loaded micelles (doxorubicin concentration from 0.5-10. mu.g/mL) were added.
After two hours of co-cultivation, the old medium was aspirated off, 200. mu.L of fresh medium was added, and cultivation was continued for 24 hours. Thereafter, the medium was removed, and 180. mu.L of fresh medium and 20. mu.L of MTT (5mg/mL) were added thereto for co-cultivation for 4 hours. Finally, removing the culture medium, adding 150 mu L of DMSO, shaking for 10min, detecting the crystal violet absorbance generated by living cells at the wavelength of 570nm, and calculating the cell survival rate;
wherein, the cytotoxicity detection results of the free 4- (hydroxymethyl) phenylboronic acid pinacol ester, the pluronic P123 blank micelle and the borate functionalized pluronic polymer blank micelle on human breast cancer cells (MCF-7) are shown in figure 10 a;
as can be seen from fig. 10 a: both blank micelles exhibited relatively good cellular compatibility.
The cytotoxicity detection results of free adriamycin pluronic 123 drug-loaded micelle and boric acid ester functionalized pluronic polymer drug-loaded micelle on human breast cancer cells (MCF-7) are shown in figure 10 b.
As can be seen from fig. 10 a: the cytotoxicity of the two drug-loaded micelles is increased along with the increase of the drug concentration, and meanwhile, the blank micelle of the borate functionalized pluronic polymer has the strongest cell killing capability.
The cytotoxicity test results of free 4- (hydroxymethyl) phenylboronic acid pinacol ester, Pluronic P123 blank micelles and boric acid ester functionalized Pluronic polymer blank micelles on human breast cancer adriamycin-resistant cells (MCF-7/ADR) are shown in FIG. 10 c.
From FIG. 10c, it can be seen that: the two blank micelles have weak killing capability on cells and good biological safety.
The cytotoxicity detection results of free doxorubicin, pluronic P123 drug-loaded micelles, and the boronate functionalized pluronic polymer drug-loaded micelles on human breast cancer doxorubicin-resistant cells (MCF-7/ADR) are shown in FIG. 10 d.
From FIG. 10d, it can be seen that: in drug-resistant cells, the killing capacity of free adriamycin on cells is obviously inhibited, and the inhibition effect can be reversed by the pluronic P123 drug-loaded micelle and the pluronic polymer drug-loaded micelle functionalized by boric acid ester, namely, the drug resistance is reversed. Meanwhile, the reverse capability of the borate functionalized pluronic polymer drug-loaded micelle is the strongest.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A borate functionalized pluronic polymer is characterized by having a structure shown as a formula I:
2. a method of making the boronate functionalized pluronic polymer according to claim 1, wherein the boronate functionalized pluronic polymer of formula i is synthesized by the following route:
3. the method of making a boronate functionalized pluronic polymer according to claim 2 wherein the method of making a boronate functionalized pluronic polymer of formula i comprises the steps of:
s1 preparation of carboxy-modified Pluronic of formula I-2:
sequentially adding pluronic P123 shown as a formula I-1, adipic anhydride and anhydrous triethylamine into a reactor, adding 20mL of organic chlorine solvent, reacting at room temperature for 12 hours, then finishing the reaction, removing the solvent by rotary evaporation, dissolving the product with ethanol, adding the product into a dialysis bag, dialyzing for 24 hours, and freeze-drying to obtain the carboxyl modified pluronic shown as a formula I-2;
preparation of S2, boronic ester functionalized pluronic Polymer of formula I:
and (2) sequentially adding the carboxyl-modified pluronic shown as the formula I-2, the 4- (hydroxymethyl) phenylboronic acid pinacol ester shown as the formula I-3, DCC and DMAP which are prepared in the step S1 into a reactor, adding 20mL of organic chlorine solvent, introducing nitrogen for protection, reacting at normal temperature for 24 hours, and filtering, concentrating and separating by column chromatography to obtain the functional pluronic polymer shown as the formula I.
4. The method for preparing a boronic ester functionalized pluronic polymer according to claim 3, wherein in step S1 the pluronic P123, adipic anhydride and anhydrous triethylamine shown as I-1 are mixed in a molar ratio of 1: 3: 3, and sequentially adding the components into the reactor.
5. The method of making a boronate functionalized pluronic polymer according to claim 3, wherein the carboxy modified pluronic of formula i-2 in step S2, the 4- (hydroxymethyl) phenylboronic acid pinacol ester of formula i-3, DCC, DMAP are present in a molar ratio of 1: 2.5: 2.2: 0.5, added into the reactor in sequence.
6. The method of making a boronate functionalized pluronic polymer according to claim 3 wherein the organic chlorine solvent in steps S1 and S2 is methylene chloride.
7. The method of making a boronate functionalized pluronic polymer according to claim 3 wherein the dialysis bag in step S1 is a 3500Da molecular weight cut-off dialysis bag.
8. Use of the boronate functionalized pluronic polymer according to claim 1 in the preparation of a drug delivery system comprising the boronate functionalized pluronic polymer, an antineoplastic agent and a pharmaceutically acceptable excipient.
9. The use of a boronate functionalized pluronic polymer according to claim 8 in the preparation of a drug delivery system wherein the antineoplastic drug is doxorubicin.
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| CN103289097A (en) * | 2013-05-15 | 2013-09-11 | 浙江大学 | Pluronic-modified PAMAM (polyamide-amine) graft polymer and application |
| CN103451174A (en) * | 2013-09-22 | 2013-12-18 | 清华大学 | Enzyme-macromolecule conjugate and preparing method and application thereof |
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