CN109125739B - Multifunctional polymer micelle drug delivery system and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of targeted drug delivery, in particular to a multifunctional high-molecular micelle drug delivery system and a preparation method and application thereof.A part of side chains of a polyethylene glycol-polylysine block copolymer are chemically modified by 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride to obtain groups with sulfydryl, and then boron neutron capture treatment drugs, namely sulfydryl dodecaborane disodium salt with sulfydryl, are reacted with the side chains in an aqueous solution through disulfide bonds to form stable multifunctional high-molecular micelles through self-assembly and partial free disulfide bond crosslinking; the prepared high molecular micelle can deliver sulfhydryl-containing disodium dodecaborane or other sulfhydryl-containing drugs or prodrugs, so that the content of the drugs delivered into tumor cells is increased, and the targeted treatment effect on tumors is improved.
Description
Technical Field
The invention relates to the technical field of targeted drug delivery, in particular to a multifunctional polymer micelle drug delivery system, a preparation method and application thereof, which have the advantages of improving the stability of the drug delivery system through disulfide bond crosslinking, realizing high-efficiency drug delivery and responding to glutathione drug release from escaping inclusion bodies to tumor cells, and particularly relates to the application of radiotherapy and/or neutron capture treatment/or chemotherapy drugs in preparing and treating tumors or malignant tumors.
Background
The drug delivery system is a drug-loading system and a mode of drug preparation, and is mainly constructed by drug carrier materials (such as high molecular materials) for loading bioactive substances/drugs and delivering the bioactive substances/drugs to the focus site to exert drug effect. The drug delivery system has the important meanings of reducing the toxic and side effects of the drug, prolonging the blood circulation time, maintaining the blood concentration with the treatment effect, delivering the drug to the focus part in a targeted manner, controlling the release of the drug, reducing the exposure of the drug to normal tissues and the like. Therefore, the development of drug delivery systems is regarded as a basic technical system for future drug therapy, receives wide attention at home and abroad, and becomes a research hotspot in the interdisciplinary fields of medicine, chemistry, biological materials, macromolecules and the like. The macromolecule micelle is one of drug delivery systems, is mainly formed by self-assembly of an amphiphilic segmented copolymer, loads a drug in a core to complete the encapsulation or solubilization of the drug, and generally comprises a polyethylene glycol macromolecule at the outer layer, or can increase the stability of the macromolecule micelle by grafting hydrophobic molecules or sulfydryl on the tail end or side chain of the macromolecule to form crosslinking in the core. The polymer micelle has the advantages of controllable particle size, large drug-loading rate, convenient preparation and the like, and is a novel drug delivery system.
Malignant tumor, cancer, is one of the important causes of human death, in China, malignant tumor is the first cause of all deaths, only 2014 years, the number of new cases of malignant tumor exceeds 380 ten thousand, more than 1 ten thousand people are diagnosed as cancer every day on average, and 7 people are diagnosed as cancer every minute. Malignant tumors become important killers for the health of China, and the current state of the tumors in China has the characteristics of high incidence rate, high death rate and the like. Malignant tumors have complexity, and the current treatment aiming at the tumors mainly adopts early diagnosis, radiotherapy, chemotherapy and other comprehensive means for treatment. The biggest defect of the treatment method is that the treatment method has no selectivity on tumor cells and normal cells and causes too large side effect on the normal cells. Therefore, the research and development of a drug delivery system with tumor targeting has important application value.
Boron neutron capture therapy is a selective cancer radiation therapy in the radiation treatment of tumors by allowing tumor cells to take up boron (B-containing)10B) By thermal neutron beam irradiation, boron atom(s) ((10B) Capture thermal neutrons because10B has large thermal neutron reaction cross section, can effectively capture thermal neutrons, then generates nuclear fission reaction, generates high-energy alpha particles (alpha) and7li nucleus for generating alpha particles to destroy cancer cells, resulting in irreversible lethal damage to tumor cells, complete destruction of its genetic structure, no repair and apoptosis, and high curative effect10B) The normal cells of the drug (C) do not have harmful effects. However, the boron neutron capture therapy produces alpha particles with very short range, about 5-9 microns, near the diameter of the cell, and the damaging effects of ionizing radiation produced by these particles are limited to uptake10B cells, and therefore, in order to achieve a therapeutic effect of effectively killing cancer cells, boron (B) -containing cells are required10B) The drug is delivered to tumor cells to achieve the therapeutic effect. Other radiation therapy modalities use radioisotopes, and boron-containing compounds for boron neutron capture therapy of tumors are not non-radioactive and are safer. Boron neutron capture therapy has been successful in curing a plurality of patients at home and abroad, such as Japan, America and China, and has been successful especially for tumors which are difficult to be removed by surgery, such as melanoma, breast cancer, brain tumor and the like. To date, there have been two boron-containing compounds (B)10B) The drugs of (1), mercaptododecaborane disodium salt (BSH) and Borophenylalanine (BPA). The mercapto dodecaborane disodium salt is a boron cluster compound which has good hydrolytic and metabolic stability in a biological environment, has high boron content and can carry more boron (boron) compared with p-borophenylalanine at the same dosage level10B) Is one of the most effective boron neutron capture therapeutic drugs at present, but due to the properties of electronegativity and the like, lacks the targeting function of tumor cells/tissues, is rarely endocytosed by the tumor cells, and is difficult to achieve the effect of boron neutron capture therapy in the tumor cells10B concentration, affecting boron neutron capture therapy of tumors, having the following structural formula:
therefore, the increase of the targeting property of the medicament, namely the mercapto dodecaborane disodium salt, to tumor cells and the intracellular medicament level has important significance, and the construction of a medicament delivery system can deliver the mercapto dodecaborane disodium salt to tumor tissues and cells and increase the number of the mercapto dodecaborane disodium salt in the tumor cells10B concentration, improving the treatment effect on the tumor. At present, the treatment effect of the disodium mercaptododecaborane salt on tumors needs to be improved, and a multifunctional high-molecular micelle nano-drug delivery system for efficiently delivering disodium mercaptododecaborane salt is also lacked, so that the endocytosis of tumor cells on the disodium mercaptododecaborane salt, the targeted delivery of drugs and the boron neutron capture treatment effect are improved.
Disclosure of Invention
The invention aims to provide a multifunctional polymer micelle drug delivery system and a preparation method and application thereof, and the specific technical scheme is as follows:
the multifunctional high molecular micelle medicine delivery system is a high molecular micelle formed by self-assembling and partial free disulfide bond crosslinking of a modified polyethylene glycol-polylysine block copolymer and a boron neutron capture therapeutic medicine with sulfydryl in an aqueous solution.
The modified polyethylene glycol-polylysine block copolymer is obtained by chemically modifying partial side chains of the polyethylene glycol-polylysine block copolymer with 3, 3' -dithio-dipropionidine dimethyl ester dihydrochloride to obtain a group with sulfydryl.
The boron neutron capture therapeutic drug with the sulfydryl is a drug molecule or a prodrug containing sulfydryl;
the boron neutron capture therapeutic drug with sulfydryl is sulfydryl dodecaborane disodium salt.
The chemical reaction process comprises the following steps:
wherein the synthesis process of the mercapto dodecaborane disodium salt crosslinked block copolymer is as follows;
wherein m is the number of repeating units of polyethylene glycol, and the range of m is 3-500;
n is the total number of units of the polycation and ranges from 3 to 300;
x is the number of polylysine repeating units and ranges from 1 to 100;
y is the number of repeating units of 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride which is used for chemically modifying a polyethylene glycol-polylysine side chain and then is connected with a drug molecule mercapto-dodecaborane disodium salt through a disulfide bond, and the range of the repeating units is 1-100;
z is the number of repeating units of 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride for chemically modifying polyethylene glycol-polylysine side chains, and the range of the repeating units is 1-100.
The multifunctional high-molecular micelle drug delivery system takes a polyethylene glycol-polylysine block copolymer as a basic raw material, compounds such as 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride generate sulfydryl on a side chain to form a high-molecular block copolymer, the high-molecular block copolymer reacts with sulfydryl-containing drug molecules such as sulfydryl dodecaborane disodium salt or prodrugs through sulfydryl, finally, high-molecular micelles are formed through self-assembly, unreacted free sulfydryl can be crosslinked through disulfide bonds, and the stability of the high-molecular micelles is improved.
The sulfhydryl-containing drug is combined with the macromolecular block copolymer through disulfide bonds, and the macromolecular micelles have polycation fragments and free sulfhydryl groups for crosslinking the nucleus of the macromolecular micelle. The particle size of the finally formed high molecular micelle is between 10 and 200 nanometers.
The preparation method of the multifunctional polymer micelle drug delivery system comprises the following steps:
(1) synthesis of high molecular material
Dissolving a polyethylene glycol-polylysine block copolymer in a boric acid buffer solution, simultaneously dissolving 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride in the boric acid buffer solution, mixing the two solutions at room temperature for reaction for 45 minutes, dialyzing a reaction product in phosphate with pH of 7.4 for three times, adding dithiothreitol for reaction, dialyzing the product with a phosphoric acid buffer solution and pure water for three times, and finally, freeze-drying to obtain a modified polyethylene glycol-polylysine block copolymer high polymer material;
(2) preparation of high molecular micelle by cross-linking medicine and high molecular material
Dissolving the modified polyethylene glycol-polylysine block copolymer in HEPES buffer solution, dissolving disodium mercaptododecaborane in the HEPES buffer solution, mixing the two solutions, stirring for reaction, dialyzing the product with the HEPES buffer solution and pure water respectively, and forming the drug-loaded disodium mercaptododecaborane-soluble high-molecular micelle through self-assembly.
The molar ratio of the polyethylene glycol-polylysine block copolymer used for preparing the ingredients in the step (1) to the 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride is 1: 1, but not limited to 1: 1. the molar ratio of 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride side chain modified polyethylene glycol-polylysine block copolymer to mercapto-dodecaborane disodium used for batching in the step (2) is 1: 1, however, the feed ratio of disodium mercaptododecaborane can be increased.
The multifunctional polymer micelle drug delivery system prepared by the invention is connected with drugs or prodrugs containing sulfhydryl groups through disulfide bonds, the controlled release function of the drugs is realized by responding to high-concentration glutathione in vivo, meanwhile, amino cations in the polymers can be protonated in the endocytosis process to realize the function of escaping endosomes or lysosomes, part of free sulfhydryl groups of the last part of free polymer side chains can enhance the stability of the polymer micelle under normal physiological conditions through crosslinking micelle cores, and the disulfide bonds can be broken through responding to glutathione in tumor cells to realize the dissociation of the crosslinked polymers. Therefore, the multifunctional polymer micelle drug delivery system can be applied to drug delivery, in particular to the application of radiotherapy and/or neutron capture therapy and/or chemotherapy drugs in the preparation of drugs for treating tumors or malignant tumors. The multifunctional polymer micelle drug delivery system is used as a drug carrier and is a drug delivery system with tumor targeting; the high molecular micelle can deliver radiotherapy drugs, chemotherapy drugs, cancer suppressor genes, nucleic acids and the like, and directionally deliver the drugs or the genes to a focus part, and is a tumor-targeted high molecular micelle.
The invention has the following beneficial effects:
1. the variety of the high-molecular micelle drug delivery system is increased, the application field of the high-molecular micelle is widened, and the research and development of the drug delivery system and a novel pharmaceutical preparation are facilitated.
2. The multifunctional polymer micelle drug delivery system has the disulfide bond capable of responding to high-concentration glutathione response in tumor cells, is used for crosslinking a micelle core and crosslinking with a drug, and can realize the functions of drug controlled release and micelle dissociation; the polycation in the macromolecule micelle has the function of destroying lysosomes and endosomes in the endocytosis process of tumor cells, realizes the purpose of high-efficiency drug delivery to the tumor cells, and solves the problems that the existing drugs are difficult to target the tumor cells and insufficient in drug delivery to the tumor cells.
3. The medicine preparation is novel, has good medicine delivery effect, and can target tumor cells.
4. The preparation method is simple, and the required equipment is conventional equipment.
Drawings
FIG. 1 is a graph showing the particle size distribution of polymer micelles in example 1;
FIG. 2 is a transmission electron microscope image of the appearance of the polymer micelle represented by example 1;
FIG. 3 is the results of example 4 for characterizing the drug release of polymeric micelles in response to glutathione and dithiothreitol;
FIG. 4a is the result of comparing the relative endocytosis of tumor cells to disodium mercaptododecaborane and drug-loaded polymeric micelles by example 4 using plasma mass spectrometry;
FIG. 4b is a graph showing the relative melanoma cell endocytosis of disodium mercaptododecaborane and drug-loaded polymeric micelles by tumor cells tested by plasma mass spectrometry in example 4;
FIG. 5a is a graph of the results of boron neutron capture therapy of breast cancer cells using disodium mercaptododecaborane and drug-loaded polymeric micelles of example 4;
FIG. 5b is a graph of the results of boron neutron capture therapy of melanoma cells using disodium mercaptododecaborane and drug-loaded polymeric micelles of example 4.
Detailed Description
The invention is further described below with reference to several examples. It is to be fully understood that the following examples are illustrative only, and are not intended to limit the invention in any way.
Example 1:
in the embodiment, a macromolecular micelle drug carrier system is constructed by taking a polyethylene glycol-polylysine block copolymer and 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride as raw materials, the used drug molecules are mercapto-dodecaborane disodium salt, and the process steps are as follows:
(1) synthesis of high molecular material
50mg of 3, 3' -dithiobisallylenediaminediamine dihydrochloride side chain-modified polyethylene glycol-polylysine block copolymer was dissolved in 15ml of HEPES buffer solution (10mM, pH7.4), 34mg of pharmaceutically acceptable disodium mercaptododecaborane was dissolved in 4ml of HEPES buffer solution (10mM, pH7.4), and then the two solutions were mixed and reacted with stirring for 6 hours, and then the product was dialyzed against HEPES buffer solution and pure water for 24 hours, respectively, to form a drug-loaded disodium mercaptododecaborane-soluble polymer micelle by self-assembly.
(2) Cross-linking of medicine and high-molecular material and preparation of high-molecular micelle
100mg of 3, 3' -dithiobisallylenediaminediamine dimethyl dihydrochloride side chain-modified polyethylene glycol-polylysine block copolymer was dissolved in 10ml of HEPES buffer solution (10mM, pH7.4), 75mg of pharmaceutically acceptable disodium mercaptododecaborane was dissolved in 10ml of HEPES buffer solution (10mM, pH7.4), and then the two solutions were mixed and reacted with stirring for 1 hour, and then the product was dialyzed with the HEPES buffer solution and pure water, respectively, for 24 hours to form a drug-loaded disodium mercaptododecaborane-soluble polymer micelle by self-assembly.
The particle size of the prepared polymer micelle is between 10 and 200nm, as shown in figure 1, and the obtained polymer micelle has the characteristic of uniform particle size distribution through the observation of a projection electron microscope, as shown in figure 2.
Example 2:
in the embodiment, a macromolecular micelle drug carrier system is constructed by taking a polyethylene glycol-polylysine block copolymer and 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride as raw materials, the used drug molecules are mercapto-dodecaborane disodium salt, and the process steps are as follows:
(1) synthesis of high molecular material
100mg of polyethylene glycol-polylysine block copolymer is dissolved in 10mL of boric acid buffer solution with the concentration of 100mM and the pH value of 9.0 at the temperature of 25 ℃, 50mg of 3,3 '-dithio-dipropionidine dimethyl dihydrochloride is dissolved in 12mL of boric acid buffer solution with the concentration of 100mM and the pH value of 9.0 at the same time, the two solutions are mixed and reacted for 60 minutes at room temperature, then the reaction product is dialyzed for three times in 10mM of phosphate with the pH value of 7.4, 70mg of dithiothreitol is added for reacting for 50 minutes, then the reaction product is dialyzed for three times by using phosphoric acid buffer solution and pure water, and finally the polyethylene glycol-polylysine block copolymer modified by the side chain of the 3, 3' -dithio-dipropionidine dimethyl dihydrochloride is obtained by freeze drying.
(2) Cross-linking of medicine and high-molecular material and preparation of high-molecular micelle
5mg of 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride side chain modified polyethylene glycol-polylysine block copolymer is dissolved in 1ml of HEPES buffer solution (10mM, pH7.4), sulfhydryl modified DNA plasmid is dissolved in 0.1ml of HEPES buffer solution (10mM, pH7.4), then the two solutions are mixed, stirred vigorously and reacted for 10 minutes, then 50 microliter of dimethyl sulfoxide is added, and the mixture is kept stand for 3-10 hours, and then the product is placed in an ultrafiltration centrifugal tube and washed and purified by pure water or HEPES buffer solution for 3-5 times to remove impurities, and finally the high molecular micelle wrapping the DNA plasmid is obtained.
Example 3:
in the embodiment, a macromolecular micelle drug carrier system is constructed by taking a polyethylene glycol-polylysine block copolymer and 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride as raw materials, the used drug molecules are mercapto-dodecaborane disodium salt, and the process steps are as follows:
(1) synthesis of high molecular material
100mg of polyethylene glycol-polylysine block copolymer is dissolved in 10mL of boric acid buffer solution with the concentration of 100mM and the pH value of 9.0 at the temperature of 25 ℃, 150mg of 3,3 '-dithio-dipropionidine dimethyl dihydrochloride is dissolved in 15mL of boric acid buffer solution with the concentration of 100mM and the pH value of 9.0, the two solutions are mixed and reacted for 120 minutes at room temperature, then the reaction product is dialyzed in 10mM of phosphate with the pH value of 7.4 for three times, then 200mg of dithiothreitol is added for reaction for 60 minutes, then the reaction product is dialyzed for three times by using phosphoric acid buffer solution and pure water, and finally the polyethylene glycol-polylysine block copolymer with the side chain modified by 3, 3' -dithio-dipropionidine dimethyl dihydrochloride is obtained by freeze drying.
(2) Cross-linking of medicine and high-molecular material and preparation of high-molecular micelle
Dissolving 5mg of 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride side chain modified polyethylene glycol-polylysine segmented copolymer in 2ml of HEPES buffer solution (10mM, pH7.4), dissolving 2mg of mercaptocamptothecin in 200 mu L of dimethyl sulfoxide, then placing the high molecular solution in ultrasound, rapidly adding mercaptocamptothecin solution, continuously performing ultrasound for 5 minutes, then standing the prepared high molecular micelle for 3-10 hours, then placing the product in an ultrafiltration centrifugal tube, washing and purifying the product with pure water or HEPES buffer solution for 3-5 times to remove impurities, and finally obtaining the high molecular micelle wrapping the DNA plasmid.
Example 4:
the application of the multifunctional polymer micelle in boron neutron capture treatment of tumors is characterized in that the polymer micelle loaded with the medicament disodium mercaptododecaborane prepared in the embodiment 1 is prepared into aqueous solution or normal saline solution, then the aqueous solution or the normal saline solution is injected into tumor-bearing mice through veins, the injection dosage is 100mg/kg, 24 hours after the medicament is injected, the tumor part is irradiated by hot neutron wires for 1 hour, and the dosage of the hot neutron wires is (1.6-2.2 multiplied by 10)12neutron/cm2) And then measuring the size of the tumor every two days to realize boron neutron capture treatment of the tumor.
The drug-loaded multifunctional polymer micelle disclosed by the invention has the advantages that the level (2-10mM) of glutathione or dithiothreitol with the same concentration under the condition of the interior of a tumor cell can be used for rapidly cutting off a disulfide bond connected between a drug and a polymer and releasing the drug from the polymer micelle, and under the normal physiological condition, the level of the glutathione content is lower (0.01mM) and the drug is not basically released, as shown in figure 3, by the mode, the targeted controlled release of the drug molecules, such as the tumor cell of disodium mercaptododecaborane, can be realized. The multifunctional polymer micelle can realize the controlled release of the sulfhydryl-containing drug or prodrug in response to the concentration of glutathione in tumor cells.
The multifunctional drug-loaded polymer micelle can deliver disodium mercaptododecaborane to tumor cells, and compared with the case that the polymer micelle is used to act on the tumor cells independently, the content of drugs in the tumor cells can be remarkably improved, for example, after the polymer micelle and the disodium mercaptododecaborane act on 4T1 breast cancer cell nucleus B16F10 melanoma cells for 24 hours, the content of the drugs in the tumor cells acted by the polymer micelle is more than 10 times that of a control group, as shown in fig. 4a and 4B. The multifunctional high molecular micelle has the functional design of escaping lysosome and endosome after being endocytosed by tumor cells, can deliver more drugs into the cells, and realizes the high-efficiency drug delivery of the targeted tumor cells.
The drug-loaded multifunctional polymer micelle can effectively kill cancer cells through boron neutron capture treatment, and the treatment effect is far better than that of disodium mercaptododecaborane, for example, after the drug-loaded polymer micelle and the disodium mercaptododecaborane (the content of drugs is 10 mu M) are respectively acted on 4T1 breast cancer cell nucleus B16F10 melanoma cells for 24 hours, the cells are irradiated by hot neutron rays for 1 hour, and then cultured for 24 hours to test the cell survival rate, the result shows that the drug-loaded multifunctional polymer micelle can kill most of tumor cells, and the disodium mercaptododecaborane hardly has a killing effect on the cells, as shown in fig. 5a and fig. 5B. The drug-loaded polymer micelle can effectively realize boron neutron capture treatment on tumors.
Claims (3)
1. The multifunctional high-molecular micelle drug delivery system is characterized in that the system is a high-molecular micelle formed by self-assembly and partial free disulfide bond crosslinking of a modified polyethylene glycol-polylysine block copolymer and a therapeutic drug with sulfydryl in an aqueous solution;
the modified polyethylene glycol-polylysine block copolymer is obtained by chemically modifying partial side chains of the polyethylene glycol-polylysine block copolymer with 3, 3' -dimethyl dithiodipropionate dihydrochloride;
the treatment drug with sulfydryl is sulfydryl dodecaborane disodium salt;
the synthesis process of the mercapto dodecaborane disodium salt crosslinked block copolymer comprises the following steps;
wherein m is the number of repeating units of polyethylene glycol, and the range of m is 3-500;
n is the total number of units of the polycation and ranges from 3 to 300;
x is the number of polylysine repeating units and ranges from 1 to 100;
y is the number of repeating units of 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride which is used for chemically modifying a polyethylene glycol-polylysine side chain and then is connected with a drug molecule mercapto-dodecaborane disodium salt through a disulfide bond, and the range of the repeating units is 1-100;
z is the number of repeating units of 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride for chemically modifying polyethylene glycol-polylysine side chains, and the range of the repeating units is 1-100.
2. The method for preparing the multifunctional polymeric micelle drug delivery system according to claim 1, comprising the steps of:
(1) synthesis of high molecular material
Dissolving a polyethylene glycol-polylysine block copolymer in a boric acid buffer solution, simultaneously dissolving 3, 3' -dithio-bis-alanine dimethyl ester dihydrochloride in the boric acid buffer solution, mixing the two solutions at room temperature for reaction for 45 minutes, dialyzing a reaction product in phosphate with pH of 7.4 for three times, adding dithiothreitol for reaction, dialyzing the product with a phosphoric acid buffer solution and pure water for three times, and finally, freeze-drying to obtain a modified polyethylene glycol-polylysine block copolymer high polymer material;
(2) preparation of high molecular micelle by cross-linking medicine and high molecular material
Dissolving the modified polyethylene glycol-polylysine block copolymer in HEPES buffer solution, dissolving disodium mercaptododecaborane serving as a medicine in the HEPES buffer solution, mixing the two solutions, stirring for reaction, dialyzing the product with the HEPES buffer solution and pure water respectively, and forming the high-molecular micelle carrying the disodium mercaptododecaborane through self-assembly.
3. The use of the multifunctional polymeric micelle drug delivery system according to claim 1 or 2, wherein the multifunctional polymeric micelle drug delivery system is used for preparing drugs for treating tumors.
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