CN111529486A - Preparation method and application of dissociable nano micelle based on pH/MMP response - Google Patents
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Abstract
The invention relates to a preparation method and application of a 'dissociable' nano micelle based on pH/MMP response, which can realize the simultaneous regulation of tumor cells and TME and provide a new drug approach for chemotherapy-immunotherapy, and the method comprises the steps of synthesizing hyaluronic acid @ R of a targeting prodrug and then maleinizing the hyaluronic acid @ R; secondly, the maleimide prodrug is connected with a pH sensitive block poly (beta-amino ester) through matrix metalloproteinase sensitive peptide to form a block with pH/MMP double sensitivity, and an immunomodulator is wrapped through self-assembly to obtain the dissociative nano micelle based on pH/MMP response.
Description
Technical Field
The invention belongs to the field of medicinal preparations, and relates to a preparation method and application of a dissociable nano micelle based on pH/MMP response, which can efficiently respond to a tumor microenvironment.
Background
Tumor development and progression are the result of tumor cell and Tumor Microenvironment (TME) interactions, and conventional monotherapy modalities are often inadequate for clinical treatment needs. Traditional chemotherapy generally targets tumor cells, but neglects the improvement of TME, and combining chemotherapy and immunotherapy will greatly improve the anti-tumor therapeutic effect.
The development of nanotechnology brings new opportunity for the drug combination to generate a synergistic anti-tumor effect. However, the conventional nano drug delivery system generally co-delivers the drug to tumor cells or indiscriminately enters various cells, not only can not exert the maximum effect of drug combination, but also can cause the deterioration of TME, and provides various ways for the tumor to escape from the recognition and attack of the immune system. Tumor tissue has characteristics that are different from other normal tissues, such as: low pH (pH 6.4-6.8), high expression Matrix Metalloproteinases (MMPs) including MMP-2, MMP-9, high expression CD44 receptor, etc. Therefore, the detachable nanoparticles are designed based on the characteristics of the tumor microenvironment to deliver different drugs to corresponding target sites, and a new treatment way is provided for anti-tumor treatment by highly adjusting TME while accurately targeting tumor cells.
Poly (beta-aminoester), abbreviated as PAE or PBAE, is a widely studied pH sensitive material, which can be protonated when in a slightly acidic environment (pH 6.4-6.8), and has a structure that is converted from hydrophobic to hydrophilic, and good biocompatibility. In addition, some MMP-sensitive peptides can be cleaved by highly expressed MMP-2 or MMP-9 in the TME. Therefore, combining PAE with MMP sensitive peptides would greatly improve the responsiveness of the nanosystems to the microenvironment. Hyaluronic Acid (HA) is a non-toxic and biodegradable natural acidic polysaccharide, HAs strong affinity to CD44 receptor, is commonly used as a tumor-targeting ligand, forms a prodrug by combining with a drug, not only can accurately deliver the drug to tumor cells to improve the curative effect, but also can reduce the killing to immune effector cells. Therefore, HA as a hydrophilic moiety and linked to PAE through MMP sensitive peptides, forms pH/MMP dual-responsive nanomicelles by self-assembly, and is highly responsive to TME.
Anthracyclines, such as doxorubicin, paclitaxel, etc., can cause cell immunogenic death (ICD), release Tumor Associated Antigens (TAAs), and increase tumor immunogenicity. By connecting the anthracycline medicine with HA to form a prodrug, the efficiency of generating ICD by tumor cells can be improved, and the damage to immune effector cells is avoided. Some immunomodulators, such as celecoxib, NLG919, GW3965, etc., can improve TME, providing favorable conditions for the generation of antitumor immune effects. Therefore, the anthracycline and HA form a prodrug, the immunomodulator is loaded in the hydrophobic inner core of the nano micelle, when the nano micelle reaches a tumor part, the HA prodrug and the immunomodulator are released under the action of low pH and MMP, the HA prodrug accurately targets tumor cells, and the immunomodulator acts on TME, so that the accurate delivery of different drugs is realized, and the anti-tumor effect of the combination of the two drugs is amplified.
Disclosure of Invention
Aiming at the situation, the invention aims to provide a preparation method and application of a dissociable nano micelle based on pH/MMP response, which can realize simultaneous regulation of tumor cells and TME and provide a new drug approach for chemotherapy-immunotherapy. The dissociable nano micelle releases the prodrug and the immunomodulator in response to the TME, so that the prodrug accurately targets tumor cells, the action effect is improved, the immunomodulator improves the TME, the drug synergistic effect is obviously improved, and technical support is provided for the combined use of different target drugs.
In order to achieve the aim, the preparation method of the dissociative nano micelle based on the pH/MMP response comprises the steps of firstly, synthesizing hyaluronic acid @ R of a targeting prodrug, and then, maleimide-aminating the hyaluronic acid @ R; secondly, connecting the maleimide-treated prodrug with pH sensitive block poly (beta-amino ester) through matrix metalloproteinase sensitive peptide to form a block with pH/MMP double sensitivity, and wrapping an immunomodulator through self-assembly to obtain the 'dissociable' nano micelle based on pH/MMP response, which comprises the following specific steps:
(1) synthesis of hyaluronic acid prodrug (HA @ R): dissolving 480-500mg of hyaluronic acid in 15-25mL of formamide solution, adding 70-80mg of 4-Dimethylaminopyridine (DMAP) and 115-125mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC & HCl), stirring at 40 ℃ for 1h, and activating carboxyl of the hyaluronic acid; then, dissolving 50mg of antitumor drug R in 1mL of N, N-Dimethylformamide (DMF) solution, continuously stirring for 24h, after the reaction is finished, placing the reaction solution in a dialysis bag with MWCO:5000Da, dialyzing for 12h in 75% ethanol solution of volume concentration, dialyzing for 12h in 50% ethanol solution of volume concentration, then dialyzing for 48h in deionized water, removing unreacted substances, and freeze-drying the obtained solution to obtain a hyaluronic acid prodrug (HA @ R), and storing at 4 ℃ for later use;
the anti-tumor drug R is paclitaxel or adriamycin of an anthracycline drug;
(2) maleimide of hyaluronic acid prodrugs: dissolving 630-650mg of 6-maleimidoformic acid, 680-700mg of N-hydroxysuccinimide (NHS) and 1.18-1.2g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) in 20-25mL of N, N-Dimethylformamide (DMF) solution, stirring at room temperature for 1h, adding 480-500mg of hyaluronic acid prodrug (HA @ R), reacting at room temperature for 24h, placing the reaction solution in a dialysis bag with MWCO:5000Da, dialyzing in deionized water for 3 days, and freeze-drying to obtain the maleimido hyaluronic acid prodrug (Mal-HA @ R);
(3) synthesis of R @ HA-polypeptide (R @ HA-peptide): taking 80-100mg of polypeptide (SH-peptide-COOH) and 480-500mg of maleimide-aminated hyaluronic acid prodrug (Mal-HA @ R), stirring at room temperature for 24h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 2 days, and freeze-drying for later use;
the polypeptide is MMP-2 sensitive peptide (SH-CPLGGLAGG-COOH, SH-GPLGVRGG-COOH) or MMP-9 sensitive peptide (SH-CGPLGLPGKGG-COOH);
(4) synthesis of pH/MMP sensitive Block: 480-480 mg of R @ HA-peptide, 70-80mg of N-hydroxysuccinimide (NHS), 115-125mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) are taken to react for 1h, and 580-600mg of PAE-NH are added2Reacting for 24h, putting the reaction solution into an MWCO:5000Da dialysis bag, dialyzing in deionized water for 3 days, and freeze-drying to obtain R @ HA-peptide-PAE, namely a pH/MMP sensitive block;
(5) preparation of pH/MMP responsive "dissociable" nanomicelles: weighing 20mg of R @ HA-peptide-PAE and 4mg of immunomodulator, dissolving in 1ml of dimethyl sulfoxide (DMSO), dropwise adding deionized water until turbidity appears, stirring at room temperature for 6h, placing reaction liquid in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 12h, and filtering the obtained solution with a 0.45 mu m filter membrane to obtain a pH/MMP (metal-organic ligand) responsive dissociable nano micelle;
the immunomodulator is celecoxib, NLG919 or GW3965 which are medicines for regulating the tumor microenvironment.
The pH/MMP response-based dissociable nano-micelle prepared by the method is applied to the preparation of tumor microenvironment responsive release drugs, the preparation of co-delivery drugs with different targets or the preparation of chemotherapy-immunotherapy enhancing drugs.
The invention provides a dissociative nano micelle based on pH/MMP response, which has the advantages of simple and convenient preparation method, easily obtained raw materials, low production cost and high responsiveness of TME, and integrates chemotherapy and immunotherapy; the nano micelle uses PAE and MMP sensitive peptide as main components, and releases a prodrug and an immunomodulator by responding TME, so that the immunomodulator has a high regulating effect on the TME; the prodrug takes HA as a part combined with the drug, so that the effect of the drug can be improved, and the damage to immune effector cells can be avoided. The pH/MMP responsive dissociable nano micelle can effectively deliver two drugs to corresponding target sites, remarkably improve the treatment effect of the two drugs, provide a new drug for chemotherapy-immunotherapy, and has great economic and social benefits.
Detailed Description
The following examples and specific examples further illustrate the embodiments of the present invention.
In particular, the invention may be embodied as set forth in the following examples.
Example 1
In a specific embodiment of the invention, a preparation method of a dissociative nanomicelle based on pH/MMP response comprises the following steps:
(1) synthesis of hyaluronic acid prodrug (HA @ R): dissolving 490mg of hyaluronic acid in 20mL of formamide solution, adding 75mg of 4-Dimethylaminopyridine (DMAP) and 120mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC & HCl), stirring at 40 ℃ for 1h, and activating carboxyl groups of hyaluronic acid; then, 50mg of antitumor drug paclitaxel is dissolved in 1mL of N, N-Dimethylformamide (DMF) solution, stirring is continued for 24h, after the reaction is finished, the reaction solution is placed in a dialysis bag with MWCO:5000Da, dialyzed for 12h in 75% ethanol solution of volume concentration, dialyzed for 12h in 50% ethanol solution of volume concentration, then dialyzed for 48h in deionized water, unreacted substances are removed, the obtained solution is freeze-dried, and the hyaluronic acid prodrug is obtained and stored for later use at 4 ℃;
(2) maleimide of hyaluronic acid prodrugs: dissolving 640mg of 6-maleimidoformic acid, 690mg of N-hydroxysuccinimide (NHS) and 1.19g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) in 22.5mL of a N, N-Dimethylformamide (DMF) solution, stirring at room temperature for 1h, adding 490mg of a hyaluronic acid prodrug, reacting at room temperature for 24h, placing the reaction solution in a dialysis bag with MWCO:5000Da, dialyzing in deionized water for 3 days, and freeze-drying to obtain a maleimido hyaluronic acid prodrug;
(3) synthesis of R @ HA-polypeptide (R @ HA-peptide): taking 90mg of MMP-2 sensitive peptide (SH-GPLGVRGG-COOH) and 490mg of maleimide-based hyaluronic acid prodrug, stirring at room temperature for 24h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 2 days, and freeze-drying for later use;
(4) synthesis of pH/MMP sensitive Block: 490mg of R @ HA-peptide, 75mg of N-hydroxysuccinimide (NHS), 120mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) were taken to react for 1h, and 590mg of PAE-NH was added2Reacting for 24h, putting the reaction solution into an MWCO:5000Da dialysis bag, dialyzing in deionized water for 3 days, and freeze-drying to obtain R @ HA-peptide-PAE, namely a pH/MMP sensitive block;
(5) preparation of pH/MMP responsive "dissociable" nanomicelles: weighing 20mg of R @ HA-peptide-PAE and 4mg of celecoxib serving as an immunomodulator, dissolving the mixture in 1ml of dimethyl sulfoxide (DMSO), dropwise adding deionized water until turbidity appears, stirring the mixture at room temperature for 6 hours, placing reaction liquid in an MWCO:5000Da dialysis bag, dialyzing the mixture in the deionized water for 12 hours, and filtering the obtained solution by using a 0.45-micrometer filter membrane to obtain the pH/MMP-responsive dissociable nano micelle.
Example 2
In a specific embodiment of the invention, a preparation method of a dissociative nanomicelle based on pH/MMP response comprises the following steps:
(1) synthesis of hyaluronic acid prodrug (HA @ R): dissolving 480mg of hyaluronic acid in 15mL of formamide solution, adding 70mg of 4-Dimethylaminopyridine (DMAP) and 115mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC & HCl), stirring at 40 ℃ for 1h, and activating carboxyl of the hyaluronic acid; then, dissolving 50mg of antitumor drug adriamycin in 1mL of N, N-Dimethylformamide (DMF) solution, continuously stirring for 24h, after the reaction is finished, placing the reaction solution in a dialysis bag with MWCO:5000Da, dialyzing for 12h in 75% ethanol solution of volume concentration, dialyzing for 12h in 50% ethanol solution of volume concentration, then dialyzing for 48h in deionized water, removing unreacted substances, freeze-drying the obtained solution to obtain the hyaluronic acid prodrug, and storing at 4 ℃ for later use;
(2) maleimide of hyaluronic acid prodrugs: dissolving 630mg of 6-maleimidoformic acid, 680mg of N-hydroxysuccinimide (NHS) and 1.18g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) in 20mL of N, N-Dimethylformamide (DMF), stirring at room temperature for 1h, adding 480mg of hyaluronic acid prodrug, reacting at room temperature for 24h, placing the reaction solution in a dialysis bag with MWCO of 5000Da, dialyzing in deionized water for 3 days, and freeze-drying to obtain a maleimido hyaluronic acid prodrug;
(3) synthesis of R @ HA-polypeptide (R @ HA-peptide): stirring 80mg of MMP-2 sensitive peptide (SH-CPLGLAGG-COOH) and 480mg of maleimide hyaluronic acid prodrug at room temperature for 24h, putting the reaction solution into an MWCO:5000Da dialysis bag, dialyzing in deionized water for 2 days, and freeze-drying for later use;
(4) synthesis of pH/MMP sensitive Block: 480mg of R @ HA-peptide, 70mg of N-hydroxysuccinimide (NHS) and 115mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) are taken to react for 1h, and 580mg of PAE-NH is added2Reacting for 24h, putting the reaction solution into an MWCO:5000Da dialysis bag, dialyzing in deionized water for 3 days, and freeze-drying to obtain R @ HA-peptide-PAE, namely a pH/MMP sensitive block;
(5) preparation of pH/MMP responsive "dissociable" nanomicelles: weighing 20mg of R @ HA-peptide-PAE and an immunomodulator NLG 9194 mg, dissolving in 1ml of dimethyl sulfoxide (DMSO), dropwise adding deionized water until turbidity appears, stirring at room temperature for 6h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 12h, and filtering the obtained solution with a 0.45-micrometer filter membrane to obtain the pH/MMP-responsive dissociable nano micelle.
Example 3
In a specific embodiment of the invention, a preparation method of a dissociative nanomicelle based on pH/MMP response comprises the following steps:
(1) synthesis of hyaluronic acid prodrug: dissolving 500mg of hyaluronic acid in 25mL of formamide solution, adding 80mg of 4-Dimethylaminopyridine (DMAP) and 125mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC & HCl), stirring at 40 ℃ for 1h, and activating carboxyl groups of the hyaluronic acid; then, 50mg of antitumor drug paclitaxel is dissolved in 1mL of N, N-Dimethylformamide (DMF) solution, stirring is continued for 24h, after the reaction is finished, the reaction solution is placed in a dialysis bag with MWCO:5000Da, dialyzed for 12h in 75% ethanol solution of volume concentration, dialyzed for 12h in 50% ethanol solution of volume concentration, then dialyzed for 48h in deionized water, unreacted substances are removed, the obtained solution is freeze-dried, and the hyaluronic acid prodrug is obtained and stored for later use at 4 ℃;
(2) maleimide of hyaluronic acid prodrugs: dissolving 650mg of 6-maleimidoformic acid, 700mg of N-hydroxysuccinimide (NHS) and 1.2g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) in a 25mLN, N-Dimethylformamide (DMF) solution, stirring at room temperature for 1h, then adding 500mg of a hyaluronic acid prodrug, reacting at room temperature for 24h, placing the reaction solution in a dialysis bag with MWCO:5000Da, dialyzing in deionized water for 3 days, and freeze-drying to obtain a maleimido hyaluronic acid prodrug;
(3) synthesis of R @ HA-polypeptide (R @ HA-peptide): stirring 100mg of MMP-9 sensitive peptide (SH-CGPLGLPGKGG-COOH) and 500mg of maleimide-based hyaluronic acid prodrug at room temperature for 24h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 2 days, and freeze-drying for later use;
(4) synthesis of pH/MMP sensitive Block: 500mg of R @ HA-peptide, 80mg of N-hydroxysuccinimide (NHS) and 125mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) were taken to react for 1h, and 600mg of PAE-NH was added2Reacting for 24h, putting the reaction solution into an MWCO:5000Da dialysis bag, dialyzing in deionized water for 3 days, and freeze-drying to obtain R @ HA-peptide-PAE, namely a pH/MMP sensitive block;
(5) preparation of pH/MMP responsive "dissociable" nanomicelles: weighing 20mg of R @ HA-peptide-PAE and an immunomodulator GW 39654 mg, dissolving in 1ml of dimethyl sulfoxide (DMSO), dropwise adding deionized water until turbidity appears, stirring at room temperature for 6h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 12h, and filtering the obtained solution with a 0.45-micrometer filter membrane to obtain the pH/MMP-responsive dissociable nano micelle.
Firstly, the method comprises the following steps: experiment of pH/MMP response 'dissociable' nano micelle in vitro stability
In vitro TME simulation is carried out to determine the stability of the nano-micelle, 4 equal parts of micelle solution are respectively added into buffer solutions of pH7.4, pH7.4+ MMP-2, pH6.5 and pH6.5+ MMP-2 for incubation for a proper time, and the change of the shape and the particle size of the nano-micelle under different environments is evaluated through a transmission electron microscope (TME). The result shows that the nano-particles under the environment of pH7.4 and pH7.4+ MMP-2 have regular shapes, are spherical and have no significant difference; the appearance of the nanoparticle in the pH6.5 environment is not obviously changed, but the particle size is obviously increased, which indicates that PAE is protonated and the inner core becomes loose; the intact morphology of the nanoparticles at pH6.5+ MMP-2 was significantly disrupted. The result shows that the nano micelle has good pH/MMP-2 response characteristics.
Experiment of pH/MMP response for in vitro induction of tumor cell ICD by 'dissociable' nano micelle
After ICD development, the cells are thinHigh expression of Calreticulin (CRT) and increased release of high mobility histone (HMGB 1) on the cell surface 4T1 cells in logarithmic growth phase were tested as 2 × 10 in vitro using 4T1 breast cancer as a tumor cell model5The density of each well is inoculated in a 6-well plate, and the plate is placed in a constant temperature incubator overnight, so that the cells are fully attached to the wall. And (3) discarding the old culture medium, adding a medicament-containing culture medium containing PBS, HA-peptide-PAE, HA-peptide-PAE/CXB, PTX @ HA-peptide-PAE and PTX @ HA-peptide-PAE/CXB, and continuing to culture for 24h, wherein the concentration of PTX is 3 mu g/mL, and the concentration of CXB is 5 mu g/mL. Then, the supernatants and cells of each group were collected, the cells were resuspended in 100. mu.L of PBS, 1. mu.L of FITC-CRT antibody was added thereto, and incubated at 4 ℃ for 30min, and the cells were washed 3 times with PBS to remove excess antibody. And finally, resuspending the cell on a flow machine by using a proper amount of PBS to detect the rate of the CRT positive cells. In addition, the content of HMGB1 in the supernatant was determined according to the HMGB1 specification. The result shows that the CRT expression amount reaches 75.2% after the action of PTX @ HA-peptide-PAE/CXB, and the secretion amount of HMGB1 is 3 times of that of a blank group, which indicates that the nano micelle causes ICD.
Experiment of in vitro induction of Dendritic Cell (DC) maturation of pH/MMP responsive 'dissociable' nanomicelles
Bone marrow cells were extracted from mice and successfully induced into immature DC cells in vitro. According to the method (II), cell supernatants of the groups after the drug action are respectively cultured with DC cells successfully induced for 24h, then the DC cells of each group are collected, CD40 and CD86 fluorescent antibody are added for incubation at 4 ℃ for 30min, and excess antibody is removed by washing 3 times with PBS. Finally, resuspending with appropriate amount of PBS, and detecting each group of CD40 on a flow machine+CD86+Expression level of DC. The results showed that PTX @ HA-peptide-PAE/CXB group CD40+CD86+The expression level of the DC is the highest, which indicates that relevant antigens and danger signal molecules released by tumor cells treated by PTX @ HA-peptide-PAE/CXB can stimulate the maturation of DCs to a greater extent.
In vivo anti-tumor experiment of pH/MMP responsive dissociable nano micelle
Taking the tumor volume of about 100mm330 mice bearing 4T1 tumors were randomly divided into 6 groups of 5 mice each. In addition, with HA and deoxycholic acid (D)OCA) is used as a material for preparing non-sensitive traditional nano preparation entrapment model drugs PTX and celecoxib (PTX @ HA-DOCA/CXB) which are used as preparation controls. The experimental groups were as follows: (1) PBS, (2) HA-peptide-PAE, (3) HA-peptide-PAE/CXB, (4) PTX @ HA-peptide-PAE, (5) PTX @ HA-DOCA/CXB, (6) PTX @ HA-peptide-PAE/CXB. The dose of PTX is 10mg/kg and CXB is 5mg/kg, and the tail vein administration is carried out every other day for 5 times of continuous administration. During dosing, mice were fed normally and weighed, and tumor volumes were monitored and recorded for each group of mice. The results show that the blank group and the single-drug treatment group have rapid tumor growth, the two drug combination groups can inhibit the tumor growth, and compared with PTX @ HA-DOCA/CXB (traditional nano-drug administration group), the PTX @ HA-peptide-PAE/CXB (dissociative nano-drug administration group) can inhibit the tumor growth more obviously. The result shows that the PTX @ HA-peptide-PAE/CXB releases the HA @ PTX prodrug and the CXB in response to the TME, so that the tumor cells are accurately targeted, the TME is highly regulated and controlled, and the anti-tumor treatment effect is remarkably enhanced.
Experiment of lung metastasis inhibition effect of pH/MMP response 'dissociable' nano micelle
And (3) establishing a mouse lung metastasis model, administering according to the groups, dissecting each group of mice after administration is finished, taking out lung tissues, and performing statistical analysis on each group of lung tissue nodules. The results show that the number of the nodules of the mice in the single-drug treatment group containing the PTX is increased, the number of the lung nodules of the mice in the two-drug combination group can be reduced, and compared with the traditional nano-preparation administration group, the number of the lung nodules of the mice in the PTX @ HA-peptide-PAE/CXB group is reduced more remarkably, and the action mode HAs certain superiority compared with the traditional administration group.
The same experiment as described above was carried out for examples 2 and 3 in addition to the experiment described above for example 1, and the same and similar results were obtained, which are not listed here.
Experiments show that compared with the prior art, the invention has the following outstanding beneficial technical effects:
(1) the pH/MMP responsive dissociable nano micelle and the pharmaceutical composition thereof utilize targeted HA and TME responsive PAE and MMP sensitive peptide as components. The nano micelle is kept stable in the in vivo circulation process, highly responds to TME after reaching a tumor part, and realizes the release of the medicine as required.
(2) According to the pH/MMP responsive dissociable nano micelle and the pharmaceutical composition thereof, HA is used as a hydrophilic part of the nano micelle and is also part of a prodrug, the micelle is disintegrated when reaching a tumor position to release the HA prodrug, and HA and CD44 highly expressed on the surface of a tumor cell have higher affinity, so that the drug can be accurately delivered to the tumor cell, the curative effect is improved, and the damage to immune effector cells is avoided.
(3) The pH/MMP responsive dissociable nano micelle and the pharmaceutical composition thereof realize the double targeting effect of tumor cells and TME by accurately delivering different drugs to corresponding target sites, remarkably enhance the effect of combining the two drugs and avoid adverse side effects in chemotherapy.
In a word, the invention provides a dissociative nano micelle based on pH/MMP response, has a simple preparation method, low production cost and high responsiveness of TME, and integrates chemotherapy and immunotherapy; the nano micelle uses PAE and MMP sensitive peptide as preparation components, and realizes the high regulation effect of the immunomodulator on TME by responding to TME to release a prodrug and the immunomodulator; the prodrug takes HA as a part combined with the drug, so that the effect of the drug can be improved, and the damage to immune effector cells can be avoided. The pH/MMP responsive dissociable nano micelle can effectively deliver two drugs to corresponding target sites, remarkably improves the treatment effect of the two drugs, is effectively used for preparing tumor microenvironment responsive release drugs, preparing co-delivery drugs with different target sites or preparing enhanced chemotherapy-immunotherapy drugs, provides new drugs for chemotherapy-immunotherapy, and has huge economic and social benefits.
Claims (7)
1. A preparation method of 'dissociable' nano-micelle based on pH/MMP response is characterized in that firstly, hyaluronic acid @ R of a targeting prodrug is synthesized, and then the targeting prodrug is subjected to maleimide treatment; secondly, connecting the maleimide-treated prodrug with pH sensitive block poly (beta-amino ester) through matrix metalloproteinase sensitive peptide to form a block with pH/MMP double sensitivity, and wrapping an immunomodulator through self-assembly to obtain the 'dissociable' nano micelle based on pH/MMP response, which comprises the following specific steps:
(1) synthesis of hyaluronic acid prodrug: dissolving 480-500mg of hyaluronic acid in 15-25mL of formamide solution, adding 70-80mg of 4-dimethylaminopyridine and 115-125mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, stirring at 40 ℃ for 1h, and activating carboxyl of the hyaluronic acid; then, dissolving 50mg of antitumor drug R in 1mL of N, N-dimethylformamide solution, continuously stirring for 24h, after the reaction is finished, placing the reaction solution in a dialysis bag with MWCO:5000Da, dialyzing for 12h in 75% ethanol solution of volume concentration, dialyzing for 12h in 50% ethanol solution of volume concentration, then dialyzing for 48h in deionized water, removing unreacted substances, and freeze-drying the obtained solution to obtain the hyaluronic acid prodrug, and storing at 4 ℃ for later use;
the anti-tumor drug R is paclitaxel or adriamycin of an anthracycline drug;
(2) maleimide of hyaluronic acid prodrugs: dissolving 630-650mg of 6-maleimidoformic acid, 680-700mg of N-hydroxysuccinimide and 1.18-1.2g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in 20-25mL of N, N-dimethylformamide solution, stirring at room temperature for 1h, then adding 480-500mg of hyaluronic acid prodrug, reacting at room temperature for 24h, placing the reaction solution in a dialysis bag with MWCO of 5000Da, dialyzing in deionized water for 3 days, and freeze-drying to obtain the maleimide hyaluronic acid prodrug;
(3) synthesis of R @ HA-polypeptide (R @ HA-peptide): taking 80-100mg of polypeptide (SH-peptide-COOH) and 480-500mg of maleimide-aminated hyaluronic acid prodrug, stirring at room temperature for 24h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 2 days, and freeze-drying for later use;
the polypeptide is MMP-2 sensitive peptide (SH-CPLGGLAGG-COOH, SH-GPLGVRGG-COOH) or MMP-9 sensitive peptide (SH-CGPLGLPGKGG-COOH);
(4) synthesis of pH/MMP sensitive Block: 500mg of R @ HA-peptide, 70-80mg of N-hydroxysuccinimide, 115-125mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are taken for reaction for 1h, and 580-600mg of PAE-NH are added2Reacting for 24h, putting the reaction solution into an MWCO:5000Da dialysis bag, dialyzing in deionized water for 3 days, and freeze-drying to obtain R @ HA-peptide-PAE, namely a pH/MMP sensitive block;
(5) preparation of pH/MMP responsive "dissociable" nanomicelles: weighing 20mg of R @ HA-peptide-PAE and 4mg of immunomodulator, dissolving in 1ml of dimethyl sulfoxide, dropwise adding deionized water until turbidity appears, stirring at room temperature for 6h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 12h, and filtering the obtained solution with a 0.45 mu m filter membrane to obtain a pH/MMP (metal-organic ligand) responsive dissociable nano micelle;
the immunomodulator is celecoxib, NLG919 or GW3965 which are medicines for regulating the tumor microenvironment.
2. The method for preparing "dissociable" nanomicelles based on pH/MMP response of claim 1, characterized in that it comprises the following steps:
(1) synthesis of hyaluronic acid prodrug: dissolving 490mg of hyaluronic acid in 20mL of formamide solution, adding 75mg of 4-dimethylaminopyridine and 120mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, stirring at 40 ℃ for 1h, and activating carboxyl of hyaluronic acid; subsequently, 50mg of antitumor drug paclitaxel is dissolved in 1mL of N, N-dimethylformamide solution, stirring is continued for 24h, after the reaction is finished, the reaction solution is placed in a dialysis bag with MWCO:5000Da, dialyzed for 12h in 75% ethanol solution of volume concentration, dialyzed for 12h in 50% ethanol solution of volume concentration, dialyzed for 48h in deionized water, unreacted substances are removed, and the obtained solution is freeze-dried to obtain a hyaluronic acid prodrug, which is stored for later use at 4 ℃;
(2) maleimide of hyaluronic acid prodrugs: dissolving 640mg of 6-maleimidoformic acid, 690mg of N-hydroxysuccinimide and 1.19g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in 22.5mL of N, N-dimethylformamide solution, stirring at room temperature for 1h, adding 490mg of hyaluronic acid prodrug, reacting at room temperature for 24h, placing the reaction solution in a dialysis bag with MWCO of 5000Da, dialyzing in deionized water for 3 days, and freeze-drying to obtain a maleimido hyaluronic acid prodrug;
(3) synthesis of R @ HA-polypeptide (R @ HA-peptide): taking 90mg of MMP-2 sensitive peptide (SH-GPLGVRGG-COOH) and 490mg of maleimide-based hyaluronic acid prodrug, stirring at room temperature for 24h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 2 days, and freeze-drying for later use;
(4) synthesis of pH/MMP sensitive Block: 490mg of R @ HA-peptide, 75mg of N-hydroxysuccinimide, 120mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are taken to react for 1h, and 590mg of PAE-NH is added2Reacting for 24h, putting the reaction solution into an MWCO:5000Da dialysis bag, dialyzing in deionized water for 3 days, and freeze-drying to obtain R @ HA-peptide-PAE, namely a pH/MMP sensitive block;
(5) preparation of pH/MMP responsive "dissociable" nanomicelles: weighing 20mg of R @ HA-peptide-PAE and 4mg of celecoxib serving as an immunomodulator, dissolving the mixture in 1ml of dimethyl sulfoxide, dropwise adding deionized water until turbidity appears, stirring the mixture at room temperature for 6 hours, placing reaction liquid in an MWCO:5000Da dialysis bag, dialyzing the mixture in the deionized water for 12 hours, and filtering the obtained solution by using a 0.45-micrometer filter membrane to obtain the pH/MMP (metal-organic phosphate) responsive dissociable nano micelle.
3. The method for preparing "dissociable" nanomicelles based on pH/MMP response of claim 1, characterized in that it comprises the following steps:
(1) synthesis of hyaluronic acid prodrug: dissolving 480mg of hyaluronic acid in 15mL of formamide solution, adding 4-dimethylaminopyridine 70mg and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride 115mg, stirring at 40 ℃ for 1h, and activating carboxyl of the hyaluronic acid; then, dissolving 50mg of antitumor drug adriamycin in 1mL of N, N-dimethylformamide solution, continuously stirring for 24h, after the reaction is finished, placing the reaction solution in a dialysis bag with MWCO:5000Da, dialyzing for 12h in 75% ethanol solution of volume concentration, dialyzing for 12h in 50% ethanol solution of volume concentration, then dialyzing for 48h in deionized water, removing unreacted substances, and freeze-drying the obtained solution to obtain the hyaluronic acid prodrug, and storing at 4 ℃ for later use;
(2) maleimide of hyaluronic acid prodrugs: dissolving 630mg of 6-maleimidoformic acid, 680mg of N-hydroxysuccinimide and 1.18g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in 20mL of N, N-dimethylformamide solution, stirring at room temperature for 1h, adding 480mg of hyaluronic acid prodrug, reacting at room temperature for 24h, placing the reaction solution in a dialysis bag with MWCO of 5000Da, dialyzing in deionized water for 3 days, and freeze-drying to obtain a maleimido hyaluronic acid prodrug;
(3) synthesis of R @ HA-polypeptide (R @ HA-peptide): stirring 80mg of MMP-2 sensitive peptide (SH-CPLGLAGG-COOH) and 480mg of maleimide hyaluronic acid prodrug at room temperature for 24h, putting the reaction solution into an MWCO:5000Da dialysis bag, dialyzing in deionized water for 2 days, and freeze-drying for later use;
(4) synthesis of pH/MMP sensitive Block: 480mg of R @ HA-peptide, 70mg of N-hydroxysuccinimide, 115mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are taken to react for 1h, and 580mg of PAE-NH are added2Reacting for 24h, putting the reaction solution into an MWCO:5000Da dialysis bag, dialyzing in deionized water for 3 days, and freeze-drying to obtain R @ HA-peptide-PAE, namely a pH/MMP sensitive block;
(5) preparation of pH/MMP responsive "dissociable" nanomicelles: weighing 20mg of R @ HA-peptide-PAE and an immunomodulator NLG 9194 mg, dissolving in 1ml of dimethyl sulfoxide, dropwise adding deionized water until turbidity appears, stirring at room temperature for 6h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 12h, and filtering the obtained solution with a 0.45-micrometer filter membrane to obtain the pH/MMP-responsive dissociable nano micelle.
4. The method for preparing "dissociable" nanomicelles based on pH/MMP response of claim 1, characterized in that it comprises the following steps:
(1) synthesis of hyaluronic acid prodrug: dissolving 500mg of hyaluronic acid in 25mL of formamide solution, adding 80mg of 4-dimethylaminopyridine and 125mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, stirring at 40 ℃ for 1h, and activating carboxyl of the hyaluronic acid; subsequently, 50mg of antitumor drug paclitaxel is dissolved in 1mL of N, N-dimethylformamide solution, stirring is continued for 24h, after the reaction is finished, the reaction solution is placed in a dialysis bag with MWCO:5000Da, dialyzed for 12h in 75% ethanol solution of volume concentration, dialyzed for 12h in 50% ethanol solution of volume concentration, dialyzed for 48h in deionized water, unreacted substances are removed, and the obtained solution is freeze-dried to obtain a hyaluronic acid prodrug, which is stored for later use at 4 ℃;
(2) maleimide of hyaluronic acid prodrugs: dissolving 650mg of 6-maleimidoformic acid, 700mg of N-hydroxysuccinimide and 1.2g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in 25mL of N, N-dimethylformamide solution, stirring at room temperature for 1h, then adding 500mg of hyaluronic acid prodrug, reacting at room temperature for 24h, then placing the reaction solution in a dialysis bag with MWCO of 5000Da, dialyzing in deionized water for 3 days, and freeze-drying to obtain a maleimido hyaluronic acid prodrug;
(3) synthesis of R @ HA-polypeptide (R @ HA-peptide): stirring 100mg of MMP-9 sensitive peptide (SH-CGPLGLPGKGG-COOH) and 500mg of maleimide-based hyaluronic acid prodrug at room temperature for 24h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 2 days, and freeze-drying for later use;
(4) synthesis of pH/MMP sensitive Block: 500mg of R @ HA-peptide, 80mg of N-hydroxysuccinimide, 125mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are taken to react for 1h, and 600mg of PAE-NH is added2Reacting for 24h, putting the reaction solution into an MWCO:5000Da dialysis bag, dialyzing in deionized water for 3 days, and freeze-drying to obtain R @ HA-peptide-PAE, namely a pH/MMP sensitive block;
(5) preparation of pH/MMP responsive "dissociable" nanomicelles: weighing 20mg of R @ HA-peptide-PAE and an immunomodulator GW 39654 mg, dissolving in 1ml of dimethyl sulfoxide, dropwise adding deionized water until turbidity appears, stirring at room temperature for 6h, placing the reaction solution in an MWCO:5000Da dialysis bag, dialyzing in deionized water for 12h, and filtering the obtained solution with a 0.45 mu m filter membrane to obtain the pH/MMP-responsive dissociable nano micelle.
5. Use of pH/MMP response based "dissociable" nanomicelles prepared by the method of claims 1-4 in the preparation of tumor microenvironment responsive release drugs.
6. Use of pH/MMP response based "dissociable" nanomicelles prepared by the method of claims 1-4 for the preparation of co-delivered drugs at different targets.
7. Use of pH/MMP response based "dissociable" nanomicelles prepared by the method of claims 1-4 for the preparation of a medicament for enhancing chemotherapy-immunotherapy.
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