CN114099705A - Hydralazine-based nano-drug for improving tumor microenvironment, and preparation and application thereof - Google Patents
Hydralazine-based nano-drug for improving tumor microenvironment, and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a hydralazine-based nano-drug for improving a tumor microenvironment, and preparation and application thereof, and belongs to the technical field of medicines. The nano-drug is micelle type nano-particles formed by self-assembling amphiphilic polymer and hydrophobic chemotherapeutic drug containing boric acid groups in water; the hydrophilic section of the amphiphilic polymer is polyethylene glycol, and the hydrophobic section of the amphiphilic polymer is a polymer formed by connecting hydralazine with a pH response type hydrazone bond. The nano-drug can efficiently release small-molecule hydralazine in the tumor acidic environment, and the hydralazine can improve the efficiency of delivering the entrapped chemotherapeutic drug to tumor tissues by expanding tumor blood vessels. In addition, the nano-drug can be loaded with chemotherapeutic drugs efficiently and released in the acidic tumor tissues in a concentrated manner, so that the treatment effect of the chemotherapeutic drugs is enhanced, the toxicity to normal tissues is low, and the tumor selectivity of the nano-drug is enhanced remarkably.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a hydralazine-based nano-medicine for improving a tumor microenvironment and enhancing a chemotherapy effect, and a preparation method and application thereof.
Background
The nanoparticles have been used as a substitute for non-targeted chemotherapeutic drugs due to their enhanced osmotic retention and retention effect (EPR effect) and effective reduction of toxic and side effects of chemotherapeutic drugs. However, the clinical transformation of these nano-drugs is greatly limited, mainly due to the complex tumor microenvironment. Tortuous and complex tumor vessels, which are a major component of the tumor microenvironment, are a major obstacle to nanoparticle delivery. Therefore, in order to enhance the therapeutic effect of nanoparticles and promote clinical transformation thereof, it is urgently required to develop a new strategy to improve the tumor microenvironment so as to enhance the therapeutic effect of nanoparticles.
During the past years, much research has focused on anti-angiogenic strategies that starve tumors of nutrients to inhibit tumor growth, such as the use of some anti-angiogenic inhibitors: sinomenine hydrochloride, bevacizumab, thalidomide, imatinib mesylate and the like. However, monotherapy with anti-angiogenic drugs is not as effective as expected, as disclosed in patent document No. 201610299834.6, which utilizes iron-metal organic framework compounds (Fe-MIL-101) to inhibit proliferation, migration and tubule formation of vascular endothelial cells, but tumor cells can still be nourished by other means, with limited therapeutic effect and clinical transformation. Therefore, anti-angiogenic strategies remain to be further improved. Meanwhile, a series of studies in recent years have focused on strategies for dilating blood vessels. Different from an anti-angiogenesis inhibitor, the vasodilator can permanently dilate tumor blood vessels, can be used for combined treatment with chemotherapeutic drugs to enhance delivery of nano-drugs, and has good clinical transformation prospect.
Hydralazine (HDZ) is a commonly used clinical antihypertensive drug and also has the effect of increasing tumor necrosis and inhibiting tumor growth. As a stable DNA methylation inhibitor, hydralazine has low toxicity in vitro and in vivo. The document "vasodialator hydrazine proteins nanoparticles in Advanced Desmoplastic liposomes" shows that Hydralazine loaded liposomes can act to dilate tumor vessels and increase the permeability of tumor tissues. However, this method still has some limitations, such as the need for repeated injections and non-specific release of hydralazine.
Bortezomib (BTZ), the first proteasome inhibitor approved for the treatment of cancer patients, is a dipeptide boronic acid analog that inhibits the proteasome of cancer cells by direct binding between its boronic acid group and a threonine residue in several protease active sites. However, bortezomib is less effective in treating many solid tumors due to significant dose-dependent toxicity of peripheral neuropathy, thrombocytopenia, and gastrointestinal disease. Also, bortezomib is limited in its pharmacokinetic properties due to its non-specific binding to proteins and rapid clearance in the blood.
Therefore, it is a problem to be solved by those skilled in the art to develop a novel nano-drug that can effectively expand tumor vessels to improve the tumor microenvironment, enhance the delivery efficiency of chemotherapeutic drugs such as bortezomib, and reduce their systemic toxicity.
Disclosure of Invention
The invention aims to provide a novel nano-drug which can improve the tumor microenvironment by expanding tumor blood vessels, thereby enhancing the efficiency of drug delivery to tumor tissues and further enhancing the treatment effect.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a hydralazine-based nano-drug for improving a tumor microenvironment, which is a micelle type nano-particle formed by self-assembling an amphiphilic polymer and a hydrophobic chemotherapeutic drug containing a boric acid group in water; the hydrophilic section of the amphiphilic polymer is polyethylene glycol, and the hydrophobic section of the amphiphilic polymer is a polymer formed by connecting hydralazine with a pH response type hydrazone bond.
According to the invention, a boric acid hydrophobic chemotherapeutic drug is wrapped by an amphiphilic polyhydralazine nano carrier, and in the process of self-assembling an amphiphilic polymer into a micelle, the hydrophobic hydralazine at the hydrophobic end of the amphiphilic polymer wraps the interior of the polymer micelle through the interaction of hydrophobic effect and boron-nitrogen coordination bonds, so that the nano drug is prepared.
When the hydrophobic segment of the amphiphilic polymer is constructed, N- (4-acetylphenyl) methacrylamide is used as a polymerizable monomer to participate in polymerization, and then hydralazine is connected to the polymer through reaction with the hydralazine to form a pH-responsive hydrazone bond. After the nano micelle enters the tumor tissue, under the acidic pH environment of the tumor tissue, hydrazone bonds are broken to release a large number of small molecules of hydralazine, and the microenvironment of the tumor is changed by expanding blood vessels, so that the delivery and release of chemotherapeutic drugs are accelerated. The process mainly occurs in acidic tumor tissues, so that the toxicity to normal healthy tissues is low, and the toxic and side effects of the system are greatly reduced.
Further, the preparation method of the amphiphilic polymer comprises the following steps: firstly, reacting p-aminoacetophenone with methacryloyl chloride under the catalysis of organic alkali to prepare N- (4-acetylphenyl) methacrylamide, then polymerizing the N- (4-acetylphenyl) methacrylamide by using a polyethylene glycol macromolecular chain transfer agent through a reversible addition-fragmentation chain transfer polymerization method or using a polyethylene glycol macromolecular chain initiator through an atom transfer radical polymerization method to prepare a polymer, and then connecting hydralazine to a polymer side chain through forming a hydrazone bond to prepare the amphiphilic polymer.
Preferably, the organic base is triethylamine.
Preferably, the polyethylene glycol macromolecular chain transfer agent is PETTC-PEG5k。
Preferably, the structural formula of the amphiphilic polymer is shown as a formula (I),
wherein y is 1-50 and m is 10-3000.
More preferably, y is 20-50, and m is 200-.
Furthermore, the chemotherapeutic drug is a hydrophobic chemotherapeutic drug with a boric acid group, and the chemotherapeutic drug can form a carbon-nitrogen coordination bond with hydralazine and is efficiently loaded in the amphiphilic nanoparticles due to hydrophilic-hydrophobic interaction. Under the acidic pH of the tumor, hydrazone bonds responding to the pH are broken, the nano micelle is disintegrated, and the chemotherapeutic drugs are quickly and efficiently released and activated. Such chemotherapeutic agents include, but are not limited to, bortezomib and ixazoib.
Further, the structural formula of the bortezomib is shown as a formula (II),
the structural formula of the ixazomide is shown as the formula (III):
the pH response polymer nano-carrier constructed by the invention has the characteristics of long circulation, high tumor accumulation and targeted drug delivery, can enhance the pharmacokinetic characteristic of chemotherapeutic drugs such as bortezomib and improve the curative effect of the chemotherapeutic drugs on solid tumors.
The invention also provides a method for preparing the hydralazine-based nano-drug for improving the tumor microenvironment. Specifically, the amphiphilic polymer and the chemotherapeutic agent are self-assembled in water to form the nano-particles by utilizing a preparation method of the polymer amphiphilic micelle, such as a solvent displacement method, a liquid membrane method, a dialysis method or an ultrasonic method.
Wherein the solvent displacement method comprises: firstly, dissolving amphiphilic polymer and hydrophobic chemotherapeutic drug in good solvent, then adding the mixed solution into water under the oscillation condition, and self-assembling the product to form the nano-drug.
Further, the mass ratio of the amphiphilic polymer to the hydrophobic chemotherapeutic drug is 2-5: 1. The entrapment efficiency of the amphiphilic polymer increases with the amount of the polymer, but the excessive amount of the polymer causes waste. Within the mass ratio range, higher drug coating rate and polymer utilization rate can be ensured.
The invention also aims to provide application of the hydralazine-based nano-drug for improving tumor microenvironment in preparation of tumor treatment drugs. The nano-drug provided by the invention can responsively release hydralazine in the acidic environment of tumor tissues, and the hydralazine can cause the change of the tumor microenvironment by expanding blood vessels, thereby accelerating the delivery and release of chemotherapeutic drugs. The chemotherapy drugs are wrapped inside the micelle type nanoparticles and are only released in the acidic tumor tissues in a concentrated manner, and the toxic and side effects on normal tissues are small, so that the nano-drug has potential application value in the development of tumor drugs.
Further, the tumor is a solid tumor. Still further, the solid tumors include, but are not limited to, breast cancer.
The invention has the following beneficial effects:
(1) the nano-drug provided by the invention can efficiently release small molecular hydralazine in the tumor acidic environment, so that the tumor microenvironment is remodeled by expanding tumor blood vessels, and the efficiency of delivering the entrapped chemotherapeutic drug to tumor tissues is further improved, therefore, the nano-drug is expected to be developed into a tumor treatment drug.
(2) The nano-drug provided by the invention can be efficiently loaded with boric acid hydrophobic chemotherapeutic drugs such as bortezomib and the like, and can be intensively released in tumor acidic tissues, so that the treatment effect of the chemotherapeutic drugs is enhanced, and the nano-drug has low toxicity to normal tissues, thereby obviously enhancing the tumor selectivity of the nano-drug.
Drawings
FIG. 1 is a schematic diagram of the formation of nano-drug PHDZ/BTZ in the example.
FIG. 2 is the dynamic light scattering diagram of PHDZ/BTZ of the nano-drug in the example.
FIG. 3 is the transmission electron micrograph of the nano-drug PHDZ/BTZ in the examples.
FIG. 4 is the HDZ release curves of the nano-drug PHDZ/BTZ at different pH values in the examples.
FIG. 5 is a drug release profile of the example nano-drug PHDZ/BTZ at different pH with the chemotherapeutic drug BTZ as an example.
FIG. 6 is the cytotoxicity of the nano-drug PHDZ/BTZ on different cell lines in the examples, wherein PHDZ is the cells treated with the carrier alone, Free HDZ + BTZ is the cells treated with two small molecule drugs, PAA/BTZ is the nano-drug treated cells formed by coating BTZ with PAA carrier not linked with HDZ, and Free BTZ is the cells treated with BTZ alone.
FIG. 7 is a representation of the apoptosis of the nano-drug PHDZ/BTZ on the 4T1 cell line in the examples.
FIG. 8 is an evaluation of the tumor suppression effect of the nano-drug PHDZ/BTZ in the example in the 4T1 tumor model of C57BL/6 mice, and is graphically represented as a tumor suppression curve.
FIG. 9 is an evaluation of the tumor suppression effect of the nano-drug PHDZ/BTZ in the example in the 4T1 tumor model in C57BL/6 mice, and is graphically shown as a photograph of the tumor at the end of the tumor suppression cycle.
FIG. 10 is an evaluation of the tumor suppression effect of the nano-drug PHDZ/BTZ in the example in the 4T1 tumor model of C57BL/6 mice, and is shown as a mouse weight change curve.
Detailed Description
The present invention is further illustrated by the following specific examples. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit or essential characteristics thereof.
The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The compounds referred to in the examples are described by the following abbreviations in English:
DCM-dichloromethane; AIBN-azobisisobutyronitrile; DMF-N, N-dimethylformamide.
Example 1
1. Preparation of nano medicine
(1) P-aminoacetophenone (4.0g,29.6mmol), methacryloyl chloride (3.6g,34.4mmol) and triethylamine (4.0g,39.6mmol) were dissolved in dichloromethane (DCM,100mL) and cooled with an ice bath. The mixture was stirred for 12 hours, then washed successively with saturated sodium bicarbonate solution, hydrochloric acid, distilled water and saturated sodium chloride solution, the solvent was removed by rotary evaporation, and N- (4-acetylphenyl) methacrylamide was isolated and purified by silica gel chromatography (N-hexane: ethyl acetate 4: 1). The final polymerizable monomer after vacuum drying was 2.8g (yield 46.7%). The reaction process is as follows:
(2) the polymer PAA was obtained by RAFT polymerization. Taking the polymerizable monomer N- (4-acetylphenyl) methacrylamide (0.30g,1.5mmol) obtained in the last step, and a macromolecular chain transfer agent PETTC-PEG5k(0.27g,0.05mmol) and AIBN (6.56mg,0.04mmol) were dissolved in dimethylformamide (DMF, 4mL) and treated with N at room temperature2Deoxygenation was carried out for 30 min and the reaction was stirred at 70 ℃ for 12 h. After the reaction is finished, the solution is precipitated in 200mL of ethyl glacial ether for 3 times and dried in vacuum to obtain the polymer PAA.
(3) PAA (0.20g) was mixed with excess hydralazine hydrochloride (0.20g,1.0mmol) in 10mL DMF. The pH of the solution was adjusted to 2 with hydrochloric acid and stirred at 70 ℃ for 6 hours. Dialysis with distilled water (Mw ═ 3000) for three days yielded the amphiphilic nanocarrier PHDZ (0.05g, 23%).
(4) Preparing PHDZ/BTZ by a coprecipitation method: PHDZ (20mg) and BTZ (10mg) were dissolved in 300. mu.L DMSO and the solution was added to 4mL deionized water with vigorous stirring. DMSO was then removed by dialysis (Mw 3500). The precipitated BTZ was removed by filtration. The self-assembly process is shown in figure 1.
2. Particle size analysis of nano-drugs
As shown in fig. 2, the average particle size of the nano-drug PHDZ/BTZ was measured to be 138.4nm (distribution coefficient PDI of 0.11) by Dynamic Light Scattering (DLS).
As shown in FIG. 3, the particle size of the nano-drug PHDZ/BTZ was observed to be around 140.0nm by Transmission Electron Microscopy (TEM), which is consistent with the particle size results measured by DLS.
3. In vitro release of HDZ and BTZ
PHDZ/BTZ (2.0mL) was sealed in a dialysis bag with a molecular weight cut-off of 3500Da and incubated in 40mL of PBS containing 2% Tween 80 at pH 7.4, 6.0, 5.0, respectively. At regular intervals, 100. mu.L of the solution outside the dialysis bag was collected and the HDZ and BTZ concentrations were measured by HPLC, respectively.
The tumor acidic pH release capacity of the drug is an important part for evaluating PHDZ/BTZ in vivo application, and the good response release capacity can ensure the full activation of the drug in a tumor area and then generate cytotoxicity and simultaneously reduce the toxicity to normal tissues.
As shown in fig. 4, 5, in PBS at pH 7.4, less than 20% of both HDZ and BTZ were released after 24h, and as pH decreased, in PBS at pH 5.0, about 40% of HDZ and about 70% of BTZ were released after 24 h.
4. Toxicity of PHDZ/BTZ on different cell lines
For the assessment of cytotoxicity on B16F10, 4T1 and NIH/3T3 Cell lines, PHDZ/BTZ, PHDZ, Free HDZ + BTZ, PAA/BTZ and Free BTZ, by using CCK8(Cell Counting Kit-8) assay. Wherein PHDZ is used for treating cells with a carrier alone, Free HDZ + BTZ is used for treating cells with two small molecule drugs, PAA/BTZ is nano drug treated cells formed by coating BTZ with HDZ-unattached carrier PAA, and Free BTZ is used for treating cells with BTZ alone.
Cells were seeded at a density of 5000 cells per well in 96-well plates and incubated overnight. The cells were exposed to serially diluted drugs and cultured for another 48 hours, and then the medium was changed to a mixed solution containing 180. mu.L of fresh medium and 20. mu.L of CCK-8. After incubation for 1.5 hours at 37 ℃, absorbance in each well was measured at 450nm using a microplate reader, and cell viability was obtained by calculating the ratio of absorbance values in the drug-added wells to the blank control.
As shown in FIG. 6, by cellular level toxicity analysis, we know that no significant inhibition of cell growth was observed in cells treated with PHDZ nanoparticles alone on the 4T1 cell line, and that the cytotoxicity of BTZ-loaded nanoparticles was significantly reduced compared to BTZ alone, which can be explained by the fact that BTZ can freely diffuse into tumor cells, whereas PHDZ/BTZ nanoparticles enter cells by endocytosis and slowly release BTZ at intracellular acidic pH. Compared to PAA/BTZ nanoparticles, PHDZ/BTZ nanoparticles showed moderate levels of cytotoxicity at high concentrations of BTZ, with similar trends also observed on B16F10 and 3T3 cell lines. The results show that the PHDZ/BTZ nanoparticles can effectively reduce the cytotoxicity of BTZ, and the drug carrier PHDZ nanoparticles have almost no cytotoxicity.
5. Apoptosis assay of PHDZ/BTZ on 4T1 cell line
Apoptosis of 4T1 cells was measured using the PI/Annexin V-FITC apoptosis kit and analyzed by flow cytometry. Specifically, cells were seeded 24 hours before the experiment in 12-well plates (5 × 10)5Individual cells/well). Then, the cells were treated with PHDZ/BTZ, PHDZ, Free HDZ + BTZ, PAA/BTZ and Free BTZ (BTZ concentration: 0.02. mu.g/mL, HDZ concentration: 0.05. mu.g/mL), respectively, and incubated for 24 hours. Thereafter, the collected cells were washed twice with cold PBS, stained with Annexin V-FITC and PI, and analyzed by flow cytometry.
As shown in fig. 7, fewer apoptotic cells were detected in cells treated with PHDZ/BTZ nanoparticles than in cells treated with free BTZ, and significantly lower levels of apoptosis were obtained in cells treated with PBS and PHDZ nanoparticles, consistent with the results of the CCK8 assay, indicating that PHDZ/BTZ nanoparticles effectively reduced BTZ cytotoxicity, indicating that the drug carrier PHDZ nanoparticles have good biosafety.
6. Evaluation of tumor-inhibiting Effect in 4T1 tumor model in C57BL/6 mice
C57BL/6 mice were injected subcutaneously with 5X 1054T1 cells. The tumor volume reaches 50mm3Left and right, mice were randomly assigned to 6 treatment groups (n ═ 5): PB (PB)S, Free BTZ, Free HDZ + BTZ, PHDZ, PAA/BTZ and PHDZ/BTZ. The HDZ equivalent dose was 5.0mg/kg, and the BTZ equivalent dose was 0.8 mg/kg. The drug was injected via tail vein once every four days for a total of 5 administrations. Tumor volume (mm) was calculated using the formula3): tumor volume (shortest diameter)2X (longest diameter) × 0.5.
By tumor suppression evaluation in the 4T1 subcutaneous tumor model in C57BL/6 mice. As shown in fig. 8-10, PHDZ/BTZ showed significantly enhanced tumor suppression compared to the BTZ group. Also, the mice lost less weight in the PHDZ/BTZ group than in the BTZ group due to the lower toxicity of the chemotherapeutic prodrug during its circulation in vivo.
Claims (9)
1. The nano-drug for improving the tumor microenvironment based on hydralazine is characterized in that the nano-drug is micelle type nano-particles formed by self-assembling amphiphilic polymer and hydrophobic chemotherapeutic drug containing boric acid groups in water; the hydrophilic section of the amphiphilic polymer is polyethylene glycol, and the hydrophobic section of the amphiphilic polymer is a polymer formed by connecting hydralazine with a pH response type hydrazone bond.
2. The hydralazine based nano-drug for improving tumor microenvironment of claim 1, wherein the preparation method of the amphiphilic polymer comprises the following steps: firstly, reacting p-aminoacetophenone with methacryloyl chloride under the catalysis of organic alkali to prepare N- (4-acetylphenyl) methacrylamide, then polymerizing the N- (4-acetylphenyl) methacrylamide by using a polyethylene glycol macromolecular chain transfer agent through a reversible addition-fragmentation chain transfer polymerization method or using a polyethylene glycol macromolecular chain initiator through an atom transfer radical polymerization method to prepare a polymer, and then connecting hydralazine to the polymer through forming a hydrazone bond to obtain the amphiphilic polymer.
3. The hydralazine-based nano-drug for improving tumor microenvironment according to claim 1, wherein the amphiphilic polymer has a structural formula shown in formula (I),
wherein y is 1-50 and m is 10-3000.
4. The hydralazine-based nano-drug for improving the microenvironment of tumors as claimed in claim 3, wherein y is 20-50, and m is 200-.
5. The hydralazine-based nanomedicine for improving the tumor microenvironment of claim 1, wherein the chemotherapeutic agent is bortezomib or ixazoib.
6. The method for preparing the hydralazine based nano-drug for improving the microenvironment of tumors according to any one of claims 1 to 5, comprising: firstly, dissolving amphiphilic polymer and hydrophobic chemotherapeutic drug in good solvent, then adding the mixed solution into water under the oscillation condition, and self-assembling the product to form the nano-drug.
7. The method of claim 6, wherein the mass ratio of amphiphilic polymer to hydrophobic chemotherapeutic agent is 2-5: 1.
8. Use of hydralazine based nanomedicine for improving the tumor microenvironment of a patient in the manufacture of a medicament for the treatment of tumors as described in any of claims 1 to 5.
9. The use of claim 8, wherein the tumor is a solid tumor.
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| CN115120573A (en) * | 2022-08-31 | 2022-09-30 | 上海浙江大学高等研究院 | High-efficiency anti-tumor nano drug-loading system containing hydralazine and preparation method thereof |
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