CN114099705B - Nanometer medicine based on hydralazine for improving tumor microenvironment and preparation and application thereof - Google Patents

Nanometer medicine based on hydralazine for improving tumor microenvironment and preparation and application thereof Download PDF

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CN114099705B
CN114099705B CN202111368689.XA CN202111368689A CN114099705B CN 114099705 B CN114099705 B CN 114099705B CN 202111368689 A CN202111368689 A CN 202111368689A CN 114099705 B CN114099705 B CN 114099705B
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唐建斌
王睿
徐晓丹
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ZJU Hangzhou Global Scientific and Technological Innovation Center
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Abstract

The invention discloses a nano-drug for improving tumor microenvironment based on hydralazine, and preparation and application thereof, and belongs to the technical field of medicines. The nano-drug is micelle type nano-particles formed by self-assembly of amphiphilic polymer and hydrophobic chemotherapeutic drug containing boric acid groups in water; the hydrophilic segment of the amphiphilic polymer is polyethylene glycol, and the hydrophobic segment is a polymer formed by connecting hydralazine with a pH-responsive hydrazone bond. The nano-drug can release small molecular hydralazine with high efficiency in the acidic environment of tumor, and the hydralazine can improve the efficiency of the entrapped chemotherapy drug to the tumor tissue by expanding tumor blood vessels. In addition, the nano-drug can be loaded with the chemotherapeutic drug at high efficiency and intensively released in the tumor acidic tissue, thereby enhancing the treatment effect of the chemotherapeutic drug, having smaller toxicity to normal tissues and remarkably enhancing the tumor selectivity of the nano-drug.

Description

Nanometer medicine based on hydralazine for improving tumor microenvironment and preparation and application thereof
Technical Field
The invention relates to the technical field of medicines, in particular to a nano-drug for improving tumor microenvironment and enhancing chemotherapy effect based on hydralazine, and a preparation method and application thereof.
Background
Nanoparticles have become a substitute for non-targeted chemotherapeutic drugs due to their enhanced osmotic retention effect (enhanced permeability and retention effect, EPR effect) and effective reduction of the toxic side effects of chemotherapeutic drugs. However, the clinical transformation of these nanomedicines is greatly limited, mainly due to the complex tumor microenvironment. As a major component of the tumor microenvironment, tortuous and complex tumor vessels are major obstacles to nanoparticle delivery. Therefore, in order to enhance the therapeutic effect of nanoparticles and promote their clinical transformation, there is a need to develop new strategies to improve tumor microenvironment and thereby enhance the therapeutic effect of nanoparticles.
Over the past several years, many studies have focused on anti-angiogenic strategies that starve tumors 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 application 201610299834.6 using an iron metal organic framework compound (Fe-MIL-101) to inhibit proliferation, migration and tubule formation of vascular endothelial cells, but tumor cells can still be nourished by other means, therapeutic effects and clinical transformations thereof are limited. Thus, anti-angiogenic strategies remain to be further improved. At the same time, a series of recent studies have turned the eye towards strategies for dilating blood vessels. Unlike anti-angiogenic inhibitors, vasodilators can permanently dilate tumor vessels, can also be used in combination therapy with chemotherapeutic drugs, enhance the delivery of nanomaterials, and have good clinical transformation prospects.
Hydralazine (HDZ) is a commonly used clinical antihypertensive drug and also has the effects 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 "Vasodilator Hydralazine Promotes Nanoparticle Penetration in Advanced Desmoplastic Tumors" shows that the hydrazinodrozine loaded liposomes can act to dilate tumor vessels and increase permeability of tumor tissue. However, this approach still has some limitations, such as the need for repeated injections and nonspecific release of hydralazine.
Bortezomib (BTZ) is the first proteasome inhibitor approved for the treatment of cancer patients, a dipeptide boronic acid analog that inhibits the proteasome of cancer cells by direct binding between its boronic acid group and threonine residues in several protease active sites. However, bortezomib has a lower therapeutic effect on many solid tumors due to significant dose-dependent toxicity, such as peripheral neuropathy, thrombocytopenia, and gastrointestinal diseases. Also, bortezomib has limited pharmacokinetic properties due to its non-specific binding to proteins and rapid clearance in blood.
Therefore, the development of a novel nano-drug which can effectively dilate tumor blood vessels to improve tumor microenvironment and enhance the delivery efficiency of chemotherapeutic drugs such as bortezomib and reduce systemic toxicity is a problem to be solved by the technicians in the field.
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 above purpose, the invention adopts the following technical scheme:
the invention provides a nano-drug for improving tumor microenvironment based on hydralazine, which is micelle type nano-particles formed by self-assembly of amphiphilic polymer and hydrophobic chemotherapeutic drug containing boric acid groups in water; the hydrophilic segment of the amphiphilic polymer is polyethylene glycol, and the hydrophobic segment is a polymer formed by connecting hydralazine with a pH-responsive hydrazone bond.
According to the invention, the boric acid hydrophobic chemotherapeutic drug is wrapped by the nano-carrier of the amphiphilic polyhydrazide, and the hydrophobic end hydrazine drozine is wrapped in the polymer micelle through the interaction of the hydrophobic interaction and the boron-nitrogen coordination bond in the process that the amphiphilic polymer is self-assembled into the micelle, so that the nano-drug is prepared.
In the invention, when constructing the hydrophobic segment of the amphiphilic polymer, N- (4-acetylphenyl) methacrylamide is taken as a polymerizable monomer to participate in polymerization, and then the hydralazine is connected to the polymer through a hydrazone bond which reacts with the hydralazine to form a pH response. When the nano micelle enters into tumor tissue, hydrazone bond is broken to release a large amount of micromolecular hydralazine under the acidic pH environment of the tumor tissue, and the change of tumor microenvironment is caused 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, under the condition of organic base catalysis, p-aminoacetophenone reacts with methacryloyl chloride to prepare N- (4-acetylphenyl) methacrylamide, then a polyethylene glycol macromolecular chain transfer agent is utilized to polymerize the N- (4-acetylphenyl) methacrylamide by a reversible addition-fragmentation chain transfer polymerization method or a polyethylene glycol macromolecular chain initiator is utilized to prepare a polymer by an atom transfer radical polymerization method, and then hydrazone bond is formed to connect hydrazine drozine to a polymer side chain to prepare the amphiphilic polymer.
Preferably, the organic base is triethylamine.
Preferably, the polyethylene glycol macromolecular chain transfer agent is PETTC-PEG 5k 。
Preferably, the structural formula of the amphiphilic polymer is shown as a formula (I),
Figure BDA0003361795340000031
wherein y=1-50 and m=10-3000.
More preferably, y=20-50, m=200-500.
Furthermore, the chemotherapeutic agent is a hydrophobic chemotherapeutic agent with a boric acid group, and can form a carbon-nitrogen coordination bond with the hydralazine and is efficiently loaded in the amphiphilic nano particle due to hydrophilic-hydrophobic interaction. At the acidic pH of the tumor, the pH-responsive hydrazone bond breaks, the nanomicelle disintegrates, and the chemotherapeutic agent is released and activated rapidly and efficiently. Such chemotherapeutic agents include, but are not limited to, bortezomib and ifenprodil Sha Zuomi.
Further, the structural formula of bortezomib is shown as a formula (II),
Figure BDA0003361795340000032
the structural formula of the I Sha Zuomi is shown as a formula (III):
Figure BDA0003361795340000041
the pH response polymer nano-carrier constructed by the invention has the characteristics of long circulation, high tumor accumulation and targeted drug delivery, and can enhance the pharmacokinetic characteristics 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 nano-drug based on the hydralazine for improving the tumor microenvironment. Specifically, the amphiphilic polymer and the chemotherapeutic agent are self-assembled in water to form nano particles by using a preparation method of the polymer amphiphilic micelle, such as a solvent replacement method, a liquid membrane method, a dialysis method or an ultrasonic method.
Wherein the solvent displacement method comprises: firstly, amphiphilic polymer and hydrophobic chemotherapeutic drug are dissolved in a good solvent, then the mixed solution is added into water under the oscillation condition, and the product self-assembles to form the nano drug.
Further, the mass ratio of the amphiphilic polymer to the hydrophobic chemotherapeutic agent is 2-5:1. The packing efficiency of the amphiphilic polymer increases with the increase of the polymer dosage, but excessive polymer dosage causes waste. In the mass ratio range, higher medicine packing rate and polymer utilization rate can be ensured.
The invention also aims to provide an application of the nano-drug based on hydralazine for improving tumor microenvironment in preparation of tumor treatment drugs. The nano-drug provided by the invention can responsively release the hydralazine in the acidic environment of tumor tissues, and the hydralazine can cause the change of tumor microenvironment by expanding blood vessels, so that the delivery and release of chemotherapeutic drugs can be accelerated. The chemotherapeutic medicine is wrapped in the micelle type nano particles and only intensively released in the tumor acidic tissues, and has small toxic and side effects on normal tissues, so that the nano medicine has potential application value in the development of tumor medicines.
Further, the tumor is a solid tumor. Still further, the solid tumors include, but are not limited to, breast cancer.
The invention has the beneficial effects that:
(1) The nano-drug provided by the invention can release the micromolecular hydralazine with high efficiency in the acidic environment of tumor, thereby remodelling the tumor microenvironment by expanding tumor blood vessels and further improving the efficiency of delivering the entrapped chemotherapeutic drug to tumor tissues, thus being hopeful to be developed into tumor therapeutic drugs.
(2) The nano-drug provided by the invention can be used for efficiently loading boric acid hydrophobic chemotherapeutic drugs such as bortezomib and the like, and is intensively released in tumor acidic tissues, so that the treatment effect of the chemotherapeutic drugs is enhanced, and the toxicity to normal tissues is smaller, so that the tumor selectivity of the nano-drug is remarkably enhanced.
Drawings
FIG. 1 is a schematic illustration of the formation of the nano-drug PHDZ/BTZ in the examples.
FIG. 2 is a dynamic light scattering diagram of PHDZ/BTZ of the nano-drug of the example.
FIG. 3 is a PHDZ/BTZ transmission electron microscope of the nano-drug in the example.
FIG. 4 shows the release profile of the nano-drug PHDZ/BTZ of the example HDZ at different pH values.
Fig. 5 is a graph showing drug release curves of the nano-drug PHDZ/BTZ using chemotherapeutic BTZ as an example at different pH in the examples.
FIG. 6 shows cytotoxicity of the nanomaterials PHDZ/BTZ on different cell lines in the examples, wherein PHDZ is cells treated with the carrier alone, free HDZ+BTZ is cells treated with two small molecule drugs, PAA/BTZ is cells treated with nanomaterials formed by coating BTZ with carrier PAA to which HDZ is not attached, free BTZ is cells treated with BTZ alone.
FIG. 7 is a characterization of apoptosis of the nano-drug PHDZ/BTZ on a 4T1 cell line in the examples.
FIG. 8 is an evaluation of the tumor inhibition effect of nano-drug PHDZ/BTZ in the 4T1 tumor model of C57BL/6 mice in the examples, which is graphically represented as tumor inhibition curve.
FIG. 9 is an evaluation of the tumor suppression effect of the nano-drug PHDZ/BTZ in the 4T1 tumor model of C57BL/6 mice in the examples, which is graphically shown as a photograph of the tumor at the end of the tumor suppression period.
FIG. 10 is an evaluation of the tumor-inhibiting effect of nano-drug PHDZ/BTZ in a 4T1 tumor model of C57BL/6 mice in the examples, which is graphically represented as a change curve of the weight of the mice.
Detailed Description
The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The english abbreviations for the compounds referred to in the examples are as follows:
DCM-dichloromethane; AIBN-azobisisobutyronitrile; DMF-N, N-dimethylformamide.
Example 1
1. Preparation of nano medicine
(1) P-aminoacetophenone (4.0 g,29.6 mmol), methacryloyl chloride (3.6 g,34.4 mmol) and triethylamine (4.0 g,39.6 mmol) were dissolved in dichloromethane (DCM, 100 mL) and cooled with an ice bath. The mixture was stirred for 12 hours, then washed with saturated sodium bicarbonate solution, hydrochloric acid, distilled water and saturated sodium chloride solution in this order, the solvent was removed by rotary evaporation, 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:
Figure BDA0003361795340000061
(2) The polymer PAA was obtained by RAFT polymerization. Taking the polymerizable monomer N- (4-acetylphenyl) methacrylamide (0.30 g,1.5 mmol) obtained in the last step, and macromolecular chain transfer agent PETTC-PEG 5k (0.27 g,0.05 mmol) and AIBN (6.56 mg,0.04 mmol) are dissolved in dimethylformamide (DMF, 4 mL) and taken up in N at room temperature 2 Deoxygenation was performed for 30 min, and the reaction was stirred at 70℃for 12 h. After the reaction was completed, the solution was precipitated 3 times in 200mL of glacial ethyl ether and dried under vacuum to obtain polymer PAA.
Figure BDA0003361795340000062
(3) PAA (0.20 g) was mixed with excess hydralazine hydrochloride (0.20 g,1.0 mmol) 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 gave amphiphilic nano-carrier PHDZ (0.05 g, 23%).
(4) PHDZ/BTZ is prepared by a coprecipitation method: PHDZ (20 mg) and BTZ (10 mg) 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 fig. 1.
2. Particle size analysis of nano-drugs
As shown in fig. 2, the average particle size of the nano-drug PHDZ/BTZ was 138.4nm (distribution coefficient pdi=0.11) as measured by Dynamic Light Scattering (DLS).
As shown in FIG. 3, the particle size of PHDZ/BTZ of the nano-drug was found to be about 140.0nm by Transmission Electron Microscopy (TEM), which is consistent with the particle size result measured by DLS.
3. In vitro release of HDZ and BTZ
PHDZ/BTZ (2.0 mL) was sealed in dialysis bags with 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. 100. Mu.L of the solution outside the dialysis bag was collected at regular intervals, and the HDZ and BTZ concentrations were measured by HPLC, respectively.
The tumor acidic pH release capability of the drug is a very important part for evaluating PHDZ/BTZ in vivo application, and the good response release capability can ensure the full activation of the drug in a tumor area and the subsequent cytotoxicity generation, and simultaneously reduce the toxicity to normal tissues.
As shown in FIGS. 4 and 5, both HDZ and BTZ released less than 20% after 24 hours in PBS at pH 7.4, and about 40% after 24 hours and about 70% after 24 hours in PBS at pH 5.0 as the pH was lowered.
4. Toxicity of PHDZ/BTZ on different cell lines
The use of CCK8 (Cell Counting Kit-8) assay was used to assess cytotoxicity of PHDZ/BTZ, PHDZ, free HDZ+BTZ, PAA/BTZ and Free BTZ on B16F10, 4T1 and NIH/3T3 cell lines. Wherein PHDZ is cells treated with carrier alone, free HDZ+BTZ is cells treated with two small molecule drugs, PAA/BTZ is cells treated with nanomedicine formed by encapsulating BTZ with carrier PAA to which HDZ is not attached, free BTZ is cells treated with BTZ alone.
Cells were seeded in 96-well plates at a density of 5000 cells per well and incubated overnight. Cells were exposed to serial dilutions of the drug and cultured for an additional 48 hours, after which the medium was replaced with a mixed solution containing 180 μl of fresh medium and 20 μl of CCK-8. After incubation for 1.5 hours at 37 ℃, the absorbance in each well was measured at 450nm using a microplate reader and cell viability was obtained by calculating the ratio of absorbance values of the dosing wells to the blank.
As shown in FIG. 6, by toxicity analysis at the cellular level, we showed that no significant inhibition of cell growth was observed in cells treated with PHDZ nanoparticles alone on the 4T1 cell line, and that BTZ-loaded nanoparticles had significantly reduced cytotoxicity compared to BTZ alone, which could be interpreted as free diffusion of BTZ into tumor cells, while PHDZ/BTZ nanoparticles entered cells by endocytosis and slow release of BTZ at intracellular acidic pH. PHDZ/BTZ nanoparticles showed moderate levels of cytotoxicity at high concentrations of BTZ compared to PAA/BTZ nanoparticles, and similar trends were also observed on B16F10 and 3T3 cell lines. The results show that the PHDZ/BTZ nano-particles can effectively reduce cytotoxicity of BTZ, and the PHDZ nano-particles as drug carriers have little cytotoxicity.
5. Apoptosis experiments of PHDZ/BTZ on 4T1 cell lines
Apoptosis of 4T1 cells was measured with PI/Annexin V-FITC apoptosis kit and analyzed by flow cytometry. Specifically, cells were seeded into 12-well plates (5×10) 24 hours prior to the experiment 5 Individual cells/well). Cells were then 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. The collected cells were then 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 compared to cells treated with free BTZ, and significantly lower levels of apoptosis were obtained in cells treated with PBS and PHDZ nanoparticles, consistent with the CCK8 assay results, 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 the 4T1 tumor model of C57BL/6 mice
C57BL/6 mice were subcutaneously injected 5X 10 5 4T1 cells. Tumor volume up to 50mm 3 Left and right, mice were randomly assigned to 6 treatment groups (n=5): PBS, 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 0.8mg/kg. The drug was injected by tail vein once every four days for 5 times. Tumor volume (mm) was calculated using the formula 3 ): tumor volume= (shortest diameter) 2 X (longest diameter) ×0.5.
By tumor inhibition assessment in the 4T1 subcutaneous tumor model of C57BL/6 mice. As shown in fig. 8-10, PHDZ/BTZ exhibited significantly enhanced tumor inhibiting effect compared to BTZ group. Meanwhile, due to the lower toxicity of the chemotherapeutic prodrug during the in vivo circulation, the weight loss of mice in PHDZ/BTZ group is smaller than that of mice in BTZ group.

Claims (8)

1. The nano medicine for improving the tumor microenvironment based on the hydralazine is characterized in that the nano medicine is micelle type nano particles formed by self-assembling an amphiphilic polymer and a hydrophobic chemotherapeutic medicine containing boric acid groups in water; the structural formula of the amphiphilic polymer is shown as a formula (I),
Figure FDA0004148353170000011
wherein y=1-50 and m=10-3000.
2. The nano-drug for improving tumor microenvironment based on hydralazine according to claim 1, wherein the preparation method of the amphiphilic polymer comprises the following steps: firstly, under the condition of organic base catalysis, reacting p-aminoacetophenone with methacryloyl chloride to prepare N- (4-acetylphenyl) methacrylamide, then using polyethylene glycol macromolecular chain transfer agent to prepare a polymer through a reversible addition-fragmentation chain transfer polymerization method or using a polyethylene glycol macromolecular chain initiator to polymerize N- (4-acetylphenyl) methacrylamide through an atom transfer radical polymerization method, and then connecting hydralazine to the polymer through forming hydrazone bond to obtain the amphiphilic polymer, wherein the polyethylene glycol macromolecular chain transfer agent is PETTC-PEG 5k 。
3. The nano-drug for improving tumor microenvironment based on hydralazine according to claim 1, wherein y = 20-50 and m = 200-500.
4. The nano-drug for improving tumor microenvironment based on hydralazine according to claim 1, wherein the chemotherapeutic drug is bortezomib or ifenprodil Sha Zuomi.
5. The method for preparing the nano-medicament for improving tumor microenvironment based on hydralazine according to any one of claims 1 to 4, which comprises the following steps: firstly, amphiphilic polymer and hydrophobic chemotherapeutic drug are dissolved in a good solvent, then the mixed solution is added into water under the oscillation condition, and the product self-assembles to form the nano drug.
6. The method of claim 5, wherein the mass ratio of amphiphilic polymer to hydrophobic chemotherapeutic agent is 2-5:1.
7. Use of a nano-drug based on hydralazine for improving tumor microenvironment according to any one of claims 1-4 for the preparation of a tumor therapeutic drug.
8. The use of claim 7, wherein the tumor is a solid tumor.
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