CN114788875A - Supermolecule nano-drug for activating Hippo pathway and preparation method and application thereof - Google Patents
Supermolecule nano-drug for activating Hippo pathway and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a supermolecule nano-medicament for targeted activation of a Hippo pathway in a cancer cell and application thereof. The double-drug-component amphiphilic Fmoc-S (FA) FFYSV-SS-PEG (FASP) is obtained by connecting a hydrophobic short peptide fragment containing double drug components with a hydrophilic polyethylene glycol fragment through a disulfide bond. The compound can form an inactive nano prodrug through intermolecular non-covalent interaction self-assembly, is subjected to morphological transformation after being taken by cancer cells, forms active peptide nanofiber in situ and releases Flufenamic Acid (FA), thereby activating a disordered Hippo pathway for combined treatment and metastasis inhibition of tumor radiotherapy and chemotherapy, and is characterized in that: simple synthesis, high repeatability, good biocompatibility and great clinical transformation potential. Meanwhile, compared with free FA, the supermolecule nano-drug obtained by the invention can be delivered through covalent connection and released in situ in response, the growth inhibition effect of FA on tumor cells is obviously improved, the toxic and side effects of FA on normal cells are reduced, and the supermolecule nano-drug has good clinical transformation and application prospects.
Description
Technical Field
The invention belongs to the field of nano biomedical materials, and relates to a preparation method of a supermolecule nano-medicament for targeted activation of a Hippo pathway in cancer cells and application of the supermolecule nano-medicament in combined treatment of tumor radiotherapy and chemotherapy.
Background
The Hippo pathway was first found in drosophila, highly conserved in mammals, and has been one of the research hotspots in the field of life medicine since its discovery. Studies have shown that the Hippo pathway plays a key role in a wide range of biological processes including organ size control, cell proliferation, cancer development, and virus-induced disease. The Hippo pathway mainly comprises three parts, namely an upstream core kinase MST-LATS module, a signal cascade transfer key factor YAP protein and a downstream transcription factor family TEADS module. When the Hippo pathway is activated by an upstream growth inhibition signal, the upstream core kinase phosphorylates YAP, further promotes ubiquitination and proteasome degradation of the YAP, prevents YAP from transferring to cell nucleus to be combined with TEADs, and finally inhibits the transcription and expression of related proliferation and survival promotion genes. The existing research shows that the YAP is highly expressed in various cancer cells caused by the maladjustment of the Hippo pathway, including breast cancer, lung cancer, pancreatic cancer, liver cancer, melanoma and the like.
In addition to directly regulating the growth and proliferation of tumor cells, YAP can also be used as a signal center of the tumor microenvironment to regulate the tumor immune microenvironment. It was shown that YAP binding to TEADs would assist tumors in evading surveillance and attack by the immune system by inducing expression of genes such as immunosuppressive cytokines, chemokines, and programmed death ligand 1 (PD-L1), activating and recruiting tumor-associated macrophages, myeloid-derived suppressor cells, regulatory T cells, and cancer-associated fibroblasts. Therefore, the Hippo signal channel in the cancer cells is activated in a targeted mode, the interaction of YAP-TEADs is effectively blocked, the dual purposes of inhibiting the growth of the tumor cells and remodeling the tumor immune microenvironment can be achieved, and a new strategy is provided for tumor chemotherapy, radiotherapy and immunotherapy.
Currently, Hippo pathway-targeted cancer therapy is still in the beginning and research focus is mainly on how to inhibit the interaction between YAP and TEADs. Related small-molecule drugs which are reported comprise verteporfin, a fluoroxime compound, a flufenamic acid compound and the like. Wherein, Flufenamic Acid (FA) is a non-steroidal small molecule drug, and researches show that the Flufenamic Acid (FA) can be combined in lipid bags of TEADs palmitoylation to influence the folding and the activity of the TEADs, thereby reducing the transcription of downstream target genes caused by the combination of YAP and the TEADs and being an effective Hippo pathway activator. However, due to the small molecular property, FA has poor water solubility, shows low bioavailability and has toxic and side effects on normal tissue cells, thereby limiting the clinical popularization and application of the FA. Tyrosine-serine-valine is a short peptide with the function of a histone deacetylase inhibitor, is expected to promote YAP phosphorylation through acetylation control on upstream core kinase of a Hippo pathway, and further realizes effective activation on the Hippo pathway under the synergistic action of FA. However, the active short peptide has the defects of poor activity and easy degradation by protease, and the stability of the active short peptide needs to be improved by a reasonable strategy.
In the past decades, the development of supramolecular nano-drug delivery systems shows good application effects in cancer targeted therapy, and particularly shows that the bioavailability and anticancer curative effect of small-molecule drugs can be effectively improved, and the toxic and side effects of the small-molecule drugs can be reduced. Research shows that the spherical nano-particles can better diffuse and permeate at tumor sites, but the intracellular retention capacity of the spherical nano-particles is relatively weak; while nanorods or nanofibers with high aspect ratios can achieve high accumulation in cells, their tumor penetration ability is relatively weak. The supermolecule nano-drug with the nano-sphere-nano fiber in-situ transformation performance can meet the dual requirements of high tumor penetration and intracellular long retention, receives more and more attention in recent years, and shows great potential in clinical transformation and application. Therefore, the supramolecular nano-drug which has in-situ conversion performance and can effectively activate the Hippo pathway is reasonably designed and developed, so that the supramolecular nano-drug has important significance for accelerating and advancing clinical cancer treatment of the targeted Hippo pathway.
Disclosure of Invention
The invention aims to develop a supermolecule nano-drug for targeted activation of a Hippo pathway in cancer cells, and the supermolecule nano-drug is applied to tumor combined treatment to improve the tumor treatment effect.
The supermolecule nano-medicament provided by the invention has the following advantages: 1) the carrier component is basically composed of amino acid and polyethylene glycol, and has good biocompatibility and biodegradability; 2) the solubility and the bioavailability of the hydrophobic drug flufenamic acid are improved; 3) can be specifically converted in situ in cancer cells and activate a Hippo pathway, and enhances the selective effect of free drugs on the tumor cells; 4) has the functions of targeted therapy and radiotherapy sensitization, and realizes the tumor synergistic treatment.
In order to achieve the purpose, the invention discloses the following technical contents:
a supermolecule nano-drug Fmoc-S (FA) FFYSV-SS-PEG with targeted activation of a Hippo pathway in a cancer cell is characterized in that a hydrophobic short peptide fragment containing double drug components and a hydrophilic polyethylene glycol fragment are covalently connected through a disulfide bond, and an obtained amphiphilic compound can be self-assembled in an aqueous solution through intermolecular non-covalent bond interaction to form a nano-prodrug with a spherical micelle in a microscopic morphology, and the chemical structure of the supermolecule nano-drug is as follows:
wherein Fmoc-S (FA) is side chain hydroxyl modified micromolecule drug FA, and amino terminal is modified Fmoc serine; FFYSV is short peptide composed of phenylalanine-tyrosine-serine-valine residue, SS is disulfide bond, and PEG is polyethylene glycol fragment.
The Fmoc-S (FA) FFYSV-SS-PEG target compound is characterized by being synthesized by the following method:
taking Fmoc-Wang-Gly resin as a carrier, adding 10-20 mL of DMF into a customized synthesis tube to fully swell the resin, then cutting off an Fmoc protecting group, sequentially adding Fmoc-PEG, Fmoc-SS and Fmoc protecting amino acids (Val, Ser, Tyr and Phe) according to a standard method for polypeptide Fmoc solid phase synthesis, and finally adding Fmoc-Ser (FA) -COOH. After the reaction is finished, 10-20 mL of 95% trifluoroacetic acid is added to react for 1-2 h, the polypeptide derivative is cut from the resin, and the target product is obtained by reversed phase HPLC separation and purification. Wherein, the preparation method of Fmoc-Ser (FA) -COOH comprises the following steps:
(1) FA (562.46 mg, 2 mmol) and Fmoc-Ser-OtBu (766.88 mg, 2 mmol) were dissolved in 10-25 mL of anhydrous dichloromethane, EDCI (575.1 mg, 3 mmol) and DMAP (36.6 mg, 0.3 mmol) were added and the reaction stirred at room temperature for 24-48 h;
(2) after dichloromethane is removed by rotary evaporation, concentrated solution is separated by a silica gel chromatographic column to obtain a light yellow intermediate product Fmoc-Ser (FA) -OtBu; and adding 5-10 mL of trifluoroacetic acid into the intermediate product, stirring at room temperature for reaction for 1-2 h to remove a protecting group, dissolving with anhydrous ether, and drying to obtain a product Fmoc-Ser (FA) -COOH.
The invention further discloses application of the supermolecule nano-medicament for targeted activation of the Hippo pathway in improving the treatment effect of the anti-tumor medicament. Wherein, the improvement of the treatment effect of the antitumor drug refers to the improvement of the specific injury killing and synergistic treatment effect of the free flufenamic acid on the tumor.
The invention also discloses application of the supermolecule nano-medicament for targeted activation of the Hippo pathway in improving the sensitivity of tumors to radiotherapy. The experimental result shows that compared with the single supermolecular nano-medicament mediated targeted therapy or gamma-ray mediated radiotherapy, the supermolecular nano-medicament and radiotherapy combined application has more remarkable tumor inhibition effect. The tumor inhibition rate is improved to about 90 percent, and the high-efficiency synergistic treatment effect of targeted therapy and radiotherapy is shown. In particular, FASP can remarkably inhibit liver metastasis of tumors, and can remarkably improve the survival rate of triple negative breast cancer after combined radiotherapy.
The present invention is described in more detail as follows:
hydrophobic short peptide fragment containing double drug components and hydrophilic polyethylene glycol fragment are connected through disulfide bond to obtain amphiphilic compound Fmoc-S (FA) FFYSV-SS-PEG. The product can form a nano prodrug with a spherical micelle nano structure through intermolecular non-covalent interaction self-assembly, and the nano prodrug is subjected to structure transformation under the action of glutathione and esterase with high expression in cancer cells to form Fmoc-SFFYSV nano fibers in situ and release free small molecule drug Flufenamic Acid (FA), so that the disordered Hippo pathway is activated through the upstream and downstream synergistic effects and is used for combined treatment and metastasis inhibition of tumors. Therefore, the peptidyl nano-drug can effectively improve the bioavailability of the free casein valine peptide and the flufenamic acid so as to achieve the effect of enhancing the anticancer activity.
The preparation method of the Fmoc-S (FA) FFYSV-SS-PEG supermolecular nano-drug comprises the following steps: 5 mg of the synthesized pure compound was weighed out and dissolved in 1 mL DMSO, and added dropwise slowly to 9 mL ddH in a ratio of 1:9 2 Dripping one drop of DMSO solvent containing polypeptide every 30 s in O/PBS, and stirring while dripping; after the dropwise addition is finished, stirring is continuously carried out for 4 hours at room temperature, and micelle is stabilized; the obtained product was dialyzed in dialysis bag (MWCO = 1000D) for 48 h, and DMSO solvent was completely removed to obtain the objective supramolecular nano-drug. Further, the Fmoc-S (FA) FFYSV-SS-PEG derivative is synthesized by the following steps: taking Fmoc-Wang-Gly resin as a carrier, adding 10-20 mL of DMF (dimethyl formamide) into a customized synthesis tube to fully swell the resin, then cutting off an Fmoc protecting group, sequentially adding Fmoc-PEG, Fmoc-SS and Fmoc protected amino acids (Val, Ser, Tyr and Phe) according to a standard method for polypeptide Fmoc solid phase synthesis, and finally adding Fmoc-Ser (FA) -COOH. After the reaction is finished, 10-20 mL of 95% trifluoroacetic acid is added to react for 1-2 h, the polypeptide derivative is cut from the resin, and the target product is obtained by reversed phase HPLC separation and purification. Further, the Fmoc-Ser (FA) -COOH preparation method comprises the following steps:
1) FA (562.46 mg, 2 mmol) and Fmoc-Ser-OtBu (766.88 mg, 2 mmol) were dissolved in 10-25 mL of anhydrous dichloromethane, EDCI (575.1 mg, 3 mmol) and DMAP (36.6 mg, 0.3 mmol) were added and the reaction stirred at room temperature for 24-48 h;
2) after dichloromethane is removed by rotary evaporation, concentrated solution is separated by a silica gel chromatographic column to obtain a light yellow intermediate product Fmoc-Ser (FA) -OtBu; and adding a proper amount of 5-10 mL of trifluoroacetic acid into the intermediate product, stirring at room temperature for reaction for 1-2 h to remove a protecting group, and dissolving and drying with anhydrous ether to obtain a product Fmoc-Ser (FA) -COOH.
The invention mainly solves the problem that the regulation of a Hippo pathway target is not ideal due to poor solubility and low bioavailability of a small molecular drug flufenamic acid, and mainly researches the morphological transformation performance of the obtained supermolecule nano-drug under the action of glutathione and esterase, the activation of the Hippo pathway in cancer cells and the application of the supermolecule nano-drug in tumor combined treatment after the hydrophobic short peptide carrier is covalently combined with the hydrophilic polyethylene glycol segment. The main difficulty is that the reasonable design of the structure of the supermolecule compound containing the flufenamic acid and the casein valine enables the supermolecule compound to be self-assembled to form a nano-medicament which can generate structural transformation in cancer cells and synergistically activate a Hippo pathway, so that the bioavailability and the curative effect of the flufenamic acid are improved.
The invention discloses a supermolecule nano-medicament for targeted activation of a Hippo pathway, a preparation method and application thereof, which have the following positive effects: the solubility and bioavailability of the small-molecule drug flufenamic acid can be greatly improved, the regulation and control of the small-molecule drug flufenamic acid on the key target gene of the Hippo pathway are enhanced through the synergistic effect of the small-molecule drug flufenamic acid and the casein valine peptide, and a novel method for targeting the Hippo pathway and treating cancers is provided.
The invention discloses a pharmaceutical composition consisting of Fomc-S (FA) FFYSV-SS-PEG nano-drugs and one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutical composition can be made into solid oral preparation, liquid oral preparation, injection, etc.
The Fomc-S (FA) FFYSV-SS-PEG nanomedicines of the invention may also be administered parenterally. The preferred form of parenteral administration is injection. The solid and liquid oral formulations comprise: tablets, enteric tablets, capsules, syrups, oral solutions, injections, and the like.
The Fomc-S (FA) FFYSV-SS-PEG nano-drug composition is prepared as follows: the compounds of the present invention are combined with pharmaceutically-acceptable solid or liquid carriers and optionally with pharmaceutically-acceptable adjuvants and excipients using standard and conventional techniques to prepare microparticles or microspheres. Solid dosage forms include tablets, capsules, sustained release tablets, sustained release pellets and the like. A solid carrier can be at least one substance that can act as a diluent, flavoring agent, solubilizing agent, lubricant, suspending agent, binder, disintegrant, and encapsulating agent. Inert solid carriers include magnesium phosphate, magnesium stearate, talc, lactose, pectin, propylene glycol, polysorbate 80, dextrin, starch, gelatin, cellulosic materials such as methyl cellulose, microcrystalline cellulose, low melting waxes, polyethylene glycols, mannitol, cocoa butter, and the like. Liquid dosage forms include solvents, suspensions such as injections, powders, and the like.
The supermolecule nano-medicament for activating the Hippo pathway prepared by the invention has the following advantages:
1) the raw materials are easy to obtain, the cost is low, the yield is high, the repeatability is good, and convenience can be provided for future practical application;
2) the preparation method is simple, and the nano-micelle has relatively stable morphology;
3) the product has good biocompatibility in vivo and is easy to clinically transform;
4) can improve the selectivity of the flufenamic acid to cancer cells and reduce the toxic and side effect of the flufenamic acid to normal cells.
Drawings
Fig. 1 is a representation of supramolecular nanomedicines: A) respectively representing TEM images of different nano-drugs before and after incubation with glutathione and pig liver esterase in PBS; B) HPLC monitoring of nano-drugsHydrolysis reaction of FASP under the action of glutathione and pig liver esterase; C) FASP at H 2 Stability in O, 0.9% NaCl, PBS and RPMI 1640+10% serum over 48 h; D) time-dependent release profile of FA after incubation of FASP under different catalytic conditions. The scale is 100 nm;
FIG. 2 shows the growth inhibitory effect of supramolecular nanomedicines and free-drug molecules on triple negative breast cancer cells (MDA-MB-231 and 4T 1) and normal cells (3T 3): A) the growth inhibition effect of different nano-drugs and drug free molecules on mouse fibroblast 3T 3; B) the growth inhibition effect of different nano-drugs and drug free molecules on mouse triple negative breast cancer cells 4T 1; C) the growth inhibition effect of different nano-drugs and drug molecules on human triple negative breast cancer cells MDA-MB-231;
FIG. 3 is the regulation of the Hippo pathway in MDA-MB-231 by supramolecular nanomedicines and free pharmaceutical molecules: A-D) after different nano-drugs and free drug molecules treat MDA-MB-231, q-PCR analyzes expression conditions of genes related to promoting proliferation and migration of a Hippo channel; E) immunoblotting shows the expression of MST1, p-YAP, YAP and AxL proteins after the nano-drug and drug molecules process MDA-MB-231; F) quantitatively analyzing the ratio of p-YAP/YAP according to the immunoblot image in E);
FIG. 4 shows the in vitro radiotherapy sensitization effect of supramolecular nano-drug and free drug molecules: A) forming an experimental result by cloning after the radiation of different doses; B) cell survival curves after different doses of radiation. ✱✱✱ p < 0.001；
FIG. 5 shows the effect of supramolecular nanomedicine and free drug molecule combined radiotherapy on MDA-MB-231 breast cancer growth and liver metastasis inhibition in nude mice: A) mouse tumor volume growth curve during treatment; B) mouse body weight change curve; C) survival curves for each group of mice; D) tumor inhibition rates in mice of different treatment groups; E) different treatment groups of mice liver transfer pictures and corresponding tissue section hematoxylin-eosin staining (H & E) results. The scale bar is 100 μm. ✱✱ p < 0.01， ✱✱✱ p < 0.001。
Detailed Description
The invention is illustrated by the following specific examples. Unless otherwise specified, all technical means used in the present invention are well known to those skilled in the art. In addition, the examples should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications can be made in the components and amounts of the materials used in these examples without departing from the spirit and scope of the invention.
The raw materials used in the invention, including flufenamic acid, Fmoc amino acid, dichloromethane, trifluoroacetic acid, triisopropyl silane, N-Dimethylformamide (DMF), methanol, N-Diisopropylethylamine (DIEA), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 4-dimethylaminopyridine, cystamine dihydrochloride, sodium bicarbonate, 1, 4-dioxane, succinic anhydride, anhydrous ether, acetone and sodium bicarbonate, are all commercially available.
Example 1
The preparation and characterization of the supramolecular nano-drug comprises Fmoc-S (FA) FFYSV-SS-PEG (abbreviated as FASP), a control material Fmoc-SFFYSV-SS-PEG (abbreviated as FSSP) without FA, a control material Fmoc-S (FA) without histone deacetylase inhibitory function peptide YSV, and a control material Fmoc-S (FA) without glutathione response FFYSV-CC-PEG (abbreviated as FACP), wherein the preparation method comprises the following specific preparation steps taking FASP nano-drug as an example:
firstly, weighing 5 mg of pure polypeptide, dissolving the pure polypeptide in 1 mL of DMSO, and slowly dripping the pure polypeptide into 9 mL of ddH according to the proportion of 1:9 2 Dripping one drop of DMSO solvent containing polypeptide in O/PBS every 30 s while stirring; after the dropwise addition is finished, continuously stirring for 4 hours at room temperature to stabilize the micelle; the resulting product dialysis bag (MWCO = 1000D) was dialyzed for 48 h, and the DMSO solvent was completely removed to obtain a FASP micelle solution.
The preparation method of the nano-micelle of the control material is the same.
And (II) respectively sucking 10 mu L of nano-micelle on a 300-mesh copper net, standing for 5 min, sucking excess liquid by using filter paper, dripping 5 mu L of uranium acetate on the copper net for dyeing for 3min, sucking excess liquid by using the filter paper, placing the filter paper in a dryer for overnight drying, and observing the micro-morphology by using a transmission electron microscope for detection. The results were as follows:
as shown in FIG. 1A, the nano-morphologies of all the nano-drugs are nanospheres with regular morphology, and after incubation with pig liver esterase (20U/mL) and glutathione (10 mM), the morphologies of all the nano-drugs except FACP are all converted from nanospheres to nanofibers.
(III) FASP (5 mg/mL) was incubated with glutathione (10 mM) and pig liver esterase (20U/mL) at 37 ℃. At the indicated time intervals, freeze-dried samples were collected and FASP was found by HPLC analysis to be completely hydrolyzed at 12 h (fig. 1B). FASP in 1640 culture medium containing 10% fetal bovine serum, H 2 Incubation in O, 0.9% NaCl and PBS for 48 h showed no significant change in particle size, confirming good stability of FASP (fig. 1C). By monitoring the specific absorption peak of FA at 350 nm, the release rate of FA from FASP under different conditions was measured, compared to FASP which releases FA completely and rapidly under the action of esterase and glutathione, catalyzed by esterase alone (FIG. 1D).
Example 2
The growth inhibition effect of the nano-drugs FASP, FSSP, FAYP, FACP and free drug FA prepared in example 1 on tumor cells and normal cells in vitro is evaluated, and the specific implementation steps are as follows:
1) 6000 MDA-MB-231, 4T1 and 3T3 cells in logarithmic growth phase were taken, inoculated into 96-well plates per well, placed at 37 ℃ and 5% CO 2 Culturing in a constant temperature incubator for 24 h;
2) diluting the nano-drugs FASP, FAYP, FACP, FSSP and free drug FA to preset concentration by using a culture medium, and incubating with cells for 48 h;
3) adding 10 mu L of CCK-8 solution into each hole under the condition of keeping out of the sun, placing the mixture in an incubator for continuous culture for 2-4 h, detecting the light absorption value at 450 nm by using an enzyme-labeling instrument, and calculating the survival rate of each cell after the action of each group of medicines with different concentrations;
4) fig. 2 shows that the nano-drug FASP can significantly improve the growth inhibition ability of FA on tumor cells, while FSSP, FAYP and FACP show weaker tumor cytotoxicity, and the nano-drug reduces the toxic and side effects of FA on normal cells to a certain extent.
Example 3
The fluorescent quantitative PCR analysis is carried out on the regulation and control of the supermolecule nano-medicament and the free medicament prepared in the embodiment 1 on the key target gene of the Hippo pathway, and the specific steps are as follows:
1) taking MDA-MB-231 cells in logarithmic growth phase, inoculating the cells in a 6 cm culture dish at a density of 10 ten thousand per well, placing the cells at 37 ℃ and 5% CO 2 Culturing in a constant temperature incubator for 24 h;
2) discarding the original culture medium, adding fresh culture medium containing 50 μ M of nano-drug and free drug, and incubating with cells in the incubator for 48 h;
3) washing the cells for 2 times by PBS, and digesting and collecting the cells by pancreatin;
4) washing with PBS for 1 time at 3000 rpm, centrifuging for 5 min, and removing supernatant;
5) adding 1 mL of 4 ℃ precooled Trizol reagent, and cracking for 5 min at room temperature;
6) adding 200 μ L chloroform, and standing for 3 min;
7）13500 rpm，15 min；
8) taking an upper layer RNA solution and adding isopropanol with the same volume for washing;
9）8℃，13500 g，15 min；
10) washing RNA with 70% ethanol, centrifuging, collecting RNA precipitate, and obtaining cDNA by using a reverse transcription kit;
11) the expression of different genes was analyzed by real-time quantitative PCR.
As shown in FIGS. 3A-D, the expression of YAP gene in FASP and FSSP treatment groups of nano-drugs was significantly reduced, and the expression of CTGF, CYR61 and NRP1 downstream genes of Hippo were all inhibited, while the expression of YAP and downstream genes in other control nano-drug groups was not significantly affected. From this result, it could be confirmed that FASP could down-regulate the expression of YAP and downstream related genes in triple negative breast cancer cells.
And (II) analyzing the regulation and control of supramolecular nano-drugs and free drugs on the expression of the Hippo pathway key proteins by immunoblotting, which comprises the following specific steps:
1) logarithmic growthStage MDA-MB-231 cells, seeded at a density of 40 ten thousand per well in 6 cm dishes at 37 ℃ with 5% CO 2 Culturing in a constant temperature incubator for 24 h;
2) discarding the original culture medium, adding a fresh culture medium containing 50 μ M of the nano-drugs and the free drugs, and incubating with cells in an incubator for 48 h;
3) washing cells with PBS for 3 times, adding RIPA lysate to lyse cells at 4 deg.C for 30 min;
4) centrifuging at 12500 rpm for 15 min at 4 deg.C;
5) quantifying, namely diluting the protein sample with 4-protein loading buffer solution to the same protein concentration after the BCA is quantified;
6) denaturation: heating at 100 deg.C for 10 min for denaturation;
7) electrophoresis: preparing 12% polyacrylamide gel at 80V-30 min; 120V-90 min;
8) film transferring: 200 mA, wet-rotating for 120 min;
9) and (3) sealing: taking out the PVDF membrane after the membrane conversion, soaking the PVDF membrane in TBST containing 5% skimmed milk powder, and shaking the shaking table for 2 hours;
10) primary antibody incubation: collecting the blocking solution, adding primary antibody diluted by the blocking solution, and incubating overnight in a shaking table at 4 ℃;
11) and (3) secondary antibody incubation: collecting primary antibody, washing the PVDF membrane with TBST for 3 times/10 min, adding secondary antibody solution diluted by confining liquid, and incubating at room temperature for 1 h;
12) and (3) developing: collecting the secondary antibody, and washing the PVDF membrane for 3 times/10 min by using TBST; ECL developer and imaging with gel imager.
13) Fig. 3E shows that compared to the control material group FACP, FAYP, FSSP and free FA, the supramolecular nano-drug FASP can phosphorylate YAP by promoting the expression of MST1 protein kinase, and decrease the total protein content of YAP, which finally results in the down-regulation of cell migration and proliferation related gene AxL downstream of Hippo pathway. As can be seen from the ratio of p-YAP/YAP in FIG. 3F, FASP significantly increased the level of p-YAP in the overall YAP protein, rendering YAP inactive. The result is consistent with the gene detection conclusion of fig. 3A-D, and further confirms that supramolecular nano-drug FASP can activate Hippo pathway in triple negative breast cancer and affect expression of downstream target genes.
Example 4
The ability of the supramolecular nano-drug and the free drug prepared in the embodiment 1 to inhibit the clone formation after cell irradiation in vitro is detected to compare the radiotherapy sensitization ability, and the specific steps are as follows:
1) taking MDA-MB-231 cells in logarithmic growth phase, inoculating the cells into a six-well plate at the density of 1000 cells per well, cross-shaking uniformly, placing at 37 ℃ and 5% CO 2 Culturing for 24 h in a constant-temperature incubator;
2) discarding the original culture medium, adding a fresh culture medium containing 20 μ M of the nano-drugs and the free drugs, and incubating with cells in an incubator for 12 h;
3) carefully washing with PBS for 1 time, adding 2 mL of fresh culture medium, and performing gamma-ray irradiation with the dosage of 0, 2, 4 and 6 Gy;
4) after irradiation, the cells were placed in an incubator for further culture. Observing the formation of clones, and stopping culturing when the cells form macroscopic clonal clusters (each clonal cell is about 50-150);
5) after the culture is stopped, carefully washing the cells for 1 time by PBS (phosphate buffer solution), adding about 300 mu L of 0.25% crystal violet dye solution (prepared by pure ethanol) into each well, fixedly dyeing the cells for 30 min at room temperature, discarding the dye solution, washing the cells in water for 2 times, reversely putting the cells on a desktop, drying the cells in the air, scanning a clone plate by using a gel imaging system, storing an image (the back face is upward), counting the number of clones by naked eyes, analyzing, drawing and calculating an SER (serial number) by using Graphpad prism and Origin software;
6) fig. 4A shows that nano-drug FASP has a more significant ability to inhibit the formation of tumor cell clonal clusters compared to free FA and control materials. As can be seen from the survival curve of FIG. 4B, the radiosensitizing ability of the nano-drug FASP is more prominent at higher doses (4 Gy and 6 Gy) of radiation.
Example 5
The effect of the supramolecular nano-drug prepared in example 1, the control group thereof and the free drug in combination with radiotherapy treatment in vivo is evaluated, and the specific experimental steps are as follows:
1) 40 BALB/c nude mice were inoculated with about 1X 10 cells per breast 7 MDA-MB-231 cells of (1), until the tumor volume is as long as 50-100 mm 3 Thereafter, the groups were randomly divided into 8 groups: irradiation group (IR, IR + FA, IR + FAYP, IR + FASP), non-irradiation group (Control, FA, FAYP, FASP);
2) according to the weight injection drug dose of the mice, 100 mu L of nano drugs or free drugs containing equal substances are respectively injected into the tail vein of each group of tumor-bearing mice: nanomaterial (20 mg/kg), FA (2.25 mg/kg); after 12 h of injection, all irradiation groups are irradiated by gamma rays with the local 6Gy dose of the tumor;
3) tumor size and mouse body weight were measured every other day starting the day before treatment, and tumor volume was according to the formula: length x width 2 Calculating and making each group of volume increase change curves;
4) the tumor growth inhibition results (fig. 5A) show that although the FASP nano-drugs in the non-irradiation group have certain chemotherapeutic effects, the long-term effects are not ideal, the tumor inhibition rate can be improved to about 90% after the FASP nano-drugs are combined with radiotherapy (fig. 5D), and the high-efficiency combined treatment effect of radiotherapy and chemotherapy is shown. The weight loss was evident in free FA and IR + FA treated mice (fig. 5B), and may be associated with some systemic toxicity of FA. From the survival curve (fig. 5C), it can be seen that the FASP has a better therapeutic effect after combined radiotherapy, and the survival rate of the triple negative breast cancer tumor-bearing mice is significantly improved. As can be seen from the optical liver photograph and the corresponding tissue section hematoxylin-eosin staining result of fig. 5E, FASP can significantly inhibit tumor liver metastasis.
Example 6
Fmoc-S (FA) FFYSV-SS-PEG (abbreviated as FASP) has the following structural formula:
FASP is used as an active ingredient, and pharmaceutically acceptable auxiliary materials are added to prepare liquid injections with various specifications by a conventional method.
The administration route of FASP includes various routes, such as injection administration, intracavity administration, etc.
(1) Preparation of injection:
(2) Preparation of tablets:
10 mg of FASP, 35 mg of microcrystalline cellulose, 45 mg of starch, 4 mg of polyvinylpyrrolidone, 4.5 mg of sodium carboxymethyl starch, 0.5 mg of magnesium stearate and 1 mg of talcum powder; the FASP active ingredient, starch and cellulose are sieved and mixed thoroughly, the polyvinylpyrrolidone solution is mixed with the above powder, sieved, the wet granules are dried at 50 deg.C, the sodium carboxymethyl starch, magnesium stearate and talc powder are sieved in advance, and then the granules are added to the above granules and tabletted.
(3) Preparation of capsules
10 mg of FASP and active ingredient auxiliary materials are respectively sieved by a 100-mesh sieve, the main drug and the auxiliary materials in the prescription amount are weighed and fully mixed, a proper amount of hydroxypropyl methylcellulose solution is added to prepare soft materials, the soft materials are sieved by a 24-mesh sieve, prepared wet granules are dried in an oven at 50-60 ℃ for about 2-3 h, magnesium stearate and talcum powder are uniformly mixed with the granules, the granules are sized, the content of intermediates is measured, and the granules are filled in No. 2 capsules.
Claims (5)
1. A supermolecule nano-drug for targeted activation of a Hippo pathway in a cancer cell is characterized in that a hydrophobic short peptide fragment containing double drug components and a hydrophilic polyethylene glycol fragment are covalently connected through a disulfide bond, and an obtained amphiphilic compound can be self-assembled in an aqueous solution through intermolecular non-covalent interaction to form a nano-prodrug with a spherical micelle in a microscopic morphology, and the supermolecule nano-drug has the following chemical structure:
wherein Fmoc-S (FA) is side chain hydroxyl modified micromolecule drug FA, and amino terminal is modified Fmoc serine; FFYSV is short peptide composed of phenylalanine-tyrosine-serine-valine residue, SS is disulfide bond, and PEG is polyethylene glycol fragment.
2. The method for preparing the supramolecular nano-drug for targeted activation of Hippo pathway in cancer cells as claimed in claim 1, characterized by comprising the following steps:
taking Fmoc-Wang-Gly resin as a carrier, adding 10-20 mL of DMF (dimethyl formamide) into a customized synthesis tube to fully swell the resin, then cutting off an Fmoc protecting group, sequentially adding Fmoc-PEG, Fmoc-SS and Fmoc protected amino acids (Val, Ser, Tyr and Phe) according to a standard method for polypeptide Fmoc solid-phase synthesis, finally adding Fmoc (Ser FA) -COOH, after the reaction is finished, adding 10-20 mL of 95% trifluoroacetic acid to react for 1-2 h, cutting the polypeptide derivative from the resin, and separating and purifying by reversed-phase HPLC to obtain a target product; wherein, the preparation method of Fmoc-Ser (FA) -COOH comprises the following steps:
dissolving 2 mmol of FA and 2 mmol of Fmoc-Ser-OtBu in 10-25 mL of anhydrous dichloromethane, adding 3 mmol of EDCI and 0.3 mmol of DMAP (stirring and reacting for 24-48 h at room temperature, removing dichloromethane by rotary evaporation, separating the concentrated solution by a silica gel chromatographic column to obtain a light yellow intermediate product Fmoc-Ser (FA) -OtBu, adding 5-10 mL of trifluoroacetic acid into the intermediate product, stirring and reacting for 1-2 h at room temperature to remove a protecting group, dissolving and drying with anhydrous ether to obtain a product Fmoc-Ser (FA) -COOH;
3. the application of the supramolecular nano-drug for targeted activation of Hippo pathway in cancer cells as claimed in claim 1 in improving antitumor drugs: the Hippo pathway is activated, namely the nano prodrug is subjected to structural transformation under the action of high-expression glutathione and esterase in cancer cells, and Fmoc-SFFYSV nano fibers are formed in situ to regulate the activity of MST1 kinase from the upstream of the Hippo pathway and promote YAP phosphorylation inactivation; free small molecule drug Flufenamic Acid (FA) released in situ enters the cell nucleus to be combined with the downstream transcription factor family TEAD of the Hippo pathway, so that the interaction of YAP-TEAD is effectively inhibited through the upstream and downstream synergistic effect, and the transcription expression of survival promoting genes is finally inhibited; the improvement of the antitumor drug means that compared with free micromolecular drug FA, the nano prodrug delivery system can improve the dissolubility and the cell availability of FA, and finally improves the growth inhibition effect of the FA on tumor cells through the synergistic effect of YSV.
4. The use according to claim 3, wherein the use for enhancing the anti-tumor effect is a significant improvement in the survival of triple negative breast cancer after FASP in combination with radiation therapy.
5. The use according to claim 3, wherein the use for enhancing the antitumor effect is to significantly inhibit metastasis of tumor liver by FASP.
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