Disclosure of Invention
Based on the prior art, the invention provides a hydrogel-liposome combined drug delivery system, and a preparation method and application thereof. The invention establishes a liposome drug-loading system for encapsulating iron death small molecule inducer (Allastine) and glioma conventional chemotherapeutic drug temozolomide, and utilizes cRGD tumor targeting peptide to modify DSPE-PEG liposome, and realizes in-situ administration after tumor resection by injectable PLGA-PEG-PLGA temperature-sensitive hydrogel. The combined administration system can specifically induce glioma cell death, improve the drug resistance of glioma to temozolomide, reduce the administration dosage and the side effect of systemic administration, and obviously improve the prognosis of a subject, thus having good practical application value.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the invention there is provided the use of an inducer of iron death in combination with temozolomide in the manufacture of a medicament for the treatment of brain glioma.
Wherein the inducer of iron death may be alastine;
specifically, the brain glioma therapeutic drug has any one or more of the following applications:
(a) Inhibit proliferation, migration and invasion of glioma;
(b) Promoting apoptosis and iron death of glioma;
(c) Reversing the chemotherapy resistance of the drug-resistant glioma to temozolomide;
(d) Inhibit expression of glioma acetyl-CoA.
In a second aspect of the present invention, there is provided a pharmaceutical composition comprising as its active ingredients at least an inducer of iron death occurrence as described above and temozolomide.
The pharmaceutical composition is used for treating brain glioma, and specifically has any one or more of the following applications:
(a) Inhibit proliferation, migration and invasion of glioma;
(b) Promoting apoptosis and iron death of glioma;
(c) Reversing the chemotherapy resistance of the drug-resistant glioma to temozolomide;
(d) Inhibit expression of glioma acetyl-CoA.
In a third aspect of the invention, there is provided a pharmaceutical formulation comprising at least the pharmaceutical composition described above, more particularly the pharmaceutical formulation may be a gel comprising liposomes.
Thus, in particular, the pharmaceutical formulation may be a hydrogel-liposome combination delivery system comprising a hydrogel having a liposome comprising the pharmaceutical composition described above loaded thereon.
In a fourth aspect of the present invention, there is provided a method for preparing the above pharmaceutical preparation, which is a hydrogel-liposome combined administration system, and which can be prepared by a reverse phase evaporation method, comprising:
dissolving hydrogenated soybean phosphatidylcholine, cholesterol, cRGD-modified DSPE-PEG and alastin in an organic solution; and (3) performing ultrasonic treatment to obtain crude liposome, mixing the crude liposome with TMZ aqueous solution to form emulsion, removing the organic solvent, and processing by an ultrasonic and extrusion method.
In a fifth aspect of the invention, there is provided the use of a pharmaceutical formulation as described above for the in situ treatment of a drug resistant glioma in an operation or for the preparation of a product for the in situ treatment of a drug resistant glioma in an operation. The pharmaceutical preparation is specifically a hydrogel-liposome combined administration system.
In a sixth aspect of the invention, there is provided a method of treating glioma, the method comprising: the above pharmaceutical formulation is injected in situ after glioma resection.
The beneficial technical effects of one or more of the technical schemes are as follows:
compared with the conventional intravenous administration and oral administration, the technical scheme provides a intraoperative hydrogel-liposome combined administration system for in-situ treatment of drug-resistant glioma. The technical scheme can specifically inhibit the malignant progress of glioma, obviously improve the chemotherapy resistance of glioma to TMZ drugs, and realize the tumor inhibition effect through iron death pathway and acetyl coenzyme A level.
Among them, the intra-operative placement reduces unnecessary trauma that may be caused by secondary surgery. The hydrogel injected in situ can continuously and slowly release the active drugs in tumor resection sites, and reduce the total drug exposure of the whole system. The liposome drug-carrying system provides entrapment possibility for Erastin which is insoluble in a conventional solvent and has low bioavailability, and provides specific tumor targeting and slow release effects. The Erastin can improve TMZ chemotherapy resistance through various mechanisms, provides a new direction for glioma drug treatment, and has good practical application value.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. Experimental methods in the following embodiments, unless specific conditions are noted, are generally in accordance with conventional methods and conditions of molecular biology within the skill of the art, and are fully explained in the literature. See, e.g., sambrook et al, molecular cloning: the techniques and conditions described in the handbook, or as recommended by the manufacturer.
The invention will be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not to be construed as limiting the invention. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the sales company; materials, reagents and the like used in the examples were commercially available unless otherwise specified.
In one exemplary embodiment of the present invention, there is provided the use of an inducer of iron death occurrence in combination with temozolomide in the manufacture of a medicament for the treatment of glioma.
Wherein the inducer of iron death may be alastine;
specifically, the brain glioma therapeutic drug has any one or more of the following applications:
(a) Inhibit proliferation, migration and invasion of glioma;
(b) Promoting apoptosis and iron death of glioma;
(c) Reversing the chemotherapy resistance of the drug-resistant glioma to temozolomide;
(d) Inhibit expression of glioma acetyl-CoA.
Wherein the mass ratio of the alastine to the temozolomide is 1:1-50, and preferably 1:10-20. At this time, the two compounds show strong synergistic effect, thereby improving the therapeutic effect.
In yet another embodiment of the present invention, there is provided a pharmaceutical composition whose active ingredients comprise at least the above inducer of iron death occurrence and temozolomide; the inducer of iron death may be alastine.
The pharmaceutical composition is used for treating brain glioma, and specifically has any one or more of the following applications:
(a) Inhibit proliferation, migration and invasion of glioma;
(b) Promoting apoptosis and iron death of glioma;
(c) Reversing the chemotherapy resistance of the drug-resistant glioma to temozolomide;
(d) Inhibit expression of glioma acetyl-CoA.
Wherein the mass ratio of the alastine to the temozolomide is 1:1-50, and preferably 1:10-20.
In yet another embodiment of the present invention, a pharmaceutical formulation is provided, which comprises at least the above pharmaceutical composition, more particularly, the pharmaceutical formulation may be a gel comprising liposomes.
Thus, in particular, the pharmaceutical formulation may be a hydrogel-liposome combination delivery system comprising a hydrogel having a liposome comprising the pharmaceutical composition described above loaded thereon.
The hydrogel is a temperature-sensitive hydrogel, so that the hydrogel is convenient to implement and use. More specifically, the hydrogel is PGLA-PEG-PLGA temperature-sensitive hydrogel; meanwhile, in order to improve the targeting of the pharmaceutical preparation, the hydrogel is also modified with cRGD peptide.
The liposome can be prepared by any known method, and in one specific embodiment of the invention, the liposome is prepared from a pharmaceutical composition and an auxiliary material, wherein the auxiliary material can be hydrogenated soybean phosphatidylcholine and cholesterol; specifically, the mass ratio of the alastine, the hydrogenated soybean phosphatidylcholine and the cholesterol is 1:1-4:1, preferably 1:4:1.
In still another embodiment of the present invention, there is provided a method for preparing the above pharmaceutical preparation, which is a hydrogel-liposome combined administration system, and which can be prepared by a reverse phase evaporation method, comprising:
dissolving hydrogenated soybean phosphatidylcholine, cholesterol, cRGD-modified DSPE-PEG and alastin in an organic solution; and (3) performing ultrasonic treatment to obtain crude liposome, mixing the crude liposome with TMZ aqueous solution to form emulsion, removing the organic solvent, and processing by an ultrasonic and extrusion method.
Wherein the mass ratio of the hydrogenated soybean phosphatidylcholine, the cholesterol, the cRGD modified DSPE-PEG and the alastine is 1-4:1:1:1; preferably 4:1:1:1.
The organic solvent is preferably chloroform.
In yet another embodiment of the present invention, the preparation method further comprises removing the unloaded drug ingredient by dialysis of the liposome prepared by the ultrasonic and extrusion method. Wherein, the dialysis is performed by a dialysis membrane, and a dialysis membrane of 0.05 μm can be used.
In yet another embodiment of the present invention, there is provided the use of the above pharmaceutical formulation for in situ treatment of drug resistant glioma in surgery or for preparing a product for in situ treatment of drug resistant glioma in surgery. The pharmaceutical preparation is specifically a hydrogel-liposome combined administration system.
In yet another embodiment of the present invention, there is provided a method of treating glioma, the method comprising: the above pharmaceutical formulation is injected in situ after glioma resection. Unnecessary wounds which may be caused by secondary operations are reduced by intra-operative placement. The hydrogel injected in situ can continuously and slowly release the active drugs in tumor resection sites, and reduce the total drug exposure of the whole system.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1
1. Liposome delivery system
Is prepared by reverse phase evaporation method. HSPC (hydrogenated soybean phosphatidylcholine), cholesterol, cRGD modified DSPE-PEG (A.V.T, shanghai, china) and Erastin (MCE, NJ, USA) were dissolved in 4 ml chloroform at a ratio of 4:1:1:1. After 2 minutes of sonication, the liposomes were incubated with ddH 2 The TMZ of O was mixed to form an emulsion, which was evaporated under continuous rotary reduced pressure for 20 minutes to remove chloroform solvent. Liposome systems are formed by ultrasonic and extrusion processes. After removal of the unsupported Erastin and TMZ with a 0.05 μm dialysis membrane, the amount of drug loaded was calculated by a spectrophotometer (TMZ is 399 nm, erastin is 276 nm). Drug synergy index was calculated by computer software, at TMZ: the combined effect (CI) of the medicines is strong synergy when ERA is 20:1-10:1. The drug loading rate of TMZ in a liposome drug delivery system prepared according to the ratio of 4:1:1:1 is 9.8+/-1.35%, and the encapsulation rate is 90.4+/-4.76%; the drug loading rate of ERA is 2.98+ -0.43%, the encapsulation efficiency is 36.88+ -2.49%, wherein the ratio of the concentration of the two drugs accords with the range of the ratio of the strong synergistic concentration.
DLC (drug loading) (%) = (drug loading weight)/(drug loading nanoparticle amount) ×100%;
DLE (%) = (drug loading weight)/(total drug administered) ×100%.
2. Cell culture
Human brain glioma cells (U251) were obtained from the Shanghai institute of bioscience cell resource center (Shanghai, china). Normal Human Astrocytes (NHA) were obtained from scientific cells of carlsbad, california, usaObtained in a research laboratory. A TMZ-resistant U251 TR-resistant cell line was established by our laboratory, specifically using progressively higher TMZ concentrations (from 50. Mu.M to 500. Mu.M) over a 6 month period to induce U251 cell resistance. To maintain the TMZ resistant phenotype, U251TR was exposed to TMZ medium (500. Mu.M) for 72 hours during the weekly period. All cells were maintained in monolayer culture in DMEM medium (BI, new York, U.S.) containing 10% fetal bovine serum (BI, new York, U.S.) and 100 units/mL penicillin, 100 μg/mL streptomycin (BI, new York, U.S.). The conditions of the humidifying incubator were 37℃and 5% CO 2 . Cell counting was done by an automated cell counter IC1000 (Ai Lite, shanghai, china).
3. Cell proliferation assay
Cell count kit-8 (CCK-8) (eastern kernel chemistry, shanghai, china) was used to determine cell viability. Cells were grown in 1X 10 cells 3 The density of individual cells/wells was seeded in 96-well culture plates. At various time points after drug treatment, the old medium was replaced with 100. Mu.L of fresh medium containing 10. Mu.L of CCK-8 solution. After incubation of the cells for 2 hours at 37 ℃, absorbance (OD) at 450nm was measured with Infinite M Nano (Tecan, switzerland).
The EdU Apollo567 in vitro imaging kit (acute boy, guangzhou, china) was used to assess cell proliferation. Cells treated with drug were treated at 1X 10 3 The density of individual cells/wells was seeded in 96-well plates. After 24 hours of EdU labeling, the percentage of EdU positive cells was observed with a 1X71 fluorescence microscope (Olympic Bass, japan).
4. Flow cytometry analysis
An Annexin V-FITC apoptosis assay kit (Norpran, nanjing, china) was used to determine apoptosis levels. Cells were grown in 2X 10 cells 5 The density of individual cells/well was seeded in 6-well culture plates. After various treatments, cells were digested with 0.25% EDTA-free trypsin (seville, chinese marchand) and washed three times with pre-chilled PBS (seville, chinese marchand). Finally, cells were labeled with Annexin V-FITC and PI dye and incubated at room temperature for 10 min in the dark. Cytoflex S (Beckman, california, U.S.A.) flow cytometer was used to record FITC/PI staining rates and calculate apoptosisLevel of apoptosis.
5. Scratch test and Transwell test
Scratch tests were used to assess the migration ability of cells. Cells were seeded at 100% density in 6-well plates and streaking was performed vertically in the center of each well with a 200 μl pipette tip. Subsequently, cells were continuously cultured in high-sugar DMEM medium without Fetal Bovine Serum (FBS), and scratches were photographed under 200-fold microscope at various time points. Scratch area and healing ratio were measured with Image J (V1.8.0) and subjected to subsequent statistical analysis.
The Transwell model was used to assess the invasive capacity of cells. Cell count of 2X 10 3 The density of individual cells/wells was seeded in an upper layer cell of an 8 μm pore size Transwell (Corning star, ma). After 12 hours of incubation, cells migrating onto the underlying cell membrane were fixed with 4% paraformaldehyde (white shark, chinese joint fertilizer) and stained with 0.25% crystal violet (seville, chinese martial). The number of migrating cells was calculated by counting the average of five random areas under a 200-fold microscope.
6. Iron death level assessment
Malondialdehyde (MDA) assay (bi yun, shanghai, china) was used to evaluate lipid peroxidation levels. Cell lysates were used for lipid peroxidation detection, i.e. the reaction of Malondialdehyde (MDA) with thiobarbituric acid (TBA). MDA levels are expressed as nmol/mg prot.
GSH and GSSG detection kits (bi yun tian, shanghai, china) were used to evaluate redox levels. 5X 10 4 Individual cells were lysed and used to measure the levels of reduced Glutathione (GSH) and oxidized glutathione disulfide (GSSG).
The enhanced JC-1 detection kit (Biyun, shanghai, china) was used to evaluate mitochondrial membrane potential (Deltaψm). JC-1 monomers and multimers can be recorded by fluorescence microscopy and flow cytometry (ratio of red/green fluorescence density).
RNA extraction and fluorescent quantitative PCR (qRT-PCR) detection
Total RNA from cells was extracted by RNA easy isolation reagent (Northenzan, nanjing, china). The concentration and purity of RNA were determined by absorbance at 260 and 280nm as measured by a Nanodrop One/OneC spectrophotometer (Thermo Scientific, wisconsin, U.S.A.). The cDNA synthesis kit (full gold, beijing, china) was used to reverse transcribe 1. Mu.g of total RNA into cDNA. qRT PCR was performed in CFX Connect real-time PCR system (Bio-rad, california, usa) using TransStart Tip Green qPCR SuperMix kit (full gold, beijing, china) system. beta-actin/GAPDH was used as reference gene and the threshold cycle number (CT) of the target gene was used to evaluate the expression level of the relevant gene after standardized analysis.
8. Western blot
The total cellular proteins were extracted using RIPA lysis buffer (bi yun, shanghai, china) containing a 1:1000 protease/phosphate inhibitor (apexio, texas, usa). BCA protein quantification kit (nuuzan, jiangsu, china) was used to determine protein concentration. 10% -12.5% SDS-PAGE (Yase, shanghai China) was used to gel electrophoretically separate 25-50. Mu.g protein mixed with loading buffer (Yase, shanghai China). Subsequently, the proteins were transferred to PVDF membranes (Millipore, ma) and incubated with primary antibodies after milk blocking. After incubation of the secondary antibody bound to horseradish peroxidase, the protein was visualized with ultrasensitive chemiluminescent (ECL) reagent (family You Bo, jiangsu, china) and detected by the image analysis system tangen 4800 (tenability, shanghai, china). GAPDH/beta-actin is used as an internal reference gene for evaluating the expression level of the relevant protein.
9. Animal experiment
We established a GL261 cell line expressing luciferase through lentivirus (Sai-jia, guangzhou, china) infection and puromycin (Soy-Bao, beijing, china) drug screening. Male C57BL/6J mice (4 weeks old) were purchased from Vetong Liwa (Beijing, china) and randomly divided into different groups (7 each). Partial craniectomy of the frontal and parietal bone areas on the right side was performed 7 days prior to in situ seeding. On day 0, GL261 luciferase cells (5. Mu.L containing 2.5X10) 6 Individual cells) were stereotactically injected into the mouse brain. The injection site was 2mm behind the fontanel, the depth was 2mm, and the injection rate was 0.5. Mu.L/15 s. After injection was completed, the microinjector was left in place for 2 minutes at a rate of 1mm per minuteAnd (5) withdrawing. On day 7 post inoculation, mice were tumor resected under a 20-fold SZX16 microscope (olympus, japan) using a 1.0mm pick-and-digger (Majestic, wyoming, uk) and 10 μl hydrogel droplets containing liposomes were injected into the resected cavity. After the hydrogel was auto-coagulated, mice were wound-anastomosed with 508 dolomite EC type alpha-cyanoacrylate medical adhesive (guangzhou, china). On day 25 post-inoculation, 2 mice of each group were sacrificed for material and H-treated&E staining and immunohistochemical staining (IHC), the body weight and survival rate of the remaining 5 mice per group will be assessed over a long period of time. Bioluminescence imaging systems (IVIS Spectrum in vivo imaging system, perkinemer, ma) were used to assess tumor size at days 6, 12 and 24.
10. Statistical analysis
Each set of data was independently repeated at least three times, the values being expressed as mean ± standard error. Statistical significance between the two sets of data was assessed by Student t test. GraphPad Prism 9 was used for statistical analysis and graphics fabrication. Survival analysis was performed using the Kaplan-Meier method and a log rank test. P values less than 0.05 are considered statistically significant (P <0.05, < P <0.01, and P < 0.001). Experimental results:
1. characterization of hydrogel-liposome drug delivery system
The liposomes were identified using a transmission electron microscope, a Zetasizer Nano particle size analyzer, nano ZS ZEN 3600. The cRGD peptide-modified liposomes after 2% phosphotungstic acid staining were observed under transmission electron microscopy to be of uniform elliptically-membrane structure (fig. 1A). The particle size was 163.+ -.19 nm (FIG. 1B), the PDI dispersion index was 0.17 and the zeta potential was-28.67 mV. The release results of the hydrogel and hydrogel-liposome in PBS show that the hydrogel can have a sustained release effect for up to 14 days (figure 1C), and the internally loaded liposome and drug can also realize the sustained release for 10-12 days (figure 1D). Immunofluorescence results showed that cRGD liposomes were able to take up drug resistant glioma cells via clathrin-mediated endocytosis, whose phagocytic capacity was inhibited by chloroquinone (fig. 1E, F, G).
2. In vitro verification of cancer inhibiting effect of combined administration system on drug-resistant glioma
For the TMZ-resistant cell line U251TR, the combination of Erastin with TMZ significantly inhibited the activity of glioma cells (fig. 2A), reduced their proliferative capacity (fig. 2B), migratory capacity (fig. 2C) and invasive capacity (fig. 2D), and significantly promoted the death of drug-resistant glioma cells (fig. 2E, F). Wherein P <0.001 compared to NC group.
3. In vivo verification of cancer inhibiting effect of combined administration system on drug-resistant glioma
For the C57BL/6J tumor-bearing mice in situ implantation model, resection of tumor tissue was visualized, and hydrogel-liposome drug delivery system (DDS, drug delivery system) was injected at the cavity during the procedure (FIG. 3A). Results of in vivo imaging of IVIS animals show that tumor volume is significantly reduced after surgical resection in NC versus Sham groups; the intraoperative drug delivery system significantly inhibited tumor growth in the DDS group compared to NC group (fig. 3B). Meanwhile, the weight and survival time of the mice in the DDS group were significantly improved (fig. 3c, d) and the growth of the residual tumor tissue was inhibited (fig. 3E) compared to the NC control group and the Sham treatment group.
4. Identification of iron death of drug-resistant glioma cells induced by combined administration
The combined administration significantly increased MDA levels of drug resistant glioma cells (fig. 4A) and decreased GSH levels (fig. 4B) compared to the administration alone and NC groups. At the same time, the combined administration inhibited the expression of GPX4, xCT and Ferritin at both the transcriptional and translational levels (fig. 4c, d). In addition, co-administration reduced mitochondrial membrane potential (fig. 4E) and enhanced intracellular peroxidation (ROS) levels (fig. 4F).
5. Combined administration induced reduction of acetyl-CoA in drug-resistant glioma cells
Downstream factors FABP7 and ACADVL were determined for co-administration effects by RNA-seq sequencing and PharmMapper target prediction (fig. 5A). The combined administration significantly reduced the transcription and translation levels of FABP7 and ACADVL compared to the group administered alone and NC group (fig. 5b, c). Since FABP7 and ACADVL are involved in the metabolism of Acetyl-CoA, the Elisa experiment demonstrated a decrease in intracellular Acetyl CoA levels in the co-administered group (fig. 5D).
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.