CN115192581B - Hydrogel-liposome combined drug delivery system and preparation method and application thereof - Google Patents

Hydrogel-liposome combined drug delivery system and preparation method and application thereof Download PDF

Info

Publication number
CN115192581B
CN115192581B CN202210860628.3A CN202210860628A CN115192581B CN 115192581 B CN115192581 B CN 115192581B CN 202210860628 A CN202210860628 A CN 202210860628A CN 115192581 B CN115192581 B CN 115192581B
Authority
CN
China
Prior art keywords
liposome
glioma
hydrogel
drug
peg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210860628.3A
Other languages
Chinese (zh)
Other versions
CN115192581A (en
Inventor
辛涛
刘倩
刘子豪
韩敏
王子肖
纪小帅
何东
王晓峰
王志海
王婵月
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
First Affiliated Hospital of Shandong First Medical University
Original Assignee
First Affiliated Hospital of Shandong First Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by First Affiliated Hospital of Shandong First Medical University filed Critical First Affiliated Hospital of Shandong First Medical University
Priority to CN202210860628.3A priority Critical patent/CN115192581B/en
Publication of CN115192581A publication Critical patent/CN115192581A/en
Application granted granted Critical
Publication of CN115192581B publication Critical patent/CN115192581B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Abstract

The invention provides a hydrogel-liposome combined drug delivery system, and a preparation method and application thereof, and belongs to the technical field of biological medicines. 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.

Description

Hydrogel-liposome combined drug delivery system and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a hydrogel-liposome combined drug delivery system, and a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Gliomas are the most common, most invasive craniocerebral malignancy in adults. The current standard treatment for gliomas is a maximally safe resection, combined with adjuvant radiation therapy and oral Temozolomide (TMZ) chemotherapy, which however only extends the life expectancy of the patient by 16 to 18 months. Because glioma cells have the characteristics of high invasiveness and high wettability, complete excision of all tumor bodies in microsurgery is often not possible. In addition, residual tumor tissue in surgery is prone to extensive chemotherapy resistance after prolonged application of TMZ, resulting in almost all patients inevitably recurrence of gliomas after surgery.
Iron death is a newly discovered iron-dependent cell death mode in recent years. Unlike apoptosis, necrosis, pyrodeath and autophagy, typical features of iron death include accumulation of cellular Reactive Oxygen Species (ROS), reduction of Glutathione (GSH) levels, and enhancement of lipid peroxidation levels. At present, induced iron death has been applied to tumor treatment, especially glioma treatment. Alastin (Erastin) was the first small molecule inducer found to be effective in inducing iron death to occur, which caused accumulation of lipid peroxides by inhibiting the activity of the cysteine-glutamate transporter (xCT, encoded by the SLC7a11 gene) and Glutathione Peroxidase (GPX). Recent studies indicate that the iron death pathway involved in SLC7a11 and GPX4 plays an indispensable role in TMZ chemotherapy resistance. However, although Erastin has great potential in glioma antagonism resistance, adjuvant chemotherapy, the solubility of Erastin in conventional solvents is extremely low, the bioavailability in vivo is poor, and the prospect of using the Erastin as a single drug in clinical application is very poor.
With the rapid development of nanotechnology, liposomes and hydrogels having high biocompatibility and low immunogenicity are widely used in Drug Delivery Systems (DDS). Compared with the traditional administration route, the DDS has the advantages of Blood Brain Barrier (BBB) penetrability, tumor targeting property, drug slow release property and the like. In addition, the DDS can obviously reduce the dosage of systemic administration and reduce the side effects brought by medicines. Liposomes are generally composed of modifiable polar phospholipid analogs, similar to phospholipid bilayer of cell membranes, which encapsulate drugs with any physicochemical properties. The hydrogel is formed by crosslinking a three-dimensional network formed by hydrophilic polymers, the internal aperture of the three-dimensional network is flexible, and medicines with different sizes and DDS (direct digital synthesizer) can be entrapped. The injectable temperature-sensitive gel can self-assemble to form a drug stent in an organism (when the temperature is higher than the lowest phase transition temperature), and the gel solidified in situ can realize slow release of entrapped drugs through biodegradation, so that the drug exposure of a whole body system is reduced to the greatest extent.
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.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a representation of the hydrogel-liposome combined delivery system of example 1 of the present invention. Representative transmission electron microscopy of liposomes. Scale = 100nm (B) liposome particle size distribution plot determined by dynamic light scattering. Wherein the liposome is cRGD peptide modified drug-loaded liposome. (C) the residual mass of the hydrogel system. (D) drug delivery system drug release kinetics profile. (E) uptake fluorescence profile of cells targeting liposomes. Wherein cy5.5 liposome loaded with cRGD peptide is red fluorescence, cytoskeletal F-actin is green fluorescence, and DAPI is blue fluorescence. (F) liposomes are taken up by cells by endocytosis. Wherein the liposome system is red fluorescence, DAPI is blue fluorescence, and Cloroquinone is clathrin endocytosis inhibitor. (G) flow analyser detects endocytosis of the liposomes. Wherein the P2 domain is a cy5.5 positive domain and the P3 domain is a cy5.5 negative domain.
FIG. 2 shows an in vitro test of the effect of the combination drug delivery system of example 1 of the present invention on the inhibition of drug-resistant glioma. (A) CCK-8 assay cell viability assessment was performed. Wherein cell viability = [ (experimental well absorbance-blank well absorbance)/(control well absorbance-experimental well absorbance) ]x100%. (B) EdU experiment cell proliferation potency assessment. Wherein the EdU positive cells are red fluorescent. (C) scratch test cell migration ability evaluation was performed. (D) Transwell experiments cell invasion ability assessment was performed. (E) Annexin V/PI for apoptosis assessment. Wherein the upper right quadrant UR domain is apoptotic cells. (F) evaluation of staining of live dead cells.
FIG. 3 shows in vivo detection of the effect of the combination drug delivery system of example 1 of the present invention on drug-resistant glioma. (A) intraoperative placement of a combination drug delivery system. (B) in vivo imaging of IVIS small animals to assess tumor volume. (C) a mouse model body weight change curve. (D) survival curves of mouse models. (E) H & E staining section.
FIG. 4 is a graph showing the detection of iron death in drug-resistant glioma cells induced by co-administration in example 1 of the present invention. (a) MDA levels (B) GSH and GSSH levels. (C) qPCR detects the expression level of GPX4, xCT and Ferritin. (D) Western Blot detects expression levels of GPX4, xCT, ferritin. (E) mitochondrial membrane potential JC-1 level. (F) oxidative stress ROS levels.
FIG. 5 is a graph showing the detection of ERA promoting a reduction in acetyl-CoA levels in example 1 of the present invention. (A) qPCR detects the expression levels of ACATVL and FABP 7. Wherein ACAVVL is PharmMapper target prediction, FABP7 is RNA-seq screening of significant difference genes. (B) Western Blot detection of expression levels of ACATVL and FABP 7. (C) Elisa method for detecting the level of acetyl-CoA.
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.

Claims (4)

1. A pharmaceutical formulation, characterized in that the pharmaceutical formulation comprises a pharmaceutical composition;
the pharmaceutical preparation is a gel containing liposome;
the pharmaceutical preparation is a hydrogel-liposome combined administration system; the drug delivery system comprises a hydrogel having a liposome comprising a pharmaceutical composition loaded thereon;
the active ingredients of the pharmaceutical composition comprise alastine and temozolomide;
the pharmaceutical composition is used for treating brain glioma, and 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) Inhibiting expression of glioma acetyl-coa;
the mass ratio of the alastine to the temozolomide is 1:10-20; the hydrogel is PGLA-PEG-PLGA temperature-sensitive hydrogel; the liposome is prepared from a pharmaceutical composition and auxiliary materials, wherein the auxiliary materials are hydrogenated soybean phosphatidylcholine, cholesterol and cRGD modified DSPE-PEG; the mass ratio of the hydrogenated soybean phosphatidylcholine, the cholesterol, the cRGD modified DSPE-PEG and the alastine is 4:1:1:1.
2. The method of preparing a pharmaceutical formulation according to claim 1, wherein the pharmaceutical formulation is a hydrogel-liposome combination delivery system, wherein the liposomes are prepared by reverse phase evaporation;
the preparation method comprises the following steps:
dissolving hydrogenated soybean phosphatidylcholine, cholesterol, cRGD-modified DSPE-PEG and alastin in an organic solvent; 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 to obtain the liposome.
3. The method of claim 2, wherein the organic solvent is chloroform; the preparation method also comprises the step of removing the unloaded medicinal components from the liposome prepared by ultrasonic and extrusion.
4. Use of a pharmaceutical formulation according to claim 1 for the in situ treatment of drug resistant glioma products in a preparative procedure.
CN202210860628.3A 2022-07-21 2022-07-21 Hydrogel-liposome combined drug delivery system and preparation method and application thereof Active CN115192581B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210860628.3A CN115192581B (en) 2022-07-21 2022-07-21 Hydrogel-liposome combined drug delivery system and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210860628.3A CN115192581B (en) 2022-07-21 2022-07-21 Hydrogel-liposome combined drug delivery system and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN115192581A CN115192581A (en) 2022-10-18
CN115192581B true CN115192581B (en) 2023-04-28

Family

ID=83583447

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210860628.3A Active CN115192581B (en) 2022-07-21 2022-07-21 Hydrogel-liposome combined drug delivery system and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN115192581B (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106214640A (en) * 2016-08-01 2016-12-14 华东师范大学 A kind of cancer target passs liposome delivery systems and the preparation method and application of medicine
CN112006991B (en) * 2020-08-26 2022-03-15 南京医科大学第二附属医院 Temozolamide magnetic temperature-sensitive liposome and preparation method thereof
CN114515285A (en) * 2022-03-11 2022-05-20 中国人民解放军陆军军医大学第一附属医院 Application of iron death inducer RSL3 in preparation of drug for inhibiting glioblastoma

Also Published As

Publication number Publication date
CN115192581A (en) 2022-10-18

Similar Documents

Publication Publication Date Title
KR101342971B1 (en) Drug carrier and drug carrier kit for inhibiting fibrosis
US20240033284A1 (en) Method of administration and treatment
EP2135600B1 (en) Targeting agent for cancer cell or cancer-associated fibroblast
Wang et al. MiR-101a loaded extracellular nanovesicles as bioactive carriers for cardiac repair
KR100794449B1 (en) Composition of cationic phospho lipid nanoparticles for effective delivery of nucleic acids
US11752219B2 (en) Substrate delivery of embedded liposomes
US11938186B2 (en) Thermosensitive hydrogel for cancer therapeutics and methods of preparation thereof
Wang et al. Targeting macrophage polarization as a promising therapeutic strategy for the treatment of osteoarthritis
Kong et al. Tumor microenvironmental responsive liposomes simultaneously encapsulating biological and chemotherapeutic drugs for enhancing antitumor efficacy of NSCLC
Li et al. Simultaneous blockage of contextual TGF-β by cyto-pharmaceuticals to suppress breast cancer metastasis
Chang et al. Antitumor effects of curcumin and glycyrrhetinic acid-modified curcumin-loaded cationic liposome by intratumoral administration
WO2019071875A1 (en) Cell autophagy inhibitor and preparation method therefor and application thereof
CN115192581B (en) Hydrogel-liposome combined drug delivery system and preparation method and application thereof
JP2003511404A (en) Liposome-encapsulated silver salt composition
EP3823634A1 (en) Neutral liposomes containing biologically active agents
CN102188379A (en) Preparation method of drug-carrying liposome
CN109806228A (en) A kind of solution containing meloxicam liposome
US20220249511A1 (en) Methods for the treatment of keloid, hypertrophic scars and/or hyperpigmentation disorders
US11311484B2 (en) Methods and compositions for treating skin afflictions
CN110551197B (en) Micro-peptide and cancer treatment drug
Zhu et al. A Synergistic Therapy With Antioxidant and Anti‐VEGF: Toward its Safe and Effective Elimination for Corneal Neovascularization
TWI548415B (en) Method for treatment of canine perianal gland tumor
US20240115503A1 (en) Intravesical delivery of hydrophilic therapeutic agents using liposomes
KR102641224B1 (en) Pharmaceutical compositions for preventing or treating SARS-CoV-2 infection
KR102249814B1 (en) Composition for inhibiting cancer metastasis comprising kalkitoxin thioamide alcohol or salt thereof as an active ingredient

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information
CB03 Change of inventor or designer information

Inventor after: Xin Tao

Inventor after: Wang Chanyue

Inventor after: Liu Qian

Inventor after: Liu Zihao

Inventor after: Han Min

Inventor after: Wang Zixiao

Inventor after: Ji Xiaoshuai

Inventor after: He Dong

Inventor after: Wang Xiaofeng

Inventor after: Wang Zhihai

Inventor before: Liu Qian

Inventor before: Wang Chanyue

Inventor before: Xin Tao

Inventor before: Liu Zihao

Inventor before: Han Min

Inventor before: Wang Zixiao

Inventor before: Ji Xiaoshuai

Inventor before: He Dong

Inventor before: Wang Xiaofeng

Inventor before: Wang Zhihai

GR01 Patent grant
GR01 Patent grant