CN113209025A - Dox and silPCAT 1-loaded yolk lipid nano-drug and preparation method and application thereof - Google Patents
Dox and silPCAT 1-loaded yolk lipid nano-drug and preparation method and application thereof Download PDFInfo
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- CN113209025A CN113209025A CN202110380664.5A CN202110380664A CN113209025A CN 113209025 A CN113209025 A CN 113209025A CN 202110380664 A CN202110380664 A CN 202110380664A CN 113209025 A CN113209025 A CN 113209025A
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- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
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- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention discloses a yolk lipid nano-drug loaded with Dox and silPCAT1, a preparation method and application thereof, wherein the nano-drug is based on yolk lipid and simultaneously loaded with chemical drug Dox and nucleic acid preparation silPCAT 1; shaking and incubating 3mg of a pre-prepared yolk lipid nano-carrier EYLNs and 33 mu g of PEI for 1 hour at room temperature, centrifuging at 15000rpm for 20 minutes, adding 500 mu L of double distilled water, and performing water bath ultrasound for 1 time every 3 minutes for 3 times; then 5nmol of silPCAT1 is added, after shaking and incubation for 30 minutes, water bath ultrasound is carried out for 1 time every 2 minutes, and 3 times in total; then adding 500 mu g of Dox, incubating for 30 minutes at room temperature, carrying out ultrasonic treatment for 5 minutes at 4 ℃, centrifuging for 5 minutes at 15000rpm, precipitating and drying to obtain the nano-drug EYLNs-Dox-silPCAT 1; the nano-drug is applied to the treatment of esophageal squamous cell carcinoma. Compared with a single chemotherapeutic drug Dox and an egg yolk liposome loaded Dox (EYLNs-Dox), the EYLNs-Dox-silPCAT1 has a stronger esophageal squamous carcinoma inhibition effect, and is an anti-esophageal squamous carcinoma nano-drug with clinical transformation application value.
Description
Technical Field
The invention belongs to the field of tumor treatment, relates to a nano-drug for targeted therapy of esophageal squamous cell carcinoma, and particularly relates to a yolk lipid nano-drug EYLNs-Dox-silPCAT1 loaded with Dox and silPCAT1, and a preparation method and application thereof.
Background
Esophageal squamous carcinoma is the 5 th major malignant tumor threatening the life health of men in China, about half of esophageal carcinoma occurs in China globally, the main tissue type is squamous cell carcinoma, and the total 5-year survival rate of patients is less than 20%. Synchronous chemoradiotherapy is still the main means for treating middle and late esophageal cancer at present, and although a plurality of molecular targeted drug clinical researches for esophageal cancer (including drugs cetuximab taking EGFR as a target, TKI drug erlotinib, drug trastuzumab taking HER2 receptor as a target, and bevacizumab taking vascular endothelial growth factor VEGF as a target) have been developed at home and abroad, the clinical curative effects are not obvious, and the curative effect of middle and late esophageal cancer is limited to a great extent, so that the search for a new esophageal cancer targeted therapeutic means still needs to be solved at present.
With the development of nanotechnology, tumor targeted therapy mediated by a nano delivery system is receiving wide attention, which not only can increase the solubility of drugs and reduce the clearance rate of the drugs, but also plays a more efficient tumor inhibition role through a passive targeting effect mediated by the high permeability and retention effect (EPR effect) of solid tumors and an active targeting effect mediated by various modifications.
Disclosure of Invention
The invention aims to provide a yolk lipid nano-drug EYLNs-Dox-silPCAT1 loaded with a chemical drug Dox and a small molecule nucleic acid preparation silPCAT 1; the second purpose of the invention is to provide a preparation method of yolk lipid nano-drug EYLNs-Dox-silPCAT 1; the third purpose of the invention is to provide the application of the nano-drug EYLNs-Dox-silPCAT1 in the treatment of esophageal squamous carcinoma.
The technical solution of the invention is as follows: the yolk lipid nano-drug EYLNs-Dox-silPCAT1 loaded with Dox and silPCAT1 is characterized in that: the nano-drug is based on yolk lipid and simultaneously loads chemical drug Dox and nucleic acid preparation silPCAT 1.
The preparation method of the yolk lipid nano-drug EYLNs-Dox-silPCAT1 loaded with Dox and silPCAT1 is characterized by comprising the following specific steps:
(1) pre-preparation of yolk lipid nanocarriers EYLNs: respectively extracting the required yolk lipid by a lipid extraction kit (Cell Biolabs), wherein the specific extraction method is carried out according to the steps provided by the kit; loading the mixed lipid into a glass ampule, drying, adding 400 mu L double distilled water, and carrying out water bath ultrasonic treatment for 5-10 minutes until the solution is transparent to obtain EYLNS;
(2) preparing an egg yolk lipid nano-drug EYLNs-Dox-silPCAT 1: shaking and incubating 3mg of a pre-prepared yolk lipid nano-carrier EYLNs and 33 mu g of PEI for 1 hour at room temperature, centrifuging at 15000rpm for 20 minutes, adding 500 mu L of double distilled water, and performing water bath ultrasound for 1 time every 3 minutes for 3 times; then 5nmol of silPCAT1 is added, after shaking and incubation for 30 minutes, water bath ultrasound is carried out for 1 time every 2 minutes, and 3 times in total; then adding 500 mu g of Dox, incubating for 30 minutes at room temperature, carrying out ultrasonic treatment for 5 minutes at 4 ℃, centrifuging for 5 minutes at 15000rpm, precipitating and drying to obtain the nano-drug EYLNs-Dox-silPCAT 1.
Furthermore, the mixed lipid is prepared by mixing lecithin PC, sphingomyelin SM, lysolecithin LPC, phosphatidylethanolamine PE, phosphatidylinositol PI and phosphatidylglycerol PG according to the mass ratio of 75.5:12.7:6.55:4.37:0.54: 0.34.
The application of the yolk lipid nano-drug EYLNs-Dox-silPCAT1 loaded with Dox and silPCAT1 is characterized in that: the application of the nano-drug in the treatment of esophageal squamous cell carcinoma is provided.
The invention has the advantages that: by taking yolk lipid nano-carrier EYLNs as a core, a nano-drug EYLNs-Dox-siLPCAT1 which simultaneously loads chemical drug Dox and nucleic acid preparation siLPCAT1 is developed, and earlier research data proves that the nano-drug realizes a more efficient esophageal squamous cell carcinoma inhibition effect through the synergistic effect of Dox and siLPCAT 1.
Drawings
FIG. 1 is the identification of nano-drug EYLNs-Dox-silPCAT1 co-loading Dox and silPCAT 1; red fluorescence is Dox, green fluorescence is FITC-silPCAT 1;
FIG. 2 is the morphology and particle size identification of the nano-drug EYLNs-Dox-silPCAT 1; a is the form of nano-drug observed by a transmission electron microscope, B is the particle size distribution of about 100nm detected by a particle sizer;
FIG. 3 shows the effect of nano-drug EYLNs-Dox-silPCAT1 on the expression of esophageal squamous carcinoma cell LPCAT 1; treating esophageal squamous carcinoma cells KYSE-150 by PBS, Dox, EYLNs-Dox and EYLNs-Dox-silPCAT1 respectively, and detecting LPCAT1 expression by a western blot method;
FIG. 4 is the in vitro esophageal squamous carcinoma cell killing activity analysis of the nano-drug EYLNs-Dox-silPCAT 1; a is the influence on the clone proliferation of the KYSE-150 cell, B is the influence on the migration function of the KYSE-150 cell, and C is the influence on the invasion capability of the KYSE-150 cell;
FIG. 5 is an in vivo anti-esophageal squamous carcinoma activity assay of the nano-drug EYLNs-Dox-silPCAT 1; a is the distribution detection of the drug in mouse esophageal squamous carcinoma xenograft tissues, B is the in vivo anti-mouse esophageal squamous carcinoma activity of a nano drug EYLNS-Dox-silPCAT1 and Dox and EYLNS-Dox compared by living body imaging, C is the growth condition of mouse esophageal squamous carcinoma, and D is the tumor body image of each group of mouse esophageal squamous carcinoma.
Detailed Description
The technical solution of the present invention is further illustrated by the following examples, but the technical solution is not limited thereto, and the adaptive modifications thereof are within the scope of the present invention.
1. Pre-preparation of yolk lipid nanocarriers EYLNs: respectively extracting the required yolk lipid by a lipid extraction kit (Cell Biolabs), wherein the specific extraction method is carried out according to the steps provided by the kit; loading the mixed lipid into a glass ampule, drying, adding 400 mu L double distilled water, and carrying out water bath ultrasonic treatment for 5-10 minutes until the solution is transparent to obtain EYLNS; the mixed lipid is prepared by mixing lecithin PC, sphingomyelin SM, lysolecithin LPC, phosphatidylethanolamine PE, phosphatidylinositol PI and phosphatidylglycerol PG in a mass ratio of 75.5:12.7:6.55:4.37:0.54: 0.34.
2. Preparation of nano-drug EYLNs-Dox-silPCAT 1: shaking and incubating 3mg of a pre-prepared yolk lipid nano-carrier EYLNs and 33 mu g of PEI for 1 hour at room temperature, centrifuging at 15000rpm for 20 minutes, adding 500 mu L of double distilled water, and performing water bath ultrasound for 1 time every 3 minutes for 3 times; then 5nmol of silPCAT1 is added, after shaking and incubation for 30 minutes, water bath ultrasound is carried out for 1 time every 2 minutes, and 3 times in total; then 500. mu.g of Dox was added and incubated at room temperature for 30 minutes, sonicated at 4 ℃ for 5 minutes, centrifuged at 15000rpm for 5 minutes, and precipitated, and the obtained NanoMedicine EYLNs-Dox-silPCAT1 was resuspended in PBS for use.
3. Dox and silPCAT1 in the nano-drug EYLNs-Dox-silPCAT1 are jointly loaded and identified: firstly, preparing siLPCAT1 and Dox carrying FITC fluorescent markers; shaking and incubating 3mg of a pre-prepared yolk lipid nano-carrier EYLNs and 33 mu g of PEI for 1 hour at room temperature, centrifuging at 15000rpm for 20 minutes, adding 500 mu L of double distilled water, and performing water bath ultrasound for 1 time every 3 minutes for 3 times; then 5nmol of siLPCAT1 carrying FITC fluorescent marker is added, after 30 minutes of shaking incubation, water bath ultrasound is carried out for 1 time every 2 minutes, and 3 times in total; then adding 500 mu g of Dox carrying FITC fluorescent marker to incubate for 30 minutes at room temperature, carrying out ultrasonic treatment at 4 ℃ for 5 minutes, centrifuging at 15000rpm for 5 minutes, and precipitating to obtain EYLNs-Dox-silPCAT1 carrying FITC fluorescent marker nano-drug; and finally, incubating the FITC fluorescence labeling nano-drug and esophageal squamous carcinoma cell KYSE-150 for 12 hours, and observing the co-loading condition of Dox and FITC-silPCAT1 by an immunofluorescence method.
FIG. 1 is a nano-drug EYLNs-Dox-silPCAT1 co-loading Dox and silPCAT1 identification, wherein: the red fluorescence was Dox and the green fluorescence was FITC-silPCAT 1.
4. The nanometer medicine EYLNs-Dox-silPCAT1 has the shape and the particle size characterization: dripping 10 mu L of the EYLNs-Dox-silPCAT1 prepared well on a carbon-coated copper net, and observing the shape of the carbon-coated copper net through a transmission electron microscope after uranyl acetate with the mass concentration of 1% is negatively dyed; dissolving 20 μ L of the nano-drug EYLNs-Dox-silPCAT1 in 400 μ L of double distilled water, transferring to a sample tube, and analyzing the particle size of the nano-drug by a particle size analyzer.
FIG. 2 is the morphology and particle size identification of the nano-drug EYLNs-Dox-silPCAT 1; wherein: a is the form of nano-drug observed by transmission electron microscope, B is the particle size distribution about 100nm detected by particle size analyzer.
5. The influence of the nano-drug EYLNs-Dox-silPCAT1 on the expression of esophageal squamous carcinoma cells LPCAT 1: culturing esophageal squamous carcinoma cells in a 6-well plate, respectively treating the cells with PBS, Dox, EYLNS-Dox and EYLNS-Dox-silPCAT1 after 24 hours, collecting the cells after 48 hours, cracking a lysate, carrying out protein denaturation by a BCA method at 100 ℃ for 10 minutes, and then carrying out SDS-PAGE electrophoresis and PVDF membrane transfer printing; blocking BSA with the mass concentration of 5%, and adding primary anti-LPCAT 1 antibody and secondary antibody for incubation; finally, the expression of ECL-colored LPCAT1 was performed.
FIG. 3 shows the effect of nano-drugs EYLNs-Dox-silPCAT1 on the expression of esophageal squamous carcinoma cell LPCAT 1; wherein, the esophageal squamous carcinoma cell KYSE-150 is respectively treated by PBS, Dox, EYLNs-Dox and EYLNs-Dox-silPCAT1, and the expression of LPCAT1 is detected by a western blot method.
6. The in vitro anti-esophageal squamous cell carcinoma activity of the nano-drug EYLNs-Dox-silPCAT 1: the clone proliferation experiment, the Transwell migration and invasion experiment are used for evaluating the in vitro anti-esophageal squamous cell carcinoma capacity of the nano-drug; clone proliferation experiments: the esophageal squamous carcinoma cells KYSE-150 are paved in 6-hole cell culture plates (500/hole), and then treated with PBS, Dox, EYLNs-Dox and EYLNs-Dox-silPCAT1 respectively, wherein the treatment is carried out 1 time every 3 days and 3 times; then fixing and staining the cell clone and then photographing; migration experiment: KYSE-150 cells (2X 10)3) Resuspending in 200 μ L DMEM medium, placing in upper chamber of transwell chamber with 8mm aperture, the lower chamber is DMEM medium containing 10% FBS by mass concentration, then treating the cells with PBS, Dox, EYLNs-Dox and EYLNs-Dox-silPCAT1 respectively, after 48 hours, wiping off the cells on the upper surface of the filter screen, fixing the cells on the lower surface, staining 0.1% crystal violet, and taking pictures and counting; invasion test: detection was performed by Matrigel-coated transwell, and the rest was the same as the migration experiment.
FIG. 4 is the in vitro esophageal squamous carcinoma cell killing activity analysis of the nano-drug EYLNs-Dox-silPCAT 1; wherein: a is the influence on the clone proliferation of KYSE-150 cells; b is the effect on the cell migration function of KYSE-150; c is the effect on the invasive potential of KYSE-150 cells.
7. The in vivo anti-esophageal squamous carcinoma curative effect analysis of the nano-drug EYLNs-Dox-silPCAT 1: respectively establishing a nude mouse esophageal squamous cell carcinoma xenograft model by esophageal squamous cell carcinoma cell KYSE-150 expressing luciferase reporter gene, wherein the tumor body grows toAbout 100mm3Mice were randomly divided into 4 groups, treated with PBS, Dox, EYLNs-Dox, and EYLNs-Dox-silPCAT1 (Dox dose was given at 5mg/kg mouse body weight), respectively, once every 6 days for 5 times total, live imaged once every 6 days, and mouse tumor size and mouse body weight were measured once every 3 days.
FIG. 5 is an in vivo anti-esophageal squamous carcinoma activity assay of the nano-drug EYLNs-Dox-silPCAT 1; wherein: a, detecting the distribution of the medicament in the mouse esophageal squamous carcinoma xenograft tissues; b, in vivo imaging comparison of the in vivo anti-mouse esophageal squamous carcinoma activity of the nano-drug EYLNs-Dox-silPCAT1 and Dox and EYLNs-Dox; c, growth condition of mouse esophageal squamous carcinoma; d, esophageal squamous carcinoma tumor body images of the mice in each group.
The results show that: the nanometer medicine EYLNs-Dox-SilPCAT1 with the grain diameter of about 100nm and the loading of Dox and SilPCAT1 is prepared; the nano-drug effectively reduces the expression of LPCAT1 in esophageal squamous carcinoma cells, thereby inhibiting the proliferation, migration and invasion capabilities of the cells; the mouse esophageal squamous carcinoma xenograft model further proves that EYLNS-Dox-silPCAT1 has more effective anti-esophageal squamous carcinoma effect than Dox and EYLNS-Dox.
Claims (4)
1. The yolk lipid nano-drug loaded with Dox and silPCAT1 is characterized in that: the nano-drug is based on yolk lipid and simultaneously loads chemical drug Dox and nucleic acid preparation silPCAT 1.
2. The preparation method of the yolk lipid nano-drug loaded with Dox and silPCAT1 is characterized by comprising the following steps: the preparation method comprises the following specific steps:
(1) pre-preparation of yolk lipid nanocarriers EYLNs: respectively extracting the required yolk lipid by a lipid extraction kit (Cell Biolabs), wherein the extraction method is carried out according to the steps provided by the kit; loading the mixed lipid into a glass ampule, drying, adding 400 mu L double distilled water, and carrying out water bath ultrasonic treatment for 5-10 minutes until the solution is transparent to obtain EYLNS;
(2) preparing an egg yolk lipid nano-drug EYLNs-Dox-silPCAT 1: shaking and incubating 3mg of a pre-prepared yolk lipid nano-carrier EYLNs and 33 mu g of PEI for 1 hour at room temperature, centrifuging at 15000rpm for 20 minutes, adding 500 mu L of double distilled water, and performing water bath ultrasound for 1 time every 3 minutes for 3 times; then 5nmol of silPCAT1 is added, after shaking and incubation for 30 minutes, water bath ultrasound is carried out for 1 time every 2 minutes, and 3 times in total; then adding 500 mu g of Dox, incubating for 30 minutes at room temperature, carrying out ultrasonic treatment for 5 minutes at 4 ℃, centrifuging for 5 minutes at 15000rpm, precipitating and drying to obtain the nano-drug EYLNs-Dox-silPCAT 1.
3. The method for preparing the Dox and silPCAT 1-loaded yolk lipid nano-drug according to claim 1, which is characterized in that: the mixed lipid is prepared by mixing lecithin PC, sphingomyelin SM, lysolecithin LPC, phosphatidylethanolamine PE, phosphatidylinositol PI and phosphatidylglycerol PG in a mass ratio of 75.5:12.7:6.55:4.37:0.54: 0.34.
4. The application of the yolk lipid nano-drug loaded with Dox and silPCAT1 is characterized in that: the application of the nano-drug in the treatment of esophageal squamous cell carcinoma is provided.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107811972A (en) * | 2017-11-03 | 2018-03-20 | 华中科技大学 | A kind of adriamycin and genomic medicine convey nano medicament carrying system and preparation method altogether |
| CN107823140A (en) * | 2017-10-30 | 2018-03-23 | 淮安市第人民医院 | The nano-medicament carrier prepared based on yolk polar lipid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107823140A (en) * | 2017-10-30 | 2018-03-23 | 淮安市第人民医院 | The nano-medicament carrier prepared based on yolk polar lipid |
| CN107811972A (en) * | 2017-11-03 | 2018-03-20 | 华中科技大学 | A kind of adriamycin and genomic medicine convey nano medicament carrying system and preparation method altogether |
Non-Patent Citations (1)
| Title |
|---|
| YALI JUN等: "Leukocyte-Mediated Combined Targeted Chemo and Gene Therapy for Esophageal Cancer", 《ACS APPL. MATER. INTERFACES》 * |
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