CN113577297B - Double cell membrane coated and loaded siEFNA1 egg yolk lipid nano-medicament as well as preparation method and application thereof - Google Patents
Double cell membrane coated and loaded siEFNA1 egg yolk lipid nano-medicament as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a double cell membrane wrapping and loading siEFNA1 egg yolk lipid nano-medicament, a preparation method and application thereof, wherein the nano-medicament takes an egg yolk lipid nano-carrier EYLNs as a core, loads siEFNA1 and wraps esophageal squamous carcinoma cell membranes and neutrophil granulocyte membranes. The siEFNA1 can inhibit the metastasis capability of esophageal squamous carcinoma cells, the encapsulation of neutrophil cell membranes can increase the tumor targeting enrichment of nano-drugs, and the encapsulation of esophageal squamous carcinoma cell membranes can effectively increase the immune response against esophageal squamous carcinoma, so that the composition has more effective esophageal squamous carcinoma resisting effect.
Description
Technical Field
The invention belongs to the field of tumor targeted therapy, relates to a nano-drug for tumor targeted therapy, and in particular relates to a yolk lipid nano-drug NEM/EYLNs-siEFNA1 which is wrapped by esophageal squamous carcinoma cell membranes and neutrophil granulocyte membranes and simultaneously loads siEFNA1, and a preparation method and application thereof.
Background
Along with development of nanotechnology, development of nano-drugs becomes a hotspot of tumor targeted therapy research. The nano-drug has both high permeability and retention effect (EPR effect) mediated passive targeting effect of solid tumor and active targeting effect mediated by various modifications to exert high-efficiency anti-tumor effect. Meanwhile, various cell membrane mediated bionic nano can effectively avoid immune clearance of the organism, effectively prolong circulation time of nano medicine in the organism and further enhance accumulation of the nano medicine in tumor parts.
Disclosure of Invention
The first object of the invention is to provide a yolk lipid nano-drug NEM/EYLNs-siEFNA1 which is wrapped by esophageal squamous carcinoma cell membranes and neutrophil granulocyte membranes and is loaded with siEFNA1 at the same time; a second object of the present invention is to provide a process for the preparation of NEM/EYLNs-siEFNA1; a third object of the present invention is to provide the use of the nano-drug NEM/EYLNs-siEFNA1 in the treatment of esophageal squamous carcinoma.
The technical scheme of the invention is as follows: the nano medicine takes yolk lipid nano EYLNs as a core, loads the siEFNA1 with obvious esophageal squamous carcinoma cell transfer inhibition effect, and wraps esophageal squamous carcinoma cell membranes and neutrophil granulocyte membranes.
Furthermore, the nano-drug is taken as a core to prepare other nano-drug systems for loading other therapeutic preparations.
The preparation method of the nano-drug comprises the following steps:
(1) Preparation of egg yolk lipid carrier eyls: 200-400. Mu.L ddH was added to split and dried lipid ampoule 2 Performing ultrasonic treatment on O and FS60 water bath for 15-20min until the materials are transparent, repeatedly filtering the materials through a filter membrane with the aperture of 50nm, and then placing the materials at the temperature of 4 ℃ for standby;
(2) Preparation of siEFNA 1-carrying nanocarriers EYLNs-siEFNA1: 3mg of the prepared EYLNs carrier and 33. Mu.g of polyetherimide PEI were incubated with shaking at room temperature for 1h, centrifuged at 15000rpm for 20min, and 500. Mu.L of ddH was added 2 O, carrying out water bath ultrasonic treatment for 1 time every 3min, and carrying out ultrasonic treatment for 3 times; then 5nmol of siEFNA1 was added and incubated at room temperature with shaking for 30min, and centrifuged at 15000rpm for 5min to remove free siEFNA1;
(3) Extraction of neutrophil membrane and esophageal squamous carcinoma cell membrane: the extraction of the neutrophil membrane is to separate the human peripheral blood neutrophil; re-suspending the cells in a homogenization buffer, and repeatedly homogenizing the cells on ice by a manual homogenizer for about 100 times; finally purifying the cell membrane by sucrose density gradient centrifugation; the extraction method of the esophageal squamous carcinoma cell KYSE-150 membrane is the same as that of the neutrophil membrane;
(4) Neutrophil and esophageal squamous carcinoma cell membranes encapsulate eyls-siEFNA 1: mixing the neutral granulocyte membrane and tumor cell membrane collected by centrifugation according to different ratios, and adding ddH 2 O is resuspended, and then is evenly mixed, and is subjected to ice bath ultrasonic treatment for 10min until the mixture is clear, thus obtaining the neutral granulocyte membrane/tumor cell membrane fusion membrane nano-particles; and incubating the fused cell membrane nano-particles with EYLNs-siEFNA1 on ice for 10min, and then performing ice bath ultrasonic treatment for 3 times and 3min each time to obtain the NKM/EYLNs-siEFNA1 nano-drug.
In the step (3), the components in the L buffer solution are as follows: mgCL of 10mmol/L 2 KCL at 1mmol/L, RNase at 10. Mu.g/mL, DNase at 10. Mu.g/mL, and 1 Xprotease inhibitor cocktail.
In the step (3), the mass percentage density gradient of the sucrose is as follows: 30%,40% and 55%.
In the step (4), the mass ratio of the mixture of the centrifugally collected neutrophil granulocyte membrane and the tumor cell membrane is as follows: 1:2,1:1,2:1.
Wherein, the nano-drug is applied to esophageal squamous carcinoma treatment.
The invention has the advantages that: the natural egg yolk lipid nano-carrier EYLNs is taken as a core, so that a nano-drug NEM/EYLNs-siEFNA1 which is wrapped by an esophageal squamous carcinoma cell membrane and a neutrophil granulocyte membrane and simultaneously loads siEFNA1 is constructed, and more effective esophageal squamous carcinoma treatment effect is expected to be realized through passive targeting effect-mediated siEFNA1 targeting delivery, neutrophil granulocyte membrane-mediated active targeting and esophageal squamous carcinoma immune response enhancement mediated by the esophageal squamous carcinoma cell membrane.
Drawings
FIG. 1 shows the extraction and identification of neutrophil and esophageal squamous carcinoma cell membranes and the identification of EYLNs vectors wrapped by fusion membranes; wherein: a, SDS-PAGE electrophoresis is used for identifying neutral granulocyte membrane protein; b, SDS-PAGE electrophoresis is used for identifying esophageal squamous carcinoma cell membrane proteins; c, SDS-PAGE electrophoresis is used for identifying esophageal squamous carcinoma cell membranes and neutrophil fusion membranes.
FIG. 2 shows laser confocal identification of EYLNs-siEFNA1 vectors wrapped by esophageal squamous carcinoma cell membranes and neutrophil granulocyte membranes in different proportions.
FIG. 3 is a graph showing tissue distribution analysis of nano-drugs wrapped by esophageal squamous carcinoma cell membranes and neutrophil granulocyte membranes in different proportions; wherein, A, fluorescence DiR marked nano-drug fluorescence intensity detection coated by three proportion films; b, detecting and quantifying the distribution of the three nano-drugs in the tissue of the esophageal squamous carcinoma subcutaneous tumor-bearing mice; and C, detecting and quantifying the distribution of the three nano-drugs in the esophageal squamous carcinoma lung metastasis model mouse tissue.
FIG. 4 is a 1:1 ratio film coated nanomedicine morphology and particle size distribution analysis; wherein, the ratio of A to 1 is the electron microscope morphology of the nano-drug wrapped by the film; and B,1:1 proportion of membrane-wrapped nano medicine particle size distribution.
FIG. 5 is a graph showing the effect of immune response and lung metastasis of esophageal squamous carcinoma after nano-drug injection into mice; wherein, A, mouse tumor cell membrane specific IgG level after nano drug injection; b, mouse tumor cell membrane specific IgG1 levels after nano drug injection; c, mouse tumor cell membrane specific IgG2a levels after nano drug injection.
FIG. 6 is an analysis of inhibition of lung metastasis of esophageal squamous carcinoma cells after nano-drug injection.
Detailed Description
The technical solutions of the present invention will be further described with reference to examples, but the technical solutions should not be construed as being limited thereto, and the adaptation modifications based thereon are all within the scope of the present invention.
1. Construction of NEM/EYLNs-siEFNA 1:
(1) Preparation of egg yolk lipid carrier eyls: 200-400. Mu.L ddH was added to split and dried lipid ampoule 2 O, carrying out water bath ultrasonic treatment (FS 60) for 15-20min until the materials are transparent, and then repeatedly filtering the materials through a filter membrane with the aperture of 50nm and then placing the materials at the temperature of 4 ℃ for standby;
(2) Preparation of siEFNA 1-carrying nanocarriers EYLNs-siEFNA1: the pre-prepared EYLNs carrier (3 mg) was incubated with polyetherimide PEI (33 μg) at room temperature with shaking for 1h, centrifuged at 15000rpm for 20min, and 500 μl ddH was added 2 O, carrying out water bath ultrasonic treatment for 1 time every 3min, and carrying out ultrasonic treatment for 3 times; then 5nmol of siEFNA1 was added and incubated with shaking at room temperature for 30min, centrifuged at 15000rpm for 5min to remove free siEFNA1；
(3) Neutrophil and esophageal squamous carcinoma cell membrane extraction: human peripheral blood neutrophils were isolated and the cells resuspended in homogenization buffer (10 mmol/L MgCL 2 1mmol/L KCL, 10. Mu.g/mL RNase, 10. Mu.g/mL DNase and 1 Xprotease inhibitor cocktail), repeatedly homogenizing on ice for about 100 times, and centrifuging to purify cell membrane by sucrose mass density gradient (30%, 40% and 55%); the extraction method of the esophageal squamous carcinoma cell KYSE-150 is the same as that of the esophageal squamous carcinoma cell KYSE-150;
(4) Neutrophil, esophageal squamous carcinoma cell membrane encapsulation eyls-siEFNA 1: the neutrophil membrane and tumor cell membrane (1:2, 1:1,2:1 ratio, respectively) collected by centrifugation were subjected to ddH 2 Mixing the O resuspended phases uniformly, and carrying out ice bath ultrasonic treatment for 10min until the O resuspended phases are clear to obtain neutral granulocyte membrane/tumor cell membrane fusion membrane nano-particles; the fused cell membrane nanoparticles were incubated with EYLNs-siEFNA1 on ice for 10min followed by 3 times of ice bath sonication for 3min each.
2. Fusion membrane-encapsulated EYLNs vector identification
(1) SDS-PAGE (SDS-PAGE) electrophoresis identification of esophageal squamous carcinoma cell membranes and neutrophil granulocyte membranes: human peripheral blood neutrophils were isolated and the cells resuspended in homogenization buffer (10 mmol/L MgCL 2 1mmol/L KCL, 10. Mu.g/mL RNase, 10. Mu.g/mL DNase and 1 Xprotease inhibitor cocktail), repeatedly homogenizing on ice for about 100 times, and centrifuging to purify cell membrane by sucrose density gradient (30%, 40% and 55%); the film extraction method of the esophageal squamous carcinoma cell KYSE-150 is the same as the above; and (3) performing SDS-PAGE electrophoresis identification on the separated cell membranes and the corresponding cell lysates.
As shown in fig. 1, esophageal squamous carcinoma cell membrane, neutrophil cell membrane and fusion membrane SDS-PAGE identification; wherein, A, esophageal squamous carcinoma cell membrane and esophageal squamous carcinoma cell lysate SDS-PAGE contrast analysis; b, comparing and analyzing neutrophil membrane and neutrophil lysate SDS-PAGE; c, SDS-PAGE analysis of fusion membranes.
(2) Laser confocal analysis of fusion of esophageal squamous carcinoma cell membrane and neutrophil cell membrane on carrier surface: PKH 67-labeled neutrophil membraneEsophageal squamous carcinoma cell membranes labeled with PHK26 (1:2, 1:1,2:1 ratio, respectively) were labeled with ddH 2 Mixing the O re-suspension phases, performing ice bath ultrasonic treatment for 10min to clear to obtain a neutrophil granulocyte membrane/tumor cell membrane fusion membrane carrier, incubating the neutrophil granulocyte membrane/tumor cell membrane fusion membrane carrier with EYLNs-siEFNA1 on ice for 10min, and performing ice bath ultrasonic treatment for 3 times for 3min each time; the carrier is respectively incubated with esophageal squamous carcinoma cell KYSE-150 for 12h, after PBS is washed 3 times, 2% PFA is fixed at room temperature for 10min,0.2%Triton X-100 to act for 5min, and after DAPI staining, a laser confocal microscope is used for observing the fusion condition of two cell membranes.
As shown in FIG. 2, the red fluorescent PKH 26-labeled neutrophil membrane was well fused with the green fluorescent PKH 67-labeled esophageal squamous carcinoma cell membrane.
3. Analysis of tissue distribution of nano medicine in tumor-bearing mice
(1) Establishment of a subcutaneous tumor-bearing model of a mouse: KYSE-150 cells in logarithmic growth phase were digested with 0.25% pancreatin, washed twice by centrifugation using serum-free medium, live cells were counted, and cell concentration was adjusted to 1X 10 by PBS 7 100. Mu.L, cells were resuspended in ice for use. After Balb/c nude mice were anesthetized, the left lower abdominal skin was sterilized and then 50. Mu.L (5X 10) 6 ) The cell suspension was inoculated subcutaneously into the left lower abdomen of the mice;
(2) Establishing a lung metastasis model of the esophageal squamous carcinoma of the mice: esophageal squamous carcinoma cells expressing the Luciferase reporter gene (5X 10) 6 100 μl) tail vein injection of Balb/c nude mice, and observation of pulmonary metastatic neoplasia in mice by a small animal in vivo imaging system 7 days after injection;
(3) Analysis of tissue distribution of nano-drug: diR-labeled NEM/EYLNs-PTX-siEFNA14 vectors are respectively injected into mice with subcutaneous tumor-bearing or lung metastasis model of esophageal cancer intravenously, the mice are euthanized by CO2 after 24 hours of injection, tissue organs such as heart, liver, spleen, kidney, lung, brain, stomach, tumor and the like of the mice are taken, and then the intensity of DiR signals in the organs is compared and analyzed through in-vivo scanning of small animals.
As shown in fig. 3, the nano-drug was analyzed in tumor-bearing mouse tissue distribution; wherein, A, three kinds of proportion different cell membrane wrap up nanometer medicament fluorescence detection and quantification; b, analyzing tissue distribution of three kinds of nano-drugs wrapped by cell membranes in different proportions in subcutaneous tumor-bearing mice with esophageal squamous carcinoma; and C, analyzing tissue distribution of three nano-drugs wrapped by cell membranes in different proportions in esophageal squamous carcinoma lung metastasis mice.
4. 1:1 cell membrane encapsulation nano-drug morphology and particle size analysis
Esophageal squamous carcinoma cell membrane and neutrophil cell membrane were mixed according to 1: mixing and wrapping EYLNs-siEFNA1 carrier in proportion, dripping the prepared carrier (10) on a copper mesh, standing for 20min at room temperature, performing negative dyeing on 2% uranyl acetate for 2min, and sucking residual liquid; the 2% uranyl acetate was negatively stained for 5min, and after residual liquid was removed, it was dried, and then observed by a transmission electron microscope (FEI TECNAI G, voltage: 120 kv).
In addition, freshly prepared NEM/EYLNs-siEFNA1 nano-drug was dissolved in approximately 500. Mu.L ddH 2 O, then put it in a detection sample cell, and analyze the particle size distribution of the carrier by a Nicomp 380Z 3000 particle size analyzer.
As shown in fig. 4, the nano-drug form and particle size distribution of 1:1 cell membrane encapsulation are detected; wherein, A, the morphology of the nano-drug is observed by an electron microscope; and B, the particle size distribution shows a particle size of about 150nm.
5. Nanometer medicinal preparation for stimulating immune response of organism against esophageal squamous carcinoma
The 1:1 cell membrane-encapsulated nano-drug tail vein injection mice were injected once every 5 days for 5 total injections, and then esophageal squamous carcinoma cell membrane-specific IgG, igG1, and IgG2a levels were detected by ELISA every 5 days.
As shown in fig. 5, the level of esophageal squamous carcinoma cell membrane specific antibodies in the peripheral serum of the mouse after nano-drug injection was detected; wherein, A, the specific IgG level of esophageal squamous carcinoma cell membrane after nano-drug injection; b, esophageal squamous carcinoma cell membrane specific IgG1 levels after nano-drug injection; c, esophageal squamous carcinoma cell membrane specific IgG2a levels after nano-drug injection.
6. Analysis of lung metastasis capability of esophageal squamous carcinoma of mice after nano-drug injection
The nano-drug coated by the 1:1 cell membrane is injected into the mice by tail vein, and is injected once every 5 days for 5 times, and then the tail vein is formedInjection of luciferase expressing esophageal squamous carcinoma cell KYSE-150 (5 x 10) 6 ) In vivo imaging of small animals, esophageal squamous carcinoma lung metastasis is observed.
As shown in fig. 6, nano-drug injection can significantly inhibit lung metastasis of esophageal squamous carcinoma cells.
The results show that: the nano medicine NEM/EYLNs-siEFNA1 which has the particle size of about 150nm and loads the esophageal squamous carcinoma inhibitory siEFNA1 and wraps the esophageal squamous carcinoma cell membrane and the neutrophil granulocyte membrane is prepared, and the carrier has good clinical transformation application prospect.
Claims (3)
1. The preparation method of the double cell membrane coated and loaded siEFNA1 egg yolk lipid nano-medicament takes an egg yolk lipid nano-carrier EYLNs as a core, loads siEFNA1 for inhibiting esophageal squamous carcinoma cell metastasis, and coats the carrier with esophageal squamous carcinoma cell membranes and neutrophil granulocyte membranes; the preparation method of the nano-drug is characterized by comprising the following steps:
(1) Preparation of egg yolk lipid carrier eyls: 200-400. Mu.L ddH was added to split and dried lipid ampoule 2 Performing ultrasonic treatment on O and FS60 water bath for 15-20min until the materials are transparent, repeatedly filtering the materials through a filter membrane with the aperture of 50nm, and then placing the materials at the temperature of 4 ℃ for standby;
(2) Preparation of siEFNA 1-carrying nanocarriers EYLNs-siEFNA1: 3mg of the prepared EYLNs carrier and 33. Mu.g of polyetherimide PEI were incubated with shaking at room temperature for 1h, centrifuged at 15000rpm for 20min, and 500. Mu.L of ddH was added 2 O, carrying out water bath ultrasonic treatment for 1 time every 3min, and carrying out ultrasonic treatment for 3 times; then 5nmol of siEFNA1 was added and incubated at room temperature with shaking for 30min, and centrifuged at 15000rpm for 5min to remove free siEFNA1;
(3) Extraction of neutrophil membrane and esophageal squamous carcinoma cell membrane: the extraction of the neutrophil membrane is to separate the human peripheral blood neutrophil; re-suspending the cells in a homogenization buffer, and repeatedly homogenizing the cells on ice by a manual homogenizer for about 100 times; finally purifying the cell membrane by sucrose density gradient centrifugation; the extraction method of the esophageal squamous carcinoma cell KYSE-150 membrane is the same as that of the neutrophil membrane; the components in the L buffer are as follows: mgCL of 10mmol/L 2 ，1mmoL/L KCL, 10. Mu.g/mL RNase, 10. Mu.g/mL DNase and 1 Xprotease inhibitor cocktail; the mass percent density gradient of sucrose is: 30%,40% and 55%;
(4) Neutrophil and esophageal squamous carcinoma cell membranes encapsulate eyls-siEFNA 1: mixing the neutral granulocyte membrane and tumor cell membrane collected by centrifugation according to different ratios, and adding ddH 2 O is resuspended, and then is evenly mixed, and is subjected to ice bath ultrasonic treatment for 10min until the mixture is clear, thus obtaining the neutral granulocyte membrane/tumor cell membrane fusion membrane nano-particles; incubating the fused cell membrane nano-particles with EYLNs-siEFNA1 on ice for 10min, and then performing ice bath ultrasonic treatment for 3 times and 3min each time to obtain NKM/EYLNs-siEFNA1 nano-drug; the mass ratio of the mixture of the neutrophil membrane and the tumor cell membrane collected by centrifugation is as follows: 1:2,1:1,2:1.
2. The dual cell membrane encapsulation loaded siEFNA1 egg yolk lipid nano-drug of claim 1, wherein the drug is characterized by: and (3) taking the nano-drug as a core, and preparing other nano-drug systems loaded with other therapeutic preparations.
3. The application of the double cell membrane coated and loaded siEFNA1 egg yolk lipid nano medicament is characterized in that: the nano-drug is applied to the preparation of nano-drugs for resisting esophageal squamous carcinoma.
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