CN113577297A - Double-cell-membrane-wrapped siEFNA 1-loaded yolk lipid nano-drug, and preparation method and application thereof - Google Patents
Double-cell-membrane-wrapped siEFNA 1-loaded yolk lipid nano-drug, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a double cell membrane wrapped and loaded siEFNA1 yolk lipid nano-drug, a preparation method and application thereof, wherein the nano-drug takes yolk lipid nano-carrier EYLNs as a core, loads siEFNA1, and wraps esophageal squamous cell carcinoma cell membranes and neutral granular cell membranes at the same time. The load of the siEFNA1 can inhibit the transfer capacity of esophageal squamous cell carcinoma cells, the encapsulation of neutral granulocyte cell membranes can increase the tumor targeted enrichment of nano-drugs, and the encapsulation of esophageal squamous cell carcinoma cell membranes can effectively increase the anti-esophageal squamous cell carcinoma immune response and have more effective anti-esophageal squamous cell carcinoma effect.
Description
Technical Field
The invention belongs to the field of tumor targeted therapy, relates to a nano-drug for tumor targeted therapy, and particularly relates to an egg yolk lipid nano-drug NEM/EYLNs-siEFNA1 which is wrapped by esophageal squamous carcinoma cell membranes and neutral particle cell membranes and simultaneously loads siEFNA1, and a preparation method and application thereof.
Background
With the development of nanotechnology, the development of nano-drugs has become a hot spot in the research of tumor-targeted therapy. The nano-drug has to have both the passive targeting effect mediated by the high permeability and retention effect (EPR effect) of solid tumors and the active targeting effect mediated by various modifications so as to play a role in high-efficiency anti-tumor. Meanwhile, various cell membrane mediated bionic nano-materials can effectively escape immune clearance of an organism, effectively prolong the circulation time of the nano-drugs in the organism and further enhance the accumulation of the nano-drugs in tumor parts.
Disclosure of Invention
The invention aims to provide an egg yolk lipid nano-drug NEM/EYLNs-siEFNA1 which is wrapped by esophageal squamous carcinoma cell membranes and neutrophilic granulocyte cell membranes and simultaneously loads siEFNA 1; the second purpose of the invention is to provide a preparation method of NEM/EYLNs-siEFNA 1; the third purpose of the invention is to provide the application of the nano-drug NEM/EYLNs-siEFNA1 in the treatment of esophageal squamous cell carcinoma.
The technical solution of the invention is as follows: a yolk lipid nano-drug NEM/EYLNs-siEFNA1 wrapped by esophageal squamous carcinoma cell membranes and neutral granulocyte cell membranes and simultaneously loaded with siEFNA1 takes yolk lipid nano-EYLNs as a core, loads siEFNA1 with obvious esophageal squamous carcinoma cell transfer inhibition effect, and wraps the esophageal squamous carcinoma cell membranes and the neutral granulocyte cell membranes.
Furthermore, other nano-drug systems loaded with other therapeutic agents are prepared by taking the nano-drug as a core.
Wherein, the preparation method of the nano-drug comprises the following steps:
(1) preparation of egg yolk lipid carrier EYLNs: add 200-400. mu.L of ddH to the split and dried lipid ampoules2Performing ultrasonic treatment in FS60 water bath for 15-20min to obtain transparent material, repeatedly filtering with 50nm filter membrane, and standing at 4 deg.C;
(2) preparation of nano-carrier EYLNs-siEFNA1 carrying siEFNA 1: shaking and incubating 3mg of pre-prepared EYLNs carrier and 33 mu g of polyetherimide PEI for 1h at room temperature, centrifuging at 15000rpm for 20min, and adding 500 mu L of ddH2Performing ultrasonic treatment on the mixture for 3 times in a water bath every 3min for 1 time; then 5nmol siEFNA1 is added, and the mixture is incubated for 30min at room temperature by shaking, and centrifuged at 15000rpm for 5min to remove free siEFNA 1;
(3) extraction of neutrophilic granulocyte cell membranes and esophageal squamous carcinoma cell membranes: the extraction of the neutrophil membrane is to separate human peripheral blood neutrophil; resuspending the cells in a homogenization buffer, and then repeatedly homogenizing the cells on ice for about 100 times by using a manual homogenizer; finally, cell membranes are separated and purified by sucrose density gradient centrifugation; the method for extracting the esophageal squamous carcinoma cell KYSE-150 membrane is the same as that for extracting a neutrophilic granulocyte membrane;
(4) neutral granulocyte cell membrane, esophageal squamous carcinoma cell membrane wrapped EYLNS-siEFNA 1: mixing the centrifugally collected neutrophilic granulocyte cell membrane and the tumor cell membrane according to different proportions respectively and then adding ddH2O, re-suspending, mixing uniformly, and performing ice bath ultrasound for 10min until the mixture is clear to obtain neutrophilic granulocyte cell membrane/tumor cell membrane fusion membrane nanoparticles; and incubating the fused cell membrane nanoparticles and EYLNs-siEFNA1 on ice for 10min, and carrying out ice bath ultrasound for 3 times, wherein each time is 3min, so as to obtain the NKM/EYLNs-siEFNA1 nano-drug.
In the step (3), the buffer solution L comprises the following components: 10mmol/L MgCL2KCL of 1mmol/L, RNase of 10. mu.g/mL, DNase of 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 mixing mass ratio of the neutrophile granulocyte cell membranes and the tumor cell membranes collected by centrifugation is as follows: 1:2,1:1,2:1.
Wherein, the nano-drug is applied to the treatment of esophageal squamous cell carcinoma.
The invention has the advantages that: by taking natural yolk lipid nano-carrier EYLNs as a core, the nano-medicament NEM/EYLNs-siEFNA1 which is wrapped by esophageal squamous carcinoma cell membranes and neutral granular cell membranes and simultaneously loads siEFNA1 is constructed, so that more effective treatment effect on esophageal squamous carcinoma is realized through siEFNA1 targeted delivery mediated by the passive targeting action of the EYLNs carrier, active targeting mediated by the neutral granular cell membranes and enhancement of anti-esophageal squamous carcinoma immune response mediated by the esophageal squamous carcinoma membrane.
Drawings
FIG. 1 shows the extraction and identification of neutrophilic granulosa cell membranes, esophageal squamous carcinoma cell membranes, and the identification of EYLNs vectors enveloped by fusion membranes; wherein: a, identifying a neutrophile granulocyte cell membrane protein by SDS-PAGE electrophoresis; b, SDS-PAGE electrophoresis identification of esophageal squamous carcinoma cell membrane protein; and C, SDS-PAGE electrophoresis to identify the esophageal squamous carcinoma cell membrane and the neutrophil fusion membrane.
FIG. 2 shows confocal laser identification of EYLNs-siEFNA1 vectors wrapped by esophageal squamous carcinoma cell membranes and neutrophil cell membranes in different proportions.
FIG. 3 is the tissue distribution analysis of the nano-drug wrapped by the cell membranes of esophageal squamous carcinoma and the cell membranes of neutrophilic granulocytes in different proportions; detecting the fluorescence intensity of the nano-drug coated by the three proportional membranes marked by the fluorescent DiR; b, detecting and quantifying the distribution of the three nano-drugs in the tissues of the esophageal squamous cell carcinoma subcutaneous tumor-bearing mice; and C, detecting and quantifying the distribution of the three nano-drugs in tissues of the esophageal squamous cell carcinoma lung metastasis model mouse.
FIG. 4 is the analysis of the shape and particle size distribution of the nano-drug wrapped by the membrane in a ratio of 1: 1; wherein, A, the electron microscope shape of the nano-drug wrapped by the film with the ratio of 1: 1; b, the particle size distribution of the nano-drugs wrapped by the film with the ratio of 1: 1.
FIG. 5 shows the effect of the nano-drug on the immune response and lung metastasis ability of esophageal squamous cell carcinoma after the injection of the nano-drug into mice; wherein, A, the mouse tumor cell membrane specific IgG level after nano-drug injection; b, mouse tumor cell membrane specific IgG1 level after nano-drug injection; and C, the level of mouse tumor cell membrane specific IgG2a after nano-drug injection.
FIG. 6 is the analysis of the inhibition effect of nano-drug injection on lung metastasis ability of esophageal squamous cell 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. Construction of NEM/EYLNs-siEFNA 1:
(1) yolk lipidPreparation of vector EYLNs: add 200-400. mu.L of ddH to the split and dried lipid ampoules2O, performing water bath ultrasound (FS60) for 15-20min until the solution is transparent, repeatedly filtering the solution through a filter membrane with the aperture of 50nm, and placing the solution at 4 ℃ for later use;
(2) preparation of nano-carrier EYLNs-siEFNA1 carrying siEFNA 1: pre-prepared EYLNs (3mg) vector was incubated with polyetherimide PEI (33. mu.g) at room temperature with shaking for 1h, centrifuged at 15000rpm for 20min and 500. mu.L ddH added2Performing ultrasonic treatment on the mixture for 3 times in a water bath every 3min for 1 time; then 5nmol siEFNA1 was added and incubated with shaking at room temperature for 30min, and centrifuged at 15000rpm for 5min to remove free siEFNA 1;
(3) extracting cell membranes of neutrophils and esophageal squamous carcinoma: human peripheral blood neutrophils were isolated and the cells resuspended in homogenization buffer (10mmol/L MgCL)2KCL of 1mmol/L, RNase of 10 mu g/mL, DNase of 10 mu g/mL and 1 Xprotease inhibitor cocktail), then repeatedly homogenizing for about 100 times on ice by a manual homogenizer, and finally centrifugally separating and purifying cell membranes by sucrose mass density gradient (30%, 40% and 55%); the extraction method of the esophageal squamous carcinoma cell KYSE-150 membrane is the same as the above;
(4) neutrophilic granulocyte, esophageal squamous carcinoma cell membrane-wrapped EYLNs-siEFNA 1: the neutrophilic granulosa cell membrane and the tumor cell membrane (respectively according to the proportion of 1:2,1:1 and 2: 1) collected by centrifugation are mixed with ddH2Mixing the phases after O heavy suspension, and performing ice bath ultrasonic treatment for 10min until the mixture is clear to obtain neutrophilic granulocyte cell membrane/tumor cell membrane fusion membrane nanoparticles; the fused cell membrane nanoparticles were incubated with EYLNs-siEFNA1 on ice for 10min and sonicated in ice for 3 times, 3min each.
2. Fusion membrane-encapsulated EYLNS vector identification
(1) SDS-PAGE (sodium dodecyl sulfate-PAGE) electrophoretic identification of esophageal squamous carcinoma cell membranes and neutral granulocyte cell membranes: human peripheral blood neutrophils were isolated and the cells resuspended in homogenization buffer (10mmol/L MgCL)2KCL of 1mmol/L, RNase of 10 mu g/mL, DNase of 10 mu g/mL and 1 Xprotease inhibitor cocktail), then repeatedly homogenizing for about 100 times on ice by a manual homogenizer, and finally centrifugally separating and purifying cell membranes by sucrose density gradient (30%, 40% and 55%); membrane extraction of esophageal squamous carcinoma cell KYSE-150The method is the same as above; and respectively carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic identification on the separated cell membranes and corresponding cell lysates.
As shown in figure 1, the esophageal squamous carcinoma cell membrane, the neutrophil membrane and the fusion membrane are identified by SDS-PAGE; wherein, A, the cell membrane of the esophageal squamous carcinoma cell and the esophageal squamous carcinoma cell lysate are subjected to SDS-PAGE comparative analysis; b, performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) comparative analysis on the neutrophil membrane and the neutrophil lysate; c, fusion membrane SDS-PAGE electrophoretic analysis.
(2) The fusion of esophageal squamous carcinoma cell membranes and neutrophilic granulocyte cell membranes on the surface of the carrier is analyzed by laser confocal analysis: PKH 67-labeled neutrophile granulocyte cell membrane and PHK 26-labeled esophageal squamous carcinoma cell membrane (in a ratio of 1:2,1:1 and 2:1 respectively) are mixed in ddH2Mixing the O-suspended phase and the O-suspended phase uniformly, carrying out ice bath ultrasound for 10min to be clear to obtain a neutrophilic granulocyte cell membrane/tumor cell membrane fusion membrane carrier, incubating the neutrophilic granulocyte cell membrane/tumor cell membrane fusion membrane carrier on EYLNs-siEFNA1 ice for 10min, and carrying out ice bath ultrasound for 3 times, wherein each time is 3 min; respectively incubating the carrier with esophageal squamous carcinoma cells KYSE-150 for 12h, washing with PBS for 3 times, fixing with 2% PFA at room temperature for 10min, reacting with 0.2% Triton X-100 for 5min, and observing fusion condition of two cell membranes with a laser confocal microscope after DAPI staining.
As shown in FIG. 2, the red fluorescent PKH 26-labeled neutrophil membrane fused well with the green fluorescent PKH 67-labeled esophageal squamous carcinoma cell membrane.
3. Distribution analysis of nano-drug in tumor-bearing mouse tissue
(1) Establishing a mouse subcutaneous tumor-bearing model: 0.25% pancreatin digested KYSE-150 cells in logarithmic growth phase, washed twice by centrifugation using serum-free medium, viable cells were counted, and cell concentration was adjusted to 1X 10 by PBS7Per 100. mu.L, the cells were resuspended on ice until use. Anaesthetizing Balb/c nude mouse, disinfecting left lower abdomen skin, and collecting 50 μ L (5 × 10)6) The cell suspension was inoculated subcutaneously into the left lower abdomen of the mice;
(2) establishing a mouse esophageal squamous carcinoma lung metastasis model: esophageal squamous carcinoma cells (5 multiplied by 10) expressing Luciferase reporter gene 6100 mu L) injecting Balb/c nude mice into tail vein, and observing the formation condition of the mouse lung metastasis tumor by a living body imaging system of the mouse 7 days after the injection;
(3) analyzing the tissue distribution of the nano-drug: the DiR-labeled NEM/EYLNs-PTX-siEFNA14 vector is respectively injected into a mouse with esophageal cancer subcutaneous tumor or lung metastasis model intravenously, the mouse is euthanized by CO2 after 24h of injection, the heart, the liver, the spleen, the kidney, the lung, the brain, the stomach and intestine, the tumor and other tissue organs of the mouse are taken, then the mouse is scanned by a small animal living body, and the DiR signal intensity in each organ is compared and analyzed.
As shown in fig. 3, the distribution of the nano-drug in tumor-bearing mouse tissues is analyzed; a, performing fluorescence detection and quantification on three kinds of nano-drugs wrapped by cell membranes in different proportions; b, analyzing the tissue distribution of the three cell membrane-wrapped nano-drugs with different ratios in the esophageal squamous cell carcinoma subcutaneous tumor-bearing mouse; and C, analyzing the distribution of the three cell membrane-wrapped nano-drugs in different ratios in tissues of the esophageal squamous cell carcinoma lung metastasis mouse.
4. 1:1 analysis of shape and particle size of nano-drug wrapped by cell membrane
The esophageal squamous carcinoma cell membrane and the neutrophil granulosa cell membrane are mixed according to the ratio of 1: mixing and wrapping EYLNs-siEFNA1 carrier in a ratio of 1, dripping the prepared carrier (10) on a copper net, standing for 20min at room temperature, carrying out negative dyeing on 2% uranyl acetate for 2min, and absorbing residual liquid; 2% uranyl acetate was stained for 5min, and after the residual liquid was aspirated, air-dried, followed by observation by a transmission electron microscope (FEI TECNAI G2, voltage: 120 kv).
In addition, freshly prepared NEM/EYLNs-siEFNA1 nanopharmaceutical was dissolved in approximately 500. mu.L of ddH2And O, then placing the sample in a detection sample pool, and analyzing the particle size distribution of the carrier by a Nicomp380Z3000 particle size analyzer.
As shown in fig. 4, detecting the shape and particle size distribution of the 1:1 cell membrane-coated nano-drug; wherein, A, the shape of the nano-drug is observed by an electron microscope; b, the particle size distribution shows a particle size of about 150 nm.
5. The nanometer medicinal preparation has effect in stimulating immune response of human body to esophageal squamous carcinoma
Injecting the nano-drug coated by 1:1 cell membranes into a mouse through tail vein, injecting the nano-drug once every 5 days for 5 times, and detecting the levels of the cell membrane specific IgG, IgG1 and IgG2a of the esophageal squamous carcinoma by an ELISA method every 5 days.
As shown in fig. 5, the level of specific antibodies of esophageal squamous cell carcinoma cell membranes in peripheral blood serum of mice after nano-drug injection is detected; wherein, A, the esophageal squamous carcinoma cell membrane specific IgG level after nano-drug injection; b, the esophageal squamous carcinoma cell membrane specific IgG1 level after nano-drug injection; c, the esophageal squamous carcinoma cell membrane specific IgG2a level after nano-drug injection.
6. Analysis of lung metastasis capacity of mouse esophageal squamous carcinoma after nano-drug injection
Injecting 1:1 cell membrane-coated nanometer medicine into mice via caudal vein, injecting once every 5 days for 5 times, and injecting luciferase-expressing esophageal squamous carcinoma cell KYSE-150(5 x 10)6) And the esophageal squamous carcinoma lung metastasis condition is observed by small animal living body imaging.
As shown in figure 6, the nano-drug injection can significantly inhibit esophageal squamous cell carcinoma cell lung metastasis.
The results show that: the nano-drug NEM/EYLNs-siEFNA1 which has the grain diameter of about 150nm and is loaded with the esophageal squamous cell carcinoma inhibitory siEFNA1 and wraps the esophageal squamous cell carcinoma cell membranes and neutral particle cell membranes is prepared, and the vector has a good clinical transformation application prospect.
Claims (7)
1. The double cell membrane wrapped and loaded siEFNA1 yolk lipid nano-drug is characterized in that: the nano-drug takes yolk lipid nano-carrier EYLNs as a core, loads siEFNA1 for inhibiting esophageal squamous cell carcinoma cell metastasis, and wraps the carrier with esophageal squamous cell carcinoma cell membranes and neutrophilic granulocyte cell membranes.
2. The preparation method of the double cell membrane wrapped siEFNA1 egg yolk lipid nano-drug according to claim 1, which is characterized in that: the preparation method comprises the following steps:
(1) preparation of egg yolk lipid carrier EYLNs: add 200-400. mu.L of ddH to the split and dried lipid ampoules2Performing ultrasonic treatment in FS60 water bath for 15-20min to obtain transparent material, repeatedly filtering with 50nm filter membrane, and standing at 4 deg.C;
(2) preparation of nano-carrier EYLNs-siEFNA1 carrying siEFNA 1:shaking and incubating 3mg of pre-prepared EYLNs carrier and 33 mu g of polyetherimide PEI for 1h at room temperature, centrifuging at 15000rpm for 20min, and adding 500 mu L of ddH2Performing ultrasonic treatment on the mixture for 3 times in a water bath every 3min for 1 time; then 5nmol siEFNA1 is added, and the mixture is incubated for 30min at room temperature by shaking, and centrifuged at 15000rpm for 5min to remove free siEFNA 1;
(3) extraction of neutrophilic granulocyte cell membranes and esophageal squamous carcinoma cell membranes: the extraction of the neutrophil membrane is to separate human peripheral blood neutrophil; resuspending the cells in a homogenization buffer, and then repeatedly homogenizing the cells on ice for about 100 times by using a manual homogenizer; finally, cell membranes are separated and purified by sucrose density gradient centrifugation; the method for extracting the esophageal squamous carcinoma cell KYSE-150 membrane is the same as that for extracting a neutrophilic granulocyte membrane;
(4) neutral granulocyte cell membrane, esophageal squamous carcinoma cell membrane wrapped EYLNS-siEFNA 1: mixing the centrifugally collected neutrophilic granulocyte cell membrane and the tumor cell membrane according to different proportions respectively and then adding ddH2O, re-suspending, mixing uniformly, and performing ice bath ultrasound for 10min until the mixture is clear to obtain neutrophilic granulocyte cell membrane/tumor cell membrane fusion membrane nanoparticles; and incubating the fused cell membrane nanoparticles and EYLNs-siEFNA1 on ice for 10min, and carrying out ice bath ultrasound for 3 times, wherein each time is 3min, so as to obtain the NKM/EYLNs-siEFNA1 nano-drug.
3. The preparation method of the double cell membrane wrapped siEFNA1 egg yolk lipid nano-drug according to claim 2, which is characterized in that: in the step (3), the buffer solution L comprises the following components: 10mmol/L MgCL2KCL of 1mmol/L, RNase of 10. mu.g/mL, DNase of 10. mu.g/mL and 1 Xprotease inhibitor cocktail.
4. The preparation method of the double cell membrane wrapped siEFNA1 egg yolk lipid nano-drug according to claim 2, which is characterized in that: in the step (3), the mass percentage density gradient of the sucrose is as follows: 30%, 40% and 55%.
5. The preparation method of the double cell membrane wrapped siEFNA1 egg yolk lipid nano-drug according to claim 2, which is characterized in that: in the step (4), the mixing mass ratio of the neutrophile granulocyte cell membranes and the tumor cell membranes collected by centrifugation is as follows: 1:2,1:1,2:1.
6. The double cell membrane wrapped siEFNA1 egg yolk lipid nano-drug of claim 1, which is characterized in that: the nano-drug is taken as a core to prepare other nano-drug systems loaded with other therapeutic preparations.
7. The application of the double cell membrane wrapped and loaded siEFNA1 yolk lipid nano-drug is characterized in that: the nanometer medicinal preparation can be used for treating esophageal squamous carcinoma.
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