CN109893660B - Bionic nano-carrier for treating brain glioma and preparation method thereof - Google Patents
Bionic nano-carrier for treating brain glioma and preparation method thereof Download PDFInfo
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Abstract
The invention provides a bionic nano-carrier for treating brain glioma and a preparation method thereof, belonging to the field of biomedical engineering. An Ang-RBCm-CA/siRNA consists of an Angiopep-2(Ang) polypeptide modified erythrocyte membrane (Ang-RBCm) and a citraconic anhydride grafted polylysine and polyethyleneimine-siRNA complex (CA/siRNA). Ang-RBCm-CA/siRNA can effectively load siRNA and protect siRNA from degradation. The Ang-RBCm-CA/siRNA has the advantages of prolonged blood circulation time and strong blood brain barrier penetration capability, can be efficiently absorbed by the U87MG brain glial cells over-expressed by a low density lipoprotein receptor (LPR), realizes rapid charge conversion in an endosome/lysosome, effectively releases siRNA, and effectively treats in-situ U87MG brain glioma.
Description
Technical Field
The invention relates to the field of biomedical engineering, in particular to a bionic nano-carrier for treating brain glioma and a preparation method thereof.
Background
Brain Gliomas (GBMs) are the most common and aggressive tumors in the central nervous system. Almost all drugs are ineffective in treating GBM patients due to their inability to penetrate the blood-brain barrier. To improve the blood-brain barrier penetration ability and therapeutic effect of brain gliomas, we have invested a great deal of effort to design nanomedicines that can specifically target GBM cells and cross the blood-brain barrier. Studies have shown that low density lipoprotein-related protein-1 (LRP-1), overexpressed on both endothelial cells and GBM cells of the blood-brain barrier, enhances blood-brain barrier penetration and tumor cell uptake through receptor-mediated transcytosis (RMT) and receptor-mediated endocytosis mechanisms. In recent years, many polypeptides capable of specifically binding to LRP-1 are modified on the surface of nanoparticles for brain-targeted delivery of anticancer drugs. Two basic drug conjugates are currently in clinical transformation due to the high brain penetration of Angiopep-2 (Ang). Researches find that the functionalized Ang nano-drug can greatly enhance the penetration capability of the blood brain barrier of the Ang nano-drug, thereby remarkably improving the in vivo treatment effect of GBM.
Small interfering RNA (siRNA) is considered to be an effective means for treating various malignant tumors including GBM due to its ability to directly regulate gene expression and extremely low cytotoxicity. From existing reports, siRNA with specific sequences, such as: polo-like kinase (siPLK1) and B-cell lymphoma 2(siBcl-2) are effective in inhibiting the proliferation of brain glioma cells. However, the GBM treatment by siRNA is still limited by its very short half-life, poor blood brain barrier penetration, low tumor accumulation, insufficient cellular uptake and cumbersome cellular endocytosis pathway.
Disclosure of Invention
The invention aims to provide a bionic nano-carrier for treating brain glioma and a preparation method thereof.
In the embodiment of the first aspect of the application, a biomimetic nano carrier for treating brain glioma is provided, wherein the biomimetic nano carrier takes a polyethyleneimine-siRNA compound as an inner core, citraconic anhydride grafted polylysine as a middle layer with negative charges, and a cell membrane modified by targeting polypeptide as an outer shell to coat the inner core and the middle layer.
In a second aspect of the present application, there is provided a method for preparing a biomimetic nano carrier for brain glioma treatment, comprising the following steps:
mixing and dissolving distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide and targeted polypeptide in a PBS buffer solution for reaction to prepare a distearoyl phosphatidyl ethanolamine-polyethylene glycol-targeted polypeptide compound;
mixing and incubating a cell membrane sample prepared from a cell sample with a distearoyl phosphatidyl ethanolamine-polyethylene glycol-targeted polypeptide compound to obtain a polypeptide-cell membrane compound;
dissolving, mixing and incubating branched polyethyleneimine and siRNA to obtain a cation compound, mixing and incubating the cation compound and citraconic anhydride grafted polylysine to prepare a CA-siRNA ternary compound;
and mixing the polypeptide-cell membrane compound and the CA-siRNA ternary compound to prepare the bionic nano-carrier.
In the embodiment, the bionic nanocarrier Ang-RBCm-CA/siRNA can be formed by using an angiopep-2 modified erythrocyte membrane (Red Blood Cell membrane, RBCm) as a shell to wrap citraconic anhydride grafted polylysine as a middle layer and a polyethyleneimine-siRNA complex as an inner core through electrostatic interaction, is used for compressing and protecting siRNA, and can effectively treat brain glioma.
Small interfering RNA (siRNA) is considered to be an effective means for treating various malignant tumors including GBM due to its ability to directly regulate gene expression and extremely low cytotoxicity.
The siRNA with specific sequence can effectively inhibit the proliferation of glioma cells.
The siRNA may be any one of SiPLK1, siBcl2, siVEGF and siEGFR, and the cancer cells are interfered by the siRNA.
In some embodiments of the foregoing second aspect, the targeting polypeptide comprises one of Angiopep-2, RGD peptide, apolipoprotein E, and transferrin; when the targeting polypeptide is Angiopep-2, distearoylphosphatidylethanolamine-polyethylene glycol-maleimide is mixed with Angiopep-2 in a molar ratio of 1: 2-5.
In embodiments, the permeability of the blood-brain barrier depends on the tight junctions between brain capillary endothelial cells; there are many receptors on brain capillary endothelial cells constituting the blood brain barrier, including transferrin receptor, insulin receptor, low density Lipoprotein (LRP) receptor, etc., in which LRP1 is highly expressed on glioma cells, and a novel polypeptide Angiopep-2 consisting of a 19 amino acid molecule is a ligand of LRP1 receptor; the cell penetrating capacity and the accumulating capacity are higher than those of transferrin, lactoferrin, avidin and the like, and the blood brain barrier penetrating capacity is 50 times that of transferrin monoclonal antibody. The nanoparticles modified by Angiopep-2 are subjected to competitive inhibition by various LRP ligands, are easy to be taken up by cells, and therefore, can be used as a vector for treating glioma. The targeting polypeptide may be any one of Angiopep-2, RGD peptide, apoloprotein E and transferrin, Angiopep-2 being preferred in the present application.
Angiopep-2 forms a shell carrier more readily when mixed with distearoylphosphatidylethanolamine-polyethylene glycol-maleimide.
In some embodiments of the second aspect, the cell membrane is one of erythrocyte membrane RBCm, cancer cell membrane, macrophage membrane and erythrocyte-cancer cell hybrid membrane, and the preparation of erythrocyte membrane RBCm comprises centrifuging a blood sample to obtain erythrocytes, resuspending the erythrocytes in 0.22 x PBS-0.27 x PBS buffer for 28-35min in ice bath, washing the centrifuged product with 1 x PBS buffer, and squeezing the product with a porous membrane to obtain erythrocyte membrane RBCm.
In the examples, the cell membrane and targeting polypeptide complex can be prepared by using the cell membrane as a carrier. Preferred in the present application are erythrocyte membranes RBCm; when the erythrocyte membrane RBCm is taken as a sample, the erythrocyte membrane RBCm is prepared by taking a blood sample, then centrifuging to remove plasma and leucocyte layers, then carrying out ice bath treatment by using a hypotonic medium to break erythrocytes, then centrifuging to release hemoglobin, and extruding by using a porous membrane (preferably a polycarbonate porous membrane in the embodiment).
In some embodiments of the foregoing second aspect, the branched polyethyleneimine and the siRNA are dissolved in Hepes buffer, respectively, and incubated with mixing at a nitrogen to phosphorus ratio of 7-13:1 for 25-35 min.
In the embodiment, branched polyethyleneimine and siRNA are respectively dissolved and then mixed according to the nitrogen-phosphorus ratio of 7-13:1, so that a cation compound is favorably formed, and charge reversal is conveniently formed at the later stage. The branched polyethyleneimine was firmly bound to the siRNA by incubation for 25-35 min.
In some embodiments of the second aspect, the preparation of the citraconic anhydride grafted polylysine comprises dissolving polylysine in PBS buffer, adding citraconic anhydride and adjusting the pH to neutral, and stirring overnight at room temperature to obtain the citraconic anhydride grafted polylysine.
Polylysine is dissolved in phosphate buffer solution, namely PBS buffer solution, and then citraconic anhydride is added to form stable solution, the pH value can be slowly adjusted to be neutral through NaOH, so that the influence of the quick addition of NaOH on the stability of the solution is avoided. And by dialysis in water, citraconic anhydride which does not participate in the grafting reaction can be removed, so that the citraconic anhydride which does not participate in the reaction is prevented from influencing the subsequent reaction. After dialysis, citraconic anhydride grafted polylysine is obtained after cold drying.
In some embodiments of the foregoing second aspect, the cationic complex is mixed with citraconic anhydride grafted polylysine and incubated, wherein the ratio of the number of amino groups of the branched polyethyleneimine to the number of carboxyl groups of the citraconic acid is 1: 1-5.
In the embodiment, in the mixed incubation process, the ratio of the number of amino groups of the branched polyethyleneimine to the number of carboxyl groups of citraconic acid is controlled to be 1:1-5, the amino groups generally carry cationic groups, and the carboxyl groups are anionic groups, so that the charge balance is facilitated, and the stability of the solution is ensured.
In some embodiments of the foregoing second aspect, the time of ultrasonic mixing is 100-.
Through ultrasonic mixing for 100-150s, the red cell membrane RBCm is favorable for wrapping citraconic anhydride grafted polylysine as a middle layer, and the polyethyleneimine-siRNA complex is used as an inner core.
In some embodiments of the second aspect, the biomimetic nanocarrier is prepared by ultrasonically mixing the polypeptide-cell membrane complex and the CA-siRNA ternary complex, wherein the ultrasonic mixing frequency is 42 kHz.
In some embodiments of the foregoing second aspect, the power of the ultrasonic mixing is 95-103W.
Excessive power of ultrasound easily causes heating, affects the thermal stability of the carrier, and affects the treatment effect of the carrier.
Compared with the prior art, the invention has the beneficial effects that: an Ang-RBCm-CA/siRNA consists of an Angiopep-2(Ang) polypeptide modified erythrocyte membrane (Ang-RBCm) and a citraconic anhydride grafted polylysine and polyethyleneimine-siRNA complex (CA/siRNA). Ang-RBCm-CA/siRNA can effectively load siRNA and protect siRNA from degradation. The Ang-RBCm-CA/siRNA has the advantages of prolonged blood circulation time and strong blood brain barrier penetration capability, can be efficiently absorbed by the U87MG brain glial cells over-expressed by a low density lipoprotein receptor (LPR), realizes rapid charge conversion in an endosome/lysosome, effectively releases siRNA, and effectively treats in-situ U87MG brain glioma.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of CA-siRNA ternary complex and SA-siRNA ternary complex provided in Experimental example 1 of the present invention;
FIG. 2 is a transmission electron microscope result diagram of the nano-carrier provided in Experimental example 1 of the present invention;
FIG. 3 is a diagram showing the results of gel electrophoresis of the nanocarriers provided in Experimental example 1 of the present invention;
FIG. 4 is a graph showing the results of particle size and potential tests on nanocarriers provided in Experimental example 1 of the present invention;
FIG. 5 is a diagram of an in vitro blood brain barrier model provided in Experimental example 1 of the present invention;
FIG. 6 is a graph showing the results of the time-dependent change in the blood-brain barrier penetrating efficiency of nanocarriers provided in Experimental example 1 of the present invention;
FIG. 7 is a diagram showing the result of the endocytosis capacity of the nanocarrier according to Experimental example 1 of the present invention;
FIG. 8 is a diagram showing the results of flow cytometry detection of nanocarrier uptake by cells according to Experimental example 1 of the present invention;
FIG. 9 is a graph showing the release results of the nanocarriers provided in Experimental example 1 of the present invention;
FIG. 10 is a graph showing the result of cytotoxicity test of nanocarriers provided in Experimental example 2 of the present invention;
FIG. 11 is a diagram showing the results of gene silencing experiments using nanocarriers of Experimental example 2 of the present invention;
FIG. 12 is a diagram of a sequence-specific assay for gene silencing aspects of nanocarriers provided in Experimental example 2 of the present invention;
FIG. 13 is a Western blot of nanocarriers provided in Experimental example 2 of the invention;
FIG. 14 is a graph showing the results of pharmacokinetic experiments on nanocarriers provided in Experimental example 2 of the present invention;
FIG. 15 is a comparative example of the gene silencing mice by the nanocarrier provided in Experimental example 2 of the present invention;
FIG. 16 is a graph showing the relative expression level of silent genes in nanocarriers according to Experimental example 2 of the present invention;
FIG. 17 is a schematic diagram of the targeting effect of the nanocarrier provided in Experimental example 2 of the present invention;
FIG. 18 is a schematic diagram of qualitative distribution of different tissues of a nano-carrier in an organism according to Experimental example 2 of the present invention;
FIG. 19 is a diagram illustrating the quantitative distribution of different tissues of nanocarriers according to Experimental example 2 of the present invention;
FIG. 20 is a diagram illustrating the operation of the nano-carrier treatment according to Experimental example 3 of the present invention;
FIG. 21 is a qualitative diagram of the present invention, Experimental example 3, showing that the nanocarrier is used for treating tumor;
FIG. 22 is a graph showing the results of the inhibition of cell proliferation by nanocarriers according to Experimental example 3 of the present invention;
FIG. 23 is a schematic diagram showing the change of body weight of a mouse during a treatment process when the mouse is treated with the nanocarrier according to Experimental example 3 of the present invention;
FIG. 24 is a graph showing the survival rate of mice treated with nanocarriers of Experimental example 3;
FIG. 25 is a graph showing the tumor-suppressing effect of nanocarrier-treated mice provided in Experimental example 3 of the present invention;
FIG. 26 is a TuNNEL staining pattern of brain tissue of mice treated with nanocarriers according to Experimental example 3 of the present invention.
Note that the scale of fig. 5 to 9 is 20 μm.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a preparation method of a bionic nano-carrier for treating brain glioma, which comprises the following steps:
1. preparation of Citraconic Anhydride (CA) grafted Polylysine (PLL):
1.1 dissolving polylysine PLL in PBS buffer solution, slowly adding citraconic anhydride;
1.2, adjusting the pH value to be neutral by NaOH;
1.3 the mixture is stirred at room temperature overnight;
1.4 dialyzing in water to remove unreacted citraconic anhydride, and carrying out cold drying to obtain the citraconic anhydride grafted polylysine PLL-CA.
2. Preparation of erythrocyte membrane RBCm:
2.1 taking blood sample, centrifuging at 4 ℃ for 300s, and removing plasma and leucocyte layer to obtain red blood cells;
2.2 the red blood cells obtained were washed 3 times with cold 1 XPBS buffer and then treated with hypotonic 0.25 XPBS buffer for 28min in an ice bath;
2.3 centrifuging the solution obtained in the step 2.2 at 12000rpm for 300s, discarding the hemoglobin, washing the precipitate with 1 × PBS buffer solution for 3 times to obtain a crude erythrocyte membrane product;
2.4 the crude erythrocyte membrane is treated by ultrasonic treatment for 300s and is pressed 11 times by polycarbonate porous membranes with the particle size of 400nm and 200nm to obtain the erythrocyte membrane RBCm.
The methods for preparing the cancer cell membrane, macrophage membrane and erythrocyte-cancer cell hybrid membrane are referred to the above methods.
3. Preparation of distearoylphosphatidylethanolamine-polyethylene glycol-Ang complex (DSPE-PEG-Ang) (Angiopep-2 is used as a targeting polypeptide, and the targeting polypeptide can also be one of RGD peptide, apolipoprotein E and transferrin):
3.1 dissolving distearoylphosphatidylethanolamine-polyethylene glycol-maleimide and Angiopep-2 in a molar ratio of 1:2 in PBS buffer (pH7.4), and reacting at 37 ℃ overnight;
3.2 dialysis to remove unreacted Angiopep-2, and obtaining distearoylphosphatidylethanolamine-polyethylene glycol-Ang complex (DSPE-PEG-Ang).
4. Preparation of CA-siRNA ternary complexes:
4.1 respectively dissolving branched polyethyleneimine PEI and siRNA into Hepes buffer solution with the pH value of 7.4, mixing the solution according to the nitrogen-phosphorus ratio of 7:1, and incubating the solution at room temperature for 35 min;
4.2 incubating at room temperature to obtain a cation complex, and adding the citraconic anhydride grafted polylysine PLL-CA prepared in the step 1.4, wherein the ratio of the number of amino groups of branched polyethyleneimine PEI to the number of carboxyl groups of citraconic acid is 1: 1;
4.3 incubation for 30min at room temperature to obtain the CA-siRNA ternary complex.
5. Preparing a bionic nano carrier:
5.1 mixing the erythrocyte membrane RBCm prepared in the step 2.4 with the CA-siRNA ternary complex prepared in the step 4.3;
5.2 ultrasonic mixing treatment is carried out for 100s at the frequency of 42kHz and the power of 95W, so that the red cell membrane RBCm wraps the CA-siRNA ternary compound, and the bionic nano-carrier for treating the brain glioma is obtained.
Example 2
The embodiment provides a preparation method of a bionic nano-carrier for treating brain glioma, which comprises the following steps:
1. preparation of Citraconic Anhydride (CA) grafted Polylysine (PLL):
1.1 dissolving polylysine PLL in PBS buffer solution, slowly adding citraconic anhydride;
1.2, adjusting the pH value to be neutral by NaOH;
1.3 the mixture is stirred at room temperature overnight;
1.4 dialyzing in water to remove unreacted citraconic anhydride, and carrying out cold drying to obtain the citraconic anhydride grafted polylysine PLL-CA.
2. Preparation of erythrocyte membrane RBCm:
2.1 taking blood sample, centrifuging at 4 ℃ for 300s, and removing plasma and leucocyte layer to obtain red blood cells;
2.2 the red blood cells obtained were washed 3 times with cold 1 XPBS buffer and then treated with hypotonic 0.25 XPBS buffer for 35min in an ice bath;
2.3 centrifuging the solution obtained in the step 2.2 at 12000rpm for 300s, discarding the hemoglobin, washing the precipitate with 1 × PBS buffer solution for 3 times to obtain a crude erythrocyte membrane product;
2.4 the crude erythrocyte membrane is treated by ultrasonic treatment for 300s and is pressed 11 times by polycarbonate porous membranes with the particle size of 400nm and 200nm to obtain the erythrocyte membrane RBCm.
The methods for preparing the cancer cell membrane, macrophage membrane and erythrocyte-cancer cell hybrid membrane are referred to the above methods.
3. Preparation of distearoylphosphatidylethanolamine-polyethylene glycol-Ang complex (DSPE-PEG-Ang) (Angiopep-2 is used as a targeting polypeptide, and the targeting polypeptide can also be one of RGD peptide, apolipoprotein E and transferrin):
3.1 dissolving distearoylphosphatidylethanolamine-polyethylene glycol-maleimide and Angiopep-2 in a molar ratio of 1:5 in PBS buffer (pH7.4), and reacting at 37 ℃ overnight;
3.2 dialysis to remove unreacted Angiopep-2, and obtaining distearoylphosphatidylethanolamine-polyethylene glycol-Ang complex (DSPE-PEG-Ang).
4. Preparation of CA-siRNA ternary complexes:
4.1 respectively dissolving branched polyethyleneimine PEI and siRNA into Hepes buffer solution with the pH value of 7.4, mixing the solution according to the nitrogen-phosphorus ratio of 13:1, and incubating the solution at room temperature for 25 min;
4.2 incubating at room temperature to obtain a cation complex, and adding the citraconic anhydride grafted polylysine PLL-CA prepared in the step 1.4, wherein the ratio of the number of amino groups of branched polyethyleneimine PEI to the number of carboxyl groups of citraconic acid is 1: 3;
4.3 incubation for 30min at room temperature to obtain the CA-siRNA ternary complex.
5. Preparing a bionic nano carrier:
5.1 mixing the erythrocyte membrane RBCm prepared in the step 2.4 with the CA-siRNA ternary complex prepared in the step 4.3;
5.2 ultrasonic mixing treatment is carried out for 150s at the frequency of 42kHz and the power of 103W, so that the red cell membrane RBCm wraps the CA-siRNA ternary complex, and the bionic nano-carrier for treating the brain glioma is obtained.
Example 3
The embodiment provides a preparation method of a bionic nano-carrier for treating brain glioma, which comprises the following steps:
1. preparation of Citraconic Anhydride (CA) grafted Polylysine (PLL):
1.1 dissolving polylysine PLL in PBS buffer solution, slowly adding citraconic anhydride;
1.2, adjusting the pH value to be neutral by NaOH;
1.3 the mixture is stirred at room temperature overnight;
1.4 dialyzing in water to remove unreacted citraconic anhydride, and carrying out cold drying to obtain the citraconic anhydride grafted polylysine PLL-CA.
2. Preparation of erythrocyte membrane RBCm:
2.1 taking blood sample, centrifuging at 4 ℃ for 300s, and removing plasma and leucocyte layer to obtain red blood cells;
2.2 the red blood cells obtained were washed 3 times with cold 1 XPBS buffer and then treated with hypotonic 0.25 XPBS buffer for 35min in an ice bath;
2.3 centrifuging the solution obtained in the step 2.2 at 12000rpm for 300s, discarding the hemoglobin, washing the precipitate with 1 × PBS buffer solution for 3 times to obtain a crude erythrocyte membrane product;
2.4 the crude erythrocyte membrane is treated by ultrasonic treatment for 300s and is pressed 11 times by polycarbonate porous membranes with the particle size of 400nm and 200nm to obtain the erythrocyte membrane RBCm.
The methods for preparing the cancer cell membrane, macrophage membrane and erythrocyte-cancer cell hybrid membrane are referred to the above methods.
3. Preparation of distearoylphosphatidylethanolamine-polyethylene glycol-Ang complex (DSPE-PEG-Ang) (Angiopep-2 is used as a targeting polypeptide, and the targeting polypeptide can also be one of RGD peptide, apolipoprotein E and transferrin):
3.1 dissolving distearoylphosphatidylethanolamine-polyethylene glycol-maleimide and Angiopep-2 in a molar ratio of 1:3 in PBS buffer (pH7.4), and reacting at 37 ℃ overnight;
3.2 dialysis to remove unreacted Angiopep-2, and obtaining distearoylphosphatidylethanolamine-polyethylene glycol-Ang complex (DSPE-PEG-Ang).
4. Preparation of CA-siRNA ternary complexes:
4.1 respectively dissolving branched polyethyleneimine PEI and siRNA into Hepes buffer solution with the pH value of 7.4, mixing the solution according to the nitrogen-phosphorus ratio of 10:1, and incubating the solution at room temperature for 30 min;
4.2 incubating at room temperature to obtain a cation complex, and adding the citraconic anhydride grafted polylysine PLL-CA prepared in the step 1.4, wherein the ratio of the number of amino groups of the branched polyethyleneimine PEI to the number of carboxyl groups of the citraconic acid is 1: 5;
4.3 incubation for 30min at room temperature to obtain the CA-siRNA ternary complex.
5. Preparing a bionic nano carrier:
5.1 mixing the erythrocyte membrane RBCm prepared in the step 2.4 with the CA-siRNA ternary complex prepared in the step 4.3;
5.2 ultrasonic mixing treatment is carried out for 120s under the frequency of 42kHz and the power of 100W, so that the red cell membrane RBCm wraps the CA-siRNA ternary complex, and the bionic nano-carrier for treating the brain glioma is obtained.
Experimental example 1
In this experimental example, the biomimetic nano-carrier for the treatment of brain glioma was prepared by the preparation method provided in example 3. While succinic anhydride was used as a control for comparative experiments.
1. Preparation of Citraconic Anhydride (CA) grafted Polylysine (PLL):
1.1 dissolving polylysine PLL50mg in 2mL of PBS buffer (pH7.4), and slowly adding citraconic anhydride (87.4mg, 3.87 mmol/L);
1.2, adjusting the pH value to be neutral by NaOH (5 mol/L);
1.3 the mixture is stirred at room temperature overnight;
1.4 dialyzing in water to remove unreacted citraconic anhydride, and carrying out cold drying to obtain the citraconic anhydride grafted polylysine PLL-CA36 mg.
Synthesis of succinic anhydride grafted polylysine:
polylysine (50mg, 0.387mmol/L) was dissolved in distilled water (2mL), succinic anhydride (387mg, 3.87mmol/L) was added dropwise, and stirred at room temperature overnight. Unreacted succinic anhydride (MWCO 300) was removed by dialysis, and lyophilized to give a white powder (39 mg).
2. Preparation of erythrocyte membrane RBCm:
2.1 taking whole blood from the orbital well of female mice, centrifuging the blood sample at 4 deg.C (800rcf) for 300s, removing plasma and leukocyte layer to obtain red blood cells;
2.2 the red blood cells with cold 1 x PBS buffer washing 3 times, then use hypotonic 0.25 x PBS buffer heavy suspension ice bath treatment for 30 min;
2.3 centrifuging the solution obtained in the step 2.2 at 12000rpm for 300s, discarding the hemoglobin, washing the precipitate with 1 × PBS buffer solution for 3 times to obtain a crude erythrocyte membrane product;
2.4 the crude erythrocyte membrane is treated by ultrasonic treatment for 300s and is pressed 11 times by polycarbonate porous membranes with the particle size of 400nm and 200nm to obtain the erythrocyte membrane RBCm.
3. Preparation of distearoylphosphatidylethanolamine-polyethylene glycol-Ang complex (DSPE-PEG-Ang):
3.1 dissolving distearoylphosphatidylethanolamine-polyethylene glycol-maleimide and Angiopep-2 in a molar ratio of 1:3 in PBS buffer (pH7.4), and reacting at 37 ℃ overnight;
3.2 dialysis to remove unreacted Angiopep-2, and obtaining distearoylphosphatidylethanolamine-polyethylene glycol-Ang complex (DSPE-PEG-Ang).
The grafting rate of Ang was 96% as determined by BCA protein kit.
4. Preparation of CA-siRNA ternary complexes:
4.1 respectively dissolving branched polyethyleneimine PEI (25kDa) and siRNA into 10mmol/L Hepes buffer solution with the pH value of 7.4, mixing the solution according to the nitrogen-phosphorus ratio of 10:1, and incubating the solution at room temperature for 30 min;
4.2 incubating at room temperature to obtain a cation complex, and adding the citraconic anhydride grafted polylysine PLL-CA prepared in the step 1.4, wherein the ratio of the number of amino groups of branched polyethyleneimine PEI to the number of carboxyl groups of citraconic acid is 1: 3;
4.3 incubation for 30min at room temperature to obtain the CA-siRNA ternary complex.
The process of preparing the SA-siRNA ternary complex by using succinic anhydride is the same as the preparation method of the CA-siRNA ternary complex.
The particle size and zeta potential of the SA-siRNA ternary complex and CA-siRNA ternary complex were determined using a dynamic light scattering instrument (DLS; Nano-Zen 3600, Malvern Instruments, UK). The binding capacity of siRNA was examined by 2% (w/v) agarose gel electrophoresis.
5. Preparing a bionic nano carrier:
5.1 mixing the erythrocyte membrane RBCm prepared in the step 2.4 with the CA-siRNA ternary complex prepared in the step 4.3;
5.2 ultrasonic mixing treatment is carried out for 120s under the frequency of 42kHz and the power of 100W, so that the red cell membrane RBCm wraps the CA-siRNA ternary complex, and the bionic nano-carrier (Ang-RBCm-CA/siRNA) for treating the brain glioma is obtained.
First, Charge inversion experiment of Ang-RBCm-CA/siRNA:
the particle size of the prepared PEI-siRNA complex is measured to be 92nm, the PEI-siRNA complex has a positive charge of 25.6mv, after the PEI-siRNA complex is coated with citraconic anhydride grafted polylysine PLL-CA, the charge is changed to be-32.3 mv, and the PEI-siRNA complex and the citraconic anhydride grafted polylysine PLL-CA are successfully combined to obtain the CA-siRNA ternary complex.
The CA-siRNA ternary complex is combined with an Angiopep-2 modified erythrocyte membrane RBCm to obtain a bionic nano-carrier (Ang-RBCm-CA/siRNA), and the particle size is 168 nm. The result is shown in figure 1, and the bionic nano-carrier (Ang-RBCm-CA/siRNA) has an obvious core-shell structure.
From the observation of the transmission electron microscope in FIG. 2, it is found that the Ang-RBCm-CA/siRNA structure is destroyed under the acidic environment simulating the endosome/lysosome of the tumor cells, and the Ang-RBCm-SA/siRNA has complete results under the same conditions. Indicating that the siRNA in the Ang-RBCm-CA/siRNA composite structure can be released.
0.1mol/L acetate buffer solution is respectively added into Ang-RBCm-CA/siRNA, Ang-RBCm-SA/siRNA and free siRNA to adjust the pH value to 5.0, then heparin (0.05mg/mL) is added for incubation treatment for 3h, electrophoresis is carried out in TAE electrophoresis buffer solution for 120v, 15min, and gel imaging analysis is carried out.
As shown in FIG. 3, Ang-RBC-SA/siRNA tightly bound siRNA both under neutral conditions and under acidic environment. The size of Ang-RBC-CA/siRNA increased significantly with pH change from 7.4 to 5.0, whereas Ang-RBC-SA/siRNA changed little under the same conditions.
As shown in FIG. 4, Zeta potential results further demonstrate pH-triggered charge transfer, where Ang-RBCm-CA/siRNA rapidly converted surface charge from-32 mv to +18mv after incubation at pH 5.0, while maintaining negative charge at pH 6.5 or 5.0.
The result shows that the bionic nano-carrier Ang-RBC-CA/siRNA can be depolymerized and release siRNA through charge conversion under the acidic condition.
II, in vitro blood brain barrier penetration, cell uptake and siRNA release experiment:
bEnd.3 cells (5X 10)4Hole) in the upper chamber of the chamber (Corning, NY, USA), put in a 24-well plate (800 μ L of medium per hole) for culture, and implant mouse endothelial cells bend.3 in the Transwells chamber to establish a blood brain barrier model, as shown in fig. 5. When the transendothelial resistance (TEER) value of the bEnd.3 cells is higher than 200 omega cm2When it is, the endothelial cells are proved to be intact. The medium was changed to DMEM without FBS, then Cy 5-labeled nanoparticles (200nM) were added to the chamber and incubated in a shaker (37 ℃, 50 rpm). When cultured for 4, 12, 24h, 500. mu.L of medium was taken from the basolateral compartment and then the same volume of fresh medium was added. At the end of the experiment, TEER was measured again to monitor the integrity of the bned.3 cells. The penetration (%) of the nanocomposite was determined by measuring the fluorescence of the sample by a fluorescence spectrophotometer (Thermo Scientific, USA). Subsequently, the upper chamber of the bEnd.3 cells using trypsin digestion, 1000g centrifugal 3 minutes, using PBS washing twice, and then suspended in 500L PBS. Detection was performed using a BD FACS Calibur flow cytometer (Becton Dickinson, USA).
In flow cytometry assays, U87-luc cells were plated in 6-well plates (1X 10)6Cells/well), after 24 hours of incubation at 37 ℃, 100 μ L of HEPES solution of Ang-RBCm-CA/siRNA, Ang-RBCm-SA/siRNA and free siRNA (200nM Cy5-siRNA) was added, after 4 hours of incubation, samples were aspirated and cells were digested with 500 μ L of pancreatin. The resulting cell suspension was centrifuged at 1000 Xg for 3 minutes and washed with PBSTwo times, again dispersed in 500. mu.LPBS, tested in a flow cytometer (BD FACS Calibur, Becton Dickinson, USA) over 1 hour, obtained by circling 10000 cells with Cell Quest software.
As shown in FIG. 6, Cy 5-labeled siRNA (Cy5-siRNA) nanocomplexes are time-dependent in the process of crossing the blood-brain barrier.
The penetration effect of Ang-RBCm-CA/siRNA is equivalent to that of Ang-RBCm-SA/siRNA, and the penetration effect is obviously higher than that of non-target group RBCm-CA/siRNA, which indicates that the endocytosis effect of LRP-1 mediated cells is obvious. The reason for the enhanced blood brain barrier permeability of Ang-functionalized nanocomposites is due to their higher cellular uptake in blood brain barrier (bned.3) cells, leading to increased endocytosis.
The results of cell uptake by flow cytometry are shown in FIG. 8, where Ang-RBCm-CA/siRNA uptake in bEnd.3 cells was similar to Ang-RBCm-SA/siRNA, but was 20-fold higher than RBCm-CA/siRNA.
The endocytosis capacity of the cells was assessed by implanting U87MG cells into the lower layer of bned.3, and the results are shown in fig. 7, and the uptake of Ang-RBCm-CA/siRNA in U87MG cells was observed. The result of quantitative analysis shows that the endocytosed U87MG cell has stronger uptake of Ang-RBCm-CA/siRNA, which is 3.2 and 15.2 times higher than that of Ang-RBCm-SA/siRNA and RBCm-CA/siRNA respectively.
After 4 hours of culture in Ang-RBCm-CA/siRNA, large amounts of Cy5-siRNA entered U87MG cells and were released into the cytoplasm as shown in FIG. 9. The endocytosis and release effects of Cy5-siRNA nanocomplexes in cells were verified using Confocal Laser Scanning Microscopy (CLSM). After 4 hours of culture in Ang-RBCm-CA/siRNA, large amounts of Cy5-siRNA entered U87MG cells and were released into the cytoplasm. Cells treated by Ang-RBCm-SA/siRNA have similar blood brain barrier endocytosis capacity to Ang-RBCm-CA/siRNA, but Cy5 fluorescence is weak, and the important role of inducing RBCm cleavage by CA charge conversion in intracellular siRNA release is proved. The results prove that Ang-RBCm-CA/siRNA has excellent Blood Brain Barrier (BBB) penetrability, can effectively release siRNA and has good endosome/lysosome escape capability.
Experimental example 2
This experimental example demonstrates the cytotoxicity, gene transfection, pharmacokinetics, gene silencing, and in vitro imaging and biodistribution of biomimetic nanocarriers for brain glioma treatment.
One, cytotoxicity
After culturing U87-luc cells in 96-well culture plates (5000 cells/well) for 24h, the medium was aspirated, 90. mu.L of fresh medium and 10. mu.L of RBCm-CA/siRNA, Ang-RBCm-SA/siRNA, RBCm-CA/siRNA and free siRNA (400nM siRNA) were added, after 48h of incubation, 10. mu.L of 3- (4, 5-dimethyl-thiazolyl) -2, 5-diphenyltetrazolium bromide (MTT) solution (5mg/mL) was added, after 4h of incubation, the medium was removed and 150. mu.L of LDMSO was added to dissolve MTT-formazan produced by living cells. The microplate reader measures the absorbance at 570nm for each well, with four replicates of each experimental data (n-4). The cell survival (%) was calculated as follows:
cell viability (%) ═ OD570(sample)/OD570(control). times.100%
Wherein OD570(sample) is the absorbance, OD, of the sample set570(control) is the absorbance of the control group.
The results are shown in fig. 10, and cell viability assay (MTT) analysis indicates that these siRNA nanocomposites are non-toxic to U87MG cells at siRNA concentration of 400nM, indicating that the biomimetic nanocomposites have good biocompatibility.
Second, Gene transfection
U87-Luc stably expressing luciferase was suspended in 10% FBS-containing DMEM medium and plated in 96-well plates (8X 10)3Cells/well) for 24 h. Next, 90. mu.L of fresh medium was changed and 10. mu.L of Ang-RBCm-CA/siGl3, Ang-RBCm-CA/siScr, RBCm-CA/siGl3, Ang-RBCm-SA/siGl3 and free siGL3(400nM siRNA) were added. After 48h incubation, cells were lysed by cell lysis buffer (pecan day, china). And (3) analyzing the luminescence condition of the lysed cells at 560nm by using a multifunctional microplate reader, and further evaluating the transfection effect.
Wherein, the sense strand of the luciferase siGL3 is shown as SEQ ID NO.1, and the base sequence of the SEQ ID NO.1 is as follows: 5 '-CUU ACG CUG AGU ACU UCG AdTdT-3', the antisense chain is shown as SEQ ID NO.2, and the base sequence of SEQ ID NO.2 is: 5 '-UCG AAG UAC UCA GCG UAA GdTdT-3'); siScrramble (sense strand SEQ ID NO. 3: 5 '-UUC UCC GAA CGU GUC ACG UdTdT-3', antisense strand SEQ ID NO. 4: 5 '-ACG UGA CAC GUU CGG AGA AdTdT-3'). siRNAs targeting human PLK1(siPLK1) (sense strand SEQ ID NO. 5: 5 '-UGA AGA AGA UCA CCC UCC UUA dTdT-3', antisense strand SEQ ID NO. 6: 5 '-UAA GGA GGG UGA UCU UCU UCA dTdT-3'). FAM and Cy5 introduced the 5' end of the antisense strand.
As a result, as shown in FIG. 11, the expression of luciferase was down-regulated by about 79% in Ang-RBCm-CA/siGL3, whereas the control group RBCm-CA/siGL3 and Ang-RBCm-SA/siGL3 were induced to be down-regulated by 34%, 21% of luciferase expression. Indicating that the nanocomplexes containing non-specific sirna (siscr) hardly resulted in a reduction of bioluminescence.
Further, a real-time quantitative polymerase chain reaction (RT-PCR) experiment was performed on U87MG cells to study the effect of specific gene silencing by the nanoplex consisting of sipLK 1. A brief procedure for the qRT-PCR experiment was performed by plating U87-luc cells in DEME medium containing 10% FBS in 6-well plates ((1X 10)6Individual cells/well). After 24h of culture, the medium was changed to 100. mu.L of fresh medium containing Ang-RBC-CA/siplink 1, Ang-RBC-CA/siScrramble, RBC-CA/siplink 1, Ang-RBC-SA/siplink 1 and free siplink 1(400nM siRNA). And (3) cracking the cells by using a cell lysate, and then performing a fluorescent quantitative PCR experiment by using an SYBR Real-Time PCR kit.
Results as shown in fig. 12, Ang-RBCm-CA/siPLK1 downregulated the expression of PLK1mRNA by 61% at siPLK1 concentration of 400nM and upon transfection over 48h, significantly higher than RBCm-CA/siPLK1 (downregulation of 42%) and Ang-RBCm-SA/siPLK1 (downregulation of 21%). Ang-RBCm-CA/siScr and free sipLK1 did not result in down-regulation of PLK1mRNA, which also confirmed the sequence specificity of sipLK1 in gene silencing.
Further verifying the in vitro gene silencing effect of Ang-RBCm-CA/sipLK1 by Western blot; u87-luc cells were plated in 6-well plates at 2X 10 per well5Individual cell, 37 ℃ C, 5% CO2Cultured under the conditions of (1) for 24 hours. After incubation of cells with either Ang-RBCm-CA/sipLK1, Ang-RBCm-CA/siScr, RBCm-CA/sipLK1, Ang-RBCm-SA/sipLK1, free sipLK1 or PBS for 72 hours (400nM siRNA), the cells were washed with PBSCells were lysed 3 times with RIPA buffer (Thermo Scientific, Rockford, IL). After cell lysis, proteins were extracted and the total amount of protein was determined using BCA kit (Pierce/Thermo Scientific). Samples of the same protein amount were then added to SDS-PAGE gels, run for electrophoretic separation, and then transferred to PVDF membranes. Blocking with 5% BSA solution, adding PLK1 monoclonal antibody or internal reference beta-actin monoclonal antibody, and incubating overnight at 4 deg.C. Subsequently, horseradish catalase-labeled secondary antibody was added and incubated at room temperature for 1 hour. Finally, protein signals were detected using the Super Signal EC detection system and protein bands were analyzed using Image J software.
The results are shown in FIG. 13, and the Ang-RBCm-CA/sipLK1 group has the most obvious effect of down-regulating the PLK1 protein, and is consistent with the quantitative result of PLK1 mRNA. The Ang-RBCm-CA/sipLK1 has excellent target gene knockout effect because it can efficiently enter cells through receptor-mediated endocytosis and release siRNA through charge-transfer-induced erythrocyte membrane disruption.
Third, pharmacokinetics
Ang-RBC-CA/Cy5-siRNA and free Cy5-siRNA (2mg Cy5-siRNA equiv/kg) were injected intravenously into nude mice via tail vein (n ═ 3). At the stated time points, approximately 50. mu.L of blood was removed from the eye sockets of the BLAB/c mice and immediately after blood sample removal dissolved in 0.1mL of lysis buffer (1% Triton X-100). Cy5-siRNA was extracted by centrifugation (14.8k rpm,30min) and the amount of Cy5 in the supernatant was determined by fluorescence. The pattern of the blood circulation profile is generally two phases: a fast dispersed phase and a long-term eliminated phase. Its half life (t)1/2，αAnd t1/2,β) Obtained by quadratic exponential decay fitting with Origin 8 software. The specific formula is as follows: y is A1×exp(-x/t1)+A2×exp(-x/t2)+y0Then take t1/2,α＝0.693×t1And t1/2,β＝0.693×t2。
As shown in FIG. 14, Ang-RBCm-CA/siRNA increased blood circulation time, which eliminated half-life (t)1/2，β) About 2.2 hours, indicating that the siRNA nanocomposite can maintain high concentration and long-term blood circulation; while free siRNA is rapidly eliminated in vivo (t)1/2,β＝0.2h)。
Fourth, in situ glioma fluorescence siRNA gene silencing experiment
Ang-RBC-CA/siRNA, RBC-CA/siRNA and Ang-RBC-SA/siRNA (dose: 2mg siRNA equiv./kg) loaded with siGL3 or siScramble were intravenously injected into U87-luc tumor-bearing nude mice in situ (n ═ 3). Mice were anesthetized about 10 minutes after intraperitoneal injection of 100 μ L fluorescein at a dose of 150 mg/kg. Collected by Lumina IVIS III system in vivo bioluminescence mode. Photons in the brain region were quantified using real-time image software, and all pictures were set under the same conditions and colorimetric scale.
As a result, as shown in FIGS. 15 and 16, Ang-RBCm-CA/siGL3 significantly reduced the bioluminescence of brain gliomas compared to Ang-RBCm-CA/siGL3, and Ang-RBCm-SA/siGL 3. Quantitative analysis shows that Ang-RBCm-CA/siGL3 causes 76% and 43% reduction of brain glioma bioluminescence at 24 and 48 hours after injection respectively, which indicates that the bionic siRNA nano-composite can effectively silence luciferase genes. The non-targeting nanocomplex RBCm-CA/siGL3 in the control group reduced bioluminescence by 25%, although lower than Ang-RBCm-CA/siGL3, but with better inhibitory properties than Ang-RBCm-CA/siScr and Ang-RBCm-SA/siGL 3.
Fifth, in vitro imaging and biodistribution experiments
The in vitro imaging procedure was as follows: Ang-RBC-CA/Cy5-siRNA, Ang-RBC-SA/Cy5-siRNA and RBC-CA/Cy5-siRNA (2mg Cy5-siRNA equiv./kg) were injected into U87-luc tumor-bearing nude mice in situ via tail vein, and the light emission intensity in brain at different time points was measured using IVIS III apparatus.
The biodistribution steps are as follows: Ang-RBC-CA/Cy5-siRNA, Ang-RBC-SA/Cy5-siRNA, RBC-CA/Cy5-siRNA and naked Cy5-siRNA (2mg Cy5-siRNA equiv./kg) were injected via tail vein into U87-luc tumor-bearing nude mice in situ. Mice were sacrificed 4 hours after injection, and major organs including heart, liver, spleen, lung, kidney, brain and tumor were collected, washed, dried and weighed. The fluorescence picture is obtained by the Lumina IVIS III near infrared fluorescence imaging system. To quantify the Cy5-siRNA distribution of tumors and different organs, 600. mu.L of 1% Triton homogenate was added to the brain and other tissues for 10min, and after centrifugation at 14000rpm for 30min, Cy5 in the suspension was detected by a fluorescence spectrophotometer and calculated from a standard curve, and finally expressed as percent injected per gram of tissue (% ID/g).
As shown in FIGS. 17 and 18, Ang-RBCm-CA/siRNA showed higher tumor accumulation and retention effects than control group RBCm-CA/siRNA and Ang-RBCm-SA/siRNA. The fluorescence of Ang-RBCm-CA/siRNA in brain glioma reaches the maximum value 4 hours after injection, and the intensity lasts for 24 hours, thus the siRNA nano-composite has higher blood brain barrier penetration, tumor targeting and siRNA controlled release capability. The fluorescence of Ang-RBCm-CA/siRNA in mouse tumor is obviously stronger than that of other main tissues (such as heart, liver, spleen, lung and kidney).
As shown in FIG. 19, the fluorescence of Cy5-siRNA was weak in the control group tumors such as RBCm-CA/siRNA, Ang-RBCm-SA/siRNA and free siRNA, while that of Cy5-siRNA was strong in the kidney and liver. The fluorescent quantitation result shows that the accumulation amount of Ang-RBCm-CA/siRNA in each gram of tumor site reaches 6.78% of the injection dose, which is 2.1, 3.2 and 6.3 times higher than that of RBCm-CA/siRNA, Ang-RBCm-SA/siRNA and free siRNA respectively. The mean of the siRNA levels in the brains of the four groups of control mice was significantly reduced, especially free siRNA, indicating that the Ang-functionalized nanocomposite enhanced blood brain barrier penetration of brain glioma, accumulation and retention at tumor sites.
Experimental example 3
The experimental example is used for verifying the treatment effect of the bionic nano-carrier for treating the brain glioma and carrying out blood biochemical analysis.
Mice were weighed and randomly divided into six groups (n-8) Ang-RBC-CA/siPLK1(I), Ang-RBC-CA/sipramble (ii), RBC-CA/siPLK1(III), Ang-RBC-SA/siPLK1(IV), free siPLK1(V) and pbs (vi). Mice were injected via tail vein once every 2 days at a dose of 2mg siRNA equiv./kg. The relative body weights of the mice were normalized to their initial body weights. In addition, at day 10 treatment was terminated, one mouse per group was sacrificed, major organs removed, washed, dried and imaged by the lumine IVIS III system. Thereafter, soaked in 4% formalin and embedded in paraffin, stained by H & E and photographed by an upright microscope (Olympus BX 41). TUNEL detection was performed as indicated by the TUNEL apoptosis detection kit (Roche) and nuclei were stained with DAPI, the procedure of which is shown in figure 20.
As shown in fig. 21, the nanoplex containing siPLK1 exhibited an effect of inhibiting tumor proliferation to some extent, while free siPLK1 had no effect of inhibiting tumor, compared to the PBS control group. The bioluminescence intensity of Ang-RBCm-CA/siPLK1 was significantly lower than that of the non-targeted group RBCm-CA/siPLK1, mainly due to the active targeting ability of Ang-functionalized nanocomplexes. The significant difference between Ang-RBCm-CA/sipLK1 and Ang-RBCm-SA/sipLK1 in tumor inhibition indicates that charge-transfer-induced RBCm disruption and siRNA intracellular release have important significance in enhancing the anti-tumor effect. Notably, the rapid proliferation of mouse tumors treated with Ang-RBCm-SA/sigcr, compared to Ang-RBCm-SA/siPLK1, demonstrates the sequence specificity of siPLK1 in gene knock-out.
The results are shown in fig. 22, fig. 23, fig. 24 and fig. 25, the quantitative bioluminescence analysis results are consistent with the bioluminescence imaging, and the effective inhibition effect of Ang-RBCm-CA/siPLK1 on the proliferation of U87MG-luc in the brain is further verified. The rate of inhibition of tumor growth by Ang-RBCm-CA/siPLK1 was 82.4%, significantly higher than that of the other controls. The body weight of mice treated by Ang-RBCm-CA/sipLK1, RBCm-CA/sipLK1 and Ang-RBCm-SA/sipLK1 has no significant change, while the weight of control groups injected with Ang-RBCm-CA/siScr, free sipLK1 and PBS has rapid weight loss, which can be related to rapid proliferation and invasion of brain glioma to cause brain dysfunction. The Kaplan-Meier survival curve shows that Ang-RBCm-CA/sipLK1 significantly improves the survival period, wherein the survival time is 43 days, which is significantly longer than RBCm-CA/sipLK1(32d), Ang-RBCm-SA/sipLK1(28d), Ang-RBCm-SA/sipMr (22d), free sipLK1(16d), PBS (12 d). In vivo Western blot results show that Ang-RBCm-CA/sipLK1 can obviously inhibit the expression of PLK1 protein in glioma.
The TUNNEL staining results are shown in FIG. 26, and it can be seen that large area necrosis and apoptosis of brain glioma histiocytes were observed after Ang-RBCm-CA/sipLK1 action. In addition, the TUNEL experiment result shows that the tumor cell apoptosis (green) level of mice treated by Ang-RBCm-CA/sipLK1 is the highest, which indicates that the main reason for inhibiting the tumor is attributed to the tumor cell apoptosis induced by siRNA nano-complex.
In conclusion, the results prove that Ang-RBCm-CA/sipLK1 can effectively cross blood brain barrier and accumulate at the tumor site, and efficiently release sipLK1 in cells after being taken by U87MG-luc cells, thereby achieving the optimal effect of resisting brain glioma in vivo. It is worth noting that the bionic nano-composite can also carry and release biological agents such as DNA, protein, enzyme and the like, so that the bionic nano-composite can be applied to specific treatment of various diseases.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
SEQUENCE LISTING
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<120> a bionic nano-carrier for brain glioma treatment and a preparation method thereof
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Claims (10)
1. The bionic nano-carrier for treating the brain glioma is characterized in that a polyethyleneimine-siRNA compound is used as an inner core, citraconic anhydride grafted polylysine is used as a middle layer with negative charges, a cell membrane modified by a targeting polypeptide is used as an outer shell to coat the inner core and the middle layer, and the targeting polypeptide is Angiopep-2.
2. A method for preparing the biomimetic nanocarrier for brain glioma treatment according to claim 1, comprising the following steps:
mixing and dissolving distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide and targeted polypeptide in a PBS buffer solution for reaction to prepare a distearoyl phosphatidyl ethanolamine-polyethylene glycol-targeted polypeptide compound;
mixing and incubating a cell membrane sample prepared from a cell sample with the distearoyl phosphatidyl ethanolamine-polyethylene glycol-targeted polypeptide compound to obtain a polypeptide-cell membrane compound;
dissolving, mixing and incubating branched polyethyleneimine and siRNA to obtain a cation compound, mixing and incubating the cation compound and citraconic anhydride grafted polylysine to prepare a CA-siRNA ternary compound;
and mixing the polypeptide-cell membrane compound and the CA-siRNA ternary compound to prepare the bionic nano carrier.
3. The method for preparing a biomimetic nanocarrier for brain glioma treatment according to claim 2, wherein the distearoylphosphatidylethanolamine-polyethylene glycol-maleimide and the Angiopep-2 are mixed in a molar ratio of 1: 2-5.
4. The method of claim 2, wherein the cell membrane is one of erythrocyte membrane RBCm, cancer cell membrane, macrophage membrane and erythrocyte-cancer cell hybrid membrane, the preparation of erythrocyte membrane RBCm comprises taking blood sample, centrifuging to obtain erythrocyte, resuspending the erythrocyte in 0.22 x PBS-0.27 x PBS buffer solution in ice bath for 28-35min, washing the centrifuged product with 1 x PBS buffer solution, and extruding with porous membrane to obtain the erythrocyte membrane RBCm.
5. The method for preparing a biomimetic nano carrier for brain glioma treatment according to claim 2, wherein the branched polyethyleneimine and the siRNA are respectively dissolved in Hepes buffer solution, and the nitrogen-phosphorus ratio is 7-13:1 for 25-35 min.
6. The method for preparing the biomimetic nanocarrier for brain glioma treatment according to claim 2, wherein the preparation of the citraconic anhydride grafted polylysine comprises dissolving polylysine in a PBS buffer solution, adding citraconic anhydride, adjusting pH to be neutral, and stirring at room temperature overnight to obtain the citraconic anhydride grafted polylysine.
7. The method for preparing a biomimetic nanocarrier for brain glioma treatment according to claim 2, characterized in that the cation complex is mixed with the citraconic anhydride-grafted polylysine and incubated, wherein the ratio of the number of amino groups of the branched polyethyleneimine to the number of carboxyl groups of the citraconic acid is 1: 1-5.
8. The method for preparing a biomimetic nano-carrier for the treatment of brain glioma according to claim 2, wherein the biomimetic nano-carrier is prepared by performing ultrasonic mixing on the polypeptide-cell membrane complex and the CA-siRNA ternary complex, and the time of the ultrasonic mixing is 100-150 s.
9. The method for preparing biomimetic nano-carriers for brain glioma treatment according to claim 8, wherein the frequency of the ultrasonic mixing is 42 kHz.
10. The method for preparing biomimetic nano-carriers for brain glioma treatment according to claim 9, wherein the power of the ultrasonic mixing is 95-103W.
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