PEI and targeting peptide modified magnetic nano-carrier as well as preparation method and application thereof

Abstract

The invention discloses a PEI and targeting peptide modified magnetic nano-carrier and a preparation method and application thereof. The nano-carrier is regular in morphology and uniform in particle size and has good crystallinity and magnetic performance. E5 polypeptide and RGD cyclic peptide on the surface of the nano-carrier are respectively combined with CXCR4 protein and integrin on the surface of a tumor cell, and the nano-carrier and CXCR4 are internalized into lysosome through an integrin-mediated way, so that the target protein CXCR4 is degraded. The PEI in the nano-carrier can load and deliver siRNA into tumor cells to silence the expression of CXCR4 mRNA (messenger Ribonucleic Acid). Through nucleic acid delivery and a protein targeted degradation strategy, the CXCR4 level of tumor cells is jointly reduced, the migration ability of the tumor cells is inhibited, and meanwhile, the anti-tumor effect of M1 type polarization of macrophages is activated.

Description

Technical Field

[0001] The invention belongs to the field of biomedical nanotechnology and is a PEI and targeting peptide modified magnetic nanocarrier and a preparation method and application thereof. Background Art

[0002] CXC chemokine receptor type 4 (CXCR4) is a member of the G protein-coupled receptor (GPCR) superfamily and is specific for CXC chemokine ligand 12 (CXCL12, also known as SDF-1). The interaction between CXCR4 and its ligands affects downstream signaling pathways of cell proliferation, chemotaxis, migration, and gene expression. This interaction also regulates physiological processes including hematopoiesis, organogenesis, and tissue repair. At the same time, CXCR4 is one of the most common chemokine receptors overexpressed in more than 23 human cancers. In terms of tumors, the CXCR4-CXCL12 interaction is considered to be a characteristic of tumor invasiveness and is involved in multiple aspects of tumor progression, including proliferation, angiogenesis, metastasis, and the occurrence of drug resistance. Related studies have shown that overexpression of CXCR4 is closely related to the poor prognosis of patients with malignant lymphoma, and a variety of problems including invasion, metastasis, drug resistance, and immunosuppression are often accompanied by the phenomenon of high CXCR4 expression. Cutting off the biological effects of the CXCR4-CXCL12 biological axis can solve the above problems to a certain extent.

[0003] Antagonists targeting the CXCR4 biological axis play a new auxiliary role in tumor treatment. Although their direct killing effect on tumor cells is not significant, they can enhance the killing effect of therapeutic drugs on tumor cells by constructing targeted drugs and utilizing the targeting effect of antagonists, thereby achieving the drug's synergistic and toxicity-reducing effect. However, a single antagonist reduces the interaction with CXCL12 by binding to the CXCR4 protein, which faces problems such as off-target and easy recovery in in vivo and in vitro studies. Therefore, downregulating the level of cellular CXCR4 protein by degradation is a more effective strategy.

[0004] Traditional protein targeted degradation strategies are usually ubiquitination-based proteasome degradation, which is usually used for cytoplasmic proteins, with limited application areas, and degradation complexes with ubiquitinases are difficult to be taken up and utilized by cells. At the same time, although protein targeted degradation can quickly downregulate the expression level of specific proteins in cells, the negative feedback mechanism of the cells themselves can quickly restore the target protein level, and it is impossible to achieve a long-term downregulation effect.

[0005] Nucleic acid interference technology can deliver RNA into cells, target specific genes, achieve silencing effects at the pre-translation level or post-translation level of protein expression, and reduce protein levels. Although it targets specific genes and has a high silencing effect, RNA interference technology also has some problems. First, a safe and effective nucleic acid delivery vector is required to successfully deliver RNA into the cell; second, it takes a certain amount of time for RNA interference to take effect, and it is difficult to take effect quickly.

[0006] The utilization of nanocarriers in the body usually cannot reach 100%. During the blood circulation process, there will be losses in the liver and spleen, metabolism in the kidneys, and uptake and clearance by macrophages. The nanocarriers lost in these processes cannot play the expected role and may be toxic to normal cells. Using nanocarriers taken up by macrophages to activate macrophages to kill tumors can also provide auxiliary effects for tumor treatment. Summary of the invention

[0007] Purpose of the invention: To address the problem of poor prognosis of tumor patients caused by high expression of CXCR4, the present invention provides a magnetic nanocarrier modified with PEI and a targeting peptide, which can achieve rapid degradation of CXCR4 protein in the lysosomal pathway through surface targeting peptides RGD and E5 polypeptide, and can achieve long-term downregulation of CXCR4 levels by combining nanocarriers to deliver nucleic acid molecules, inhibit the migration ability of tumor cells, and can use nanocarriers taken up by macrophages to activate the ability of macrophages to kill tumor cells, thereby assisting in tumor treatment.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides a PEI and targeting peptide modified magnetic nanocarrier, wherein the magnetic nanocarrier uses ferroferric oxide nanoparticles as the internal magnetic core, and uses hyaluronic acid HA, polyethyleneimine PEI, polypeptide E5 and cRGD cyclic peptide as the surface modification, wherein the amino acid sequence of the cRGD cyclic peptide is: Arg-Gly-Asp-Tyr-Lys, and the amino acid sequence of the polypeptide E5 is: GGRSFFLLRRIQGCRFRNTVDD.

[0009] The present invention also includes a method for preparing the PEI and targeting peptide modified magnetic nanocarrier, comprising the following steps:

[0010] (1) adding Fe3O4@PEI nanocarrier to RGD-HA aqueous solution under ultrasonic and stirring conditions to obtain Fe3O4@PEI@HA-RGD;

[0011] (2) Fe3O4@PEI@HA-RGD was coupled with polypeptide E5 using carbodiimide EDC and N-hydroxysuccinimide NHS aqueous solution to prepare Fe3O4@PEI@HA-RGD / E5 nanocarrier.

[0012] Among them, the Fe3O4@PEI nanocarrier in step (1) is obtained by adding Fe3O4@DMSA to a PEI aqueous solution under ultrasonic and stirring conditions. The hydrodynamic size of the Fe3O4@DMSA is 20 to 40 nm, the surface Zeta potential is -20 to -30 mV, and it can catalyze hydrogen peroxide to produce hydroxyl radicals.

[0013] The mass ratio of Fe3O4@DMSA to PEI is 1:1-1:3, the molecular weight of PEI is 10000Da-25000Da, the ultrasonic power is 80-120W, the mechanical stirring speed is 500-1000rpm, and after preparation, the product is purified by dialyzing through a dialysis bag with a molecular weight of 100kD for 24-48h.

[0014] In the step (1), the RGD-HA aqueous solution is coupled with cRGD and HA by adding EDC and NHS, the molecular weight of HA is 5000, the mass ratio of HA to cRGD is 1:1 to 2:1, the coupling conditions are oscillation at 80 to 150 rpm for 16 to 24 hours at 4°C, and the cRGD coupling rate is 35% to 45%.

[0015] Wherein, in step (2), the weight ratio of EDC to NHS is greater than or equal to 2:1, wherein the concentration of EDC is 0.1 mg / mL, the concentration of NHS is 0.05 mg / mL, the coupling conditions are shaking at 80-150 rpm for 16-24 hours at 4°C, and the coupling rate of E5 polypeptide is 30%-40%.

[0016] The present invention also includes the use of the PEI and targeting peptide modified magnetic nanocarriers in the preparation of drugs for treating tumors.

[0017] The magnetic nanocarrier is loaded with RNA molecules or other drugs, the mass ratio of the magnetic nanocarrier to the RNA molecule is 1:1.5 to 1:9, and the incubation condition is to place at 4 to 7° C. for 20 to 40 minutes after mixing evenly.

[0018] The drug targets the CXCR4 membrane protein of tumor cells and utilizes the lysosomal pathway to degrade the CXCR4 protein, and combines the RNA interference function of the delivered nucleic acid to jointly downregulate the CXCR4 protein level of tumor cells to inhibit tumor cell migration.

[0019] Among them, after being taken up by macrophages, the magnetic nanocarriers can activate the M1 polarization of macrophages and enhance the killing ability of tumor cells.

[0020] The method for preparing the PEI and targeting peptide modified magnetic nanocarriers of the present invention specifically comprises the following steps:

[0021] Step 1, using high temperature pyrolysis method to prepare oleic acid modified ferroferric oxide nanoparticles, namely Fe3O4@OA;

[0022] Step 2, using a ligand replacement method to prepare Fe3O4@OA modified with dithiosuccinic acid, i.e., Fe3O4@DMSA, for the Fe3O4@OA in step 1;

[0023] Step 3, adding PEI aqueous solution to the Fe3O4@DMSA in step 2 under ultrasonic and stirring conditions to complete the preparation of the Fe3O4@PEI nanocarrier;

[0024] Step 4, adding RGD-HA aqueous solution to the Fe3O4@PEI in step 3 under ultrasonic and stirring conditions to complete the preparation of Fe3O4@PEI@HA-RGD;

[0025] Step 5, for the Fe3O4@PEI@HA-RGD in step 4, the polypeptide E5 is coupled using carbodiimide EDC and N-hydroxysuccinimide NHS aqueous solution to complete the preparation of Fe3O4@PEI@HA-RGD / E5;

[0026] Step 6, incubate the Fe3O4@PEI@HA-RGD / E5 nanocarrier in step 5 with the RNA molecule to complete the loading of the RNA molecule.

[0027] The Fe3O4@OA in step 1 of this preparation method has a diameter of 8 to 15 nm, has superparamagnetism, and a saturation magnetic intensity of 60 to 80 emu / g.

[0028] The hydrodynamic size of Fe3O4@DMSA in step 2 of this preparation method is 20 to 40 nm, and the surface Zeta potential is -20 to -30 mV, which can catalyze hydrogen peroxide to produce hydroxyl radicals.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0030] 1. The nanocarriers of the present invention have regular morphology, uniform particle size, good crystallinity and magnetic properties. The present invention uses a fast-acting but easily recoverable protein degradation strategy and a slow-acting but highly efficient RNA interference technology together for the degradation of CXCR4 membrane protein. The two strategies can complement each other and achieve fast-acting and long-term preservation of CXCR4 levels. The E5 polypeptide and RGD cyclic peptide on the surface of the magnetic nanocarrier bind to the CXCR4 protein and integrin on the surface of the tumor cells, respectively, and the nanocarrier and CXCR4 are internalized into the lysosome through an integrin-mediated pathway to degrade the target protein CXCR4.

[0031] 2. The nanocarriers prepared by the present invention can activate macrophages to kill tumor cells, and effectively utilize a part of the nanocarriers that cannot act on tumors to achieve the effect of assisting in killing tumor cells. PEI in the nanocarrier can load and deliver siRNA to tumor cells to silence CXCR4 mRNA expression. By delivering nucleic acids and protein targeted degradation strategies, the CXCR4 level of tumor cells is jointly lowered, the migration ability of tumor cells is inhibited, and the anti-tumor effect of macrophage M1 polarization is activated at the same time.

[0032] 3. The protein targeted degradation technology of the present invention uses the lysosomal pathway, which can be used for extracellular proteins and membrane proteins compared with the traditional proteasome protein degradation technology, and the targeted polypeptide can enhance the cellular uptake of the nanocarrier and enhance the degradation effect.

[0033] 4. When delivering nucleic acids, the nanocarrier prepared by the present invention can enhance the delivery effect by applying an external magnetic field or magnetic separation, and enhance the effect of RNA interference by POD enzyme activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The schematic diagram is a preparation process of the PEI and targeting peptide modified magnetic nanocarrier of the present invention.

[0035] Figure 2 a is the transmission electron microscopy image of Fe3O4@OA nanoparticles of different sizes. Figure 2 b is the particle size distribution result, and the average particle size of the nanoparticles is 11 nm.

[0036] Figure 3 a is the result of high-resolution electric penetration. Figure 3 b is the corresponding relationship diagram between the electron selected diffraction result and the FeO3 crystal plane. Figure 3 c is the corresponding relationship between the diffraction ring intensity and the peak value of the standard XRD card of ferroferric oxide.

[0037] Figure 4 a is the hysteresis loop of nanoparticles tested by vibrating sample magnetometer. Figure 4b shows the absorbance change of TMB catalyzed by the nanoparticles at 650 nm.

[0038] Figure 5 a is the hydrodynamic size of the prepared nanocarriers Fe3O4@DMSA, Fe3O4@PEI, Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5, and Fe3O4@PEI@HA-RGD / E5. Figure 5 b is the Zeta potential diagram.

[0039] Figure 6 a is the gel hysteresis electrophoresis result of nanocarrier loaded nucleic acid, Figure 6 b is the statistical graph of the grayscale values ​​of each lane.

[0040] Figure 7 The results of measuring the toxicity of Fe3O4@PEI, Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5, and Fe3O4@PEI@HA-RGD / E5 nanocarriers to different cells by CCK8 method. Figure 7 a is macrophage Raw264.7, Figure 7 b is mouse malignant lymphoma cell A20, Figure 7 c is human cervical cancer cell HeLa.

[0041] Figure 8 a is a confocal fluorescence image of different nanocarriers being taken up by Hela cells. Figure 8 b is the statistical graph of fluorescence intensity of nanoparticles.

[0042] Fig. 9 a is a confocal fluorescence image of different nanocarriers being taken up by A20 cells. Fig. 9 b is the statistical graph of fluorescence intensity of nanoparticles.

[0043] Fig.10 These are the experimental results showing that the efficiency of nucleic acid delivery by nanocarriers is affected by external magnetic field, culture medium, carrier / nucleic acid ratio and dosage.

[0044] Fig.11 The experimental results show that the efficiency of nucleic acid delivery by nanocarriers is affected by magnetic separation and enrichment.

[0045] Fig.12 a WB test results of Fe3O4@PEI, Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5, and Fe3O4@PEI@HA-RGD / E5 nanocarriers for degrading CXCR4 protein in Hela cells; 12b is a statistical chart of the changes in the relative expression level of CXCR4 protein.

[0046] Fig.13 a WB test results of Fe3O4@PEI, Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5, and Fe3O4@PEI@HA-RGD / E5 nanocarriers for the degradation of CXCR4 protein in A20 cells; 13b is a statistical chart of the changes in the relative expression level of CXCR4 protein.

[0047] Fig.14 The results are as follows: The delivered nucleic acid down-regulates the CXCR4 mRNA level in A20 cells.

[0048] Fig.15 These are the experimental results showing that CXCR4 degradation and RNA interference combined to inhibit the migration ability of A20 cells.

[0049] Fig.16 This is the result of nanocarriers activating the M1 differentiation of macrophages.

[0050] Fig.17 a is the microscopic observation result of nanocarrier promoting macrophage killing of A20 cells, and 17b is the killing rate statistical histogram. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described in detail below through specific embodiments. However, it is necessary to point out that the following embodiments are only used to describe the content of the invention and do not constitute a limitation on the protection scope of the present invention.

[0052] cRGD cyclic peptide: cyclo(Arg-Gly-Asp-d-Tyr-Lys), relative molecular weight is 619.67; Gir peptide.

[0053] E5 peptide: amino acid H-GGRSFFLLRRIQGCRFRNTVDD-OH, relative molecular weight 2613.94. Anhui Guoping.

[0054] Hyaluronic acid HA, molecular weight 5000, Xi'an Qiyue Biotechnology Co., Ltd. product number, Q00093890.

[0055] The double-stranded siRNA in the present invention was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the sequence is as follows:

[0056] 5'-GCCUCAAGAUCCUUUCCAATT-3'

[0057] 3'-TTCGGAGUUCUAGGAAAGGUU-5'.

[0058] Example 1: Preparation and characterization of physicochemical properties of Fe3O4@PEI@HA-RGD / E5

[0059] Weigh 7.06g of ferric acetylacetonate, 100mL of dibenzyl ether, and 25mL of oleic acid, mix well, and add to a 250mL three-necked flask. Fix the flask containing the reactants on an iron stand, place a digital temperature-controlled heating jacket below, introduce nitrogen into the three bottle openings above, place a thermometer, and place a condenser, and the bottle openings must be fully sealed. Under the conditions of nitrogen protection and condensation reflux, the solution is heated from room temperature to 220℃ at a rate of 3.3℃ / min according to the setting of the program temperature controller, and the temperature is maintained at 220℃ for 60 minutes for explosive nucleation. After that, continue to heat to 290℃ at a rate of 3.3℃ / min, and maintain at 290℃ for 30 minutes for crystal growth and maturation. After the reaction is completed, remove the heating device to prepare oleic acid-modified Fe3O4 magnetic nanoparticles, namely Fe3O4@OA. After the product solution is cooled to room temperature, transfer it to a dry and clean 500mL beaker and perform magnetic separation for 5-10 minutes to remove the residual dibenzyl ether and oleic acid in the system. Add 100mL of anhydrous ethanol to resuspend the product at the bottom of the beaker, perform magnetic separation for 5 minutes and remove the supernatant, repeat 3-5 times, and when the supernatant is almost clear, dissolve the product in 20mL of chloroform, seal it with a sealing film and store it in a 4℃ refrigerator away from light.

[0060] Take 800 mg of Fe3O4@OA chloroform solution calculated by the weight of iron element, evaporate to dryness in a 60℃ water bath in a fume hood, and then re-dissolve it with 100 mL of n-hexane; weigh 400 mg of dithiosuccinic acid (DMSA) and dissolve it in 200 mL of acetone solution. Mix the above two solutions evenly and add them to a 250 mL three-necked flask. Fix the flask containing the reactants on an iron stand, place a 60℃ water bath below, and seal the three bottle mouths above, install the stirring paddle of the mechanical stirrer, and place the condenser. Stir mechanically at a rate of 500 rpm, condense and reflux in a 60℃ water bath for 5 hours. During the reaction, if the liquid level of the solution drops, a mixed solution of n-hexane: acetone with a volume ratio of 1:1 needs to be added to fill the liquid level to prevent evaporation. After the reaction, add 100 mL of ultrapure water to extract the product. After the mixed solution is ultrasonically mixed evenly, magnetic separation is performed for 15 minutes to remove the supernatant. After repeating three times, the product is dissolved in 50 mL of ultrapure water. The product was placed in a 100 kD molecular weight dialysis bag and dialyzed for 48 h to purify the product. The pH of the aqueous solution of the final product Fe3O4@DMSA was adjusted to about 9 and stored in a refrigerator at 4°C.

[0061] Prepare a 1mg / mL PEI aqueous solution, take 40mL and add it to a 150mL three-necked flask. Fix the flask with the solution on an iron stand, place an ultrasonic cleaner below, seal the three bottle mouths above, install the stirring paddle of the mechanical stirrer and the hose connected to the syringe of the micro-injection pump. Take 10mL of a 1mg / mL Fe3O4@DMSA aqueous solution based on the mass of the iron element and inhale it into a 20mL syringe, and install the syringe on the syringe pump. Turn on the 100W power at room temperature to ultrasonically oscillate the three-necked flask and stir at a speed of 500rpm. The injection rate of the syringe pump is 2mL / min, and the reaction time is 1 hour. The reaction product is ultrafiltered with an ultrafiltration tube with a molecular weight cutoff of 100kD. After repeating 6 times, Fe3O4@PEI nanocarriers are obtained, and the product is stored in a refrigerator at 4℃.

[0062] Dissolve 4mg hyaluronic acid HA molecules in 10mL pure water, add 2mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1mg N-hydroxysuccinimide (NHS), mix well, place in a shaker, and shake at 120rpm for 30min at room temperature. Add 2mL of 1mg / mL cRGD aqueous solution to the mixed solution, continue to place in a shaker, and shake at 120rpm for 24h at room temperature. Use a dialysis bag with a molecular weight cutoff of 1000Da to dialyze the product with pure water for 3 days to obtain the product RGD-HA aqueous solution, and take the product RGD-HA aqueous solution and freeze-dry it for determination of nuclear magnetic resonance hydrogen spectrum. Take 1mg of Fe3O4@PEI nanocarriers based on the mass of iron element and add them to a 50mL three-necked flask containing 15mL of pure water. Fix the flask containing the solution on an iron stand, place an ultrasonic cleaner below, seal the three bottle mouths above, install the stirring paddle of the mechanical stirrer, and perform drop injection. Add 2mg of RGD-HA aqueous solution dropwise to the above solution, turn on 100W ultrasonic oscillation at room temperature and stir at 500rpm for 30min. Ultrafiltration was performed on the reaction product with an ultrafiltration tube with a molecular weight cutoff of 100kD. After 6 times, Fe3O4@PEI@HA-RGD aqueous solution was obtained, and the product was stored in a refrigerator at 4℃.

[0063] Take 1mg of Fe3O4@PEI nanocarriers based on the mass of iron element and add them to a 50mL three-necked flask containing 15mL of pure water. Fix the flask containing the solution on an iron stand, place an ultrasonic cleaner below, seal the three bottle mouths above, install the stirring paddle of the mechanical stirrer, and perform drop injection. Add 2mg of HA aqueous solution drop by drop into the above solution, turn on 100W ultrasonic oscillation at room temperature and stir at 500rpm for 30min. Ultrafiltration the reaction product with an ultrafiltration tube with a molecular weight cutoff of 100kD. Repeat 6 times to obtain Fe3O4@PEI@HA aqueous solution, which is stored in a refrigerator at 4℃.

[0064] Take 200μg E5 polypeptide powder and dissolve it in 1mL pure water, add an aqueous solution containing 100μg EDC and 50μg NHS, mix well and place it in a shaker, and shake at 120rpm for 30min at room temperature. Add the above mixed solution to an aqueous solution containing 1mg of Fe3O4@PEI@HA-RGD in terms of iron element mass, mix well, continue to place it in a shaker, and shake at 120rpm for 24h at room temperature. Use a dialysis bag with a molecular weight cutoff of 1000Da to dialyze the product with pure water for 3 days, and ultrafilter the product with an ultrafiltration tube with a molecular weight cutoff of 100kD to obtain Fe3O4@PEI@HA-RGD / E5 nanocarrier, and store the product in a refrigerator at 4℃.

[0065] Take 200μg E5 polypeptide powder and dissolve it in 1mL pure water, add an aqueous solution containing 100μg EDC and 50μg NHS, mix well and place it in a shaker, and shake at 120rpm for 30min at room temperature. Add the above mixed solution to an aqueous solution containing 1mg of Fe3O4@PEI@HA in terms of iron element mass, mix well, continue to place it in a shaker, and shake at 120rpm for 24h at room temperature. Use a dialysis bag with a molecular weight cutoff of 1000Da to dialyze the product with pure water for 3 days, and ultrafilter the product with an ultrafiltration tube with a molecular weight cutoff of 100kD to obtain Fe3O4@PEI@HA / E5 nanocarriers, and store the product in a refrigerator at 4℃.

[0066] The solution of Fe3O4 nanoparticles was diluted to 0.1 mg / mL based on the mass of the iron element and dropped onto a 200-mesh copper grid. After air drying in a fume hood, the copper grid containing the sample was placed on the sample stage of a transmission electron microscope (TEM) for testing. The operating voltage for TEM morphology, high-resolution, and selected area electron diffraction was 200 kV. The results of high-resolution TEM (HRTEM) and selected area electron diffraction (SAED) were compared with standard cards.

[0067] Figure 2a is the TEM image of Fe3O4@OA, the nanoparticles have regular morphology and uniform particle size. Figure 2 b is the statistical result of particle size distribution, and the average particle size of the nanoparticles is 11 nm. Figure 3 a is the structure of the nanoparticles under high-resolution transmission electron microscope, and the lattice diffraction fringes are clearly visible. Figure 3 b is the electron selected diffraction result, which is calibrated with the Fe3O4 standard XRD card (number PDF#19-0629) in the MDI JADE 6 software. At the same time, the Digital Micrograph software is used to compare the diffraction intensity integral at different diameters of the diffraction ring in the image with the standard XRD spacing. The results are shown in Figure 3 As shown in c, the corresponding relationship is good, proving that the prepared ferroferric oxide nanoparticles have a good crystal structure.

[0068] A solution of nanoparticles containing 20 mg of iron was vacuum dried for 48 h, and the powder was scraped with a medicine spoon and placed on the sample stage of a vibrating sample magnetometer. The external magnetic field was set in the range of -10000G to 10000G for testing.

[0069] Figure 4 a The result is the hysteresis loop of the nanoparticles measured by a vibrating sample magnetometer at room temperature under the condition of an external magnetic field from -10000G to +10000G and then back to -10000G. It approximately passes through the (0, 0) point, and the remanent magnetization and coercive force are both approximately 0, indicating that the prepared Fe3O4 nanoparticle material has superparamagnetism, and the saturation magnetization intensity is 69.13emu / g (calculated based on the mass of iron element).

[0070] After diluting the sample with pure water to a concentration of 0.1 mg / mL based on the mass of the iron element, the pH of the solution was adjusted to between 7.2 and 7.3, and the solution was added to the sample cell to detect the hydrodynamic size and Zeta potential of the sample.

[0071] The hydrodynamic sizes and zeta potentials of the nanocarriers Fe3O4@DMSA, Fe3O4@PEI, Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5, and Fe3O4@PEI@HA-RGD / E5 prepared by the above method are shown in Figure 2. Figure 5 shown. Figure 5a shows the difference in the hydrodynamic size of nanocarriers modified with different polymers. It can be seen that both PEI and HA polymers and RGD modification can increase the hydrodynamic size of nanocarriers (differences between groups in the red, blue and black bar graphs). Relatively speaking, modification with the E5 polypeptide has no significant increase in the hydrodynamic size of the nanocarrier (small differences within the blue and black bar graphs). The change in Zeta potential can further reflect the modification of the nanoparticle surface by different organic molecules. It can be seen that within the pH range of 7.2-7.3, PEI molecule modification changes the surface potential of the nanoparticles from -25.2mV to +50.37mV, while subsequent modifications with HA, RGD and E5 only partially shield the positive charge on the surface of the nanocarrier, and E5 polypeptide modification has a greater effect on the surface charge of the nanocarrier than RGD cyclic peptide modification. Overall Figure 5 The effects of different polymer modifications in a and 5b on the hydrodynamic size and surface potential of the nanocarriers can indirectly confirm that during the construction of the nanocarriers, different molecules have been gradually and successfully modified on the surface of the nanocarriers.

[0072] Example 2 Test of RNA loading capacity of Fe3O4@PEI@HA-RGD / E5 nanocarrier

[0073] Dilute the 50×TAE solution to 1×TAE with ultrapure water, use the 1×TAE solution to prepare a 4% agarose gel, add 2μL GelRed dye, cool in the mold, and place in an electrophoresis tank filled with 1×TAE. Mix the Fe3O4@PEI@HA-RGD / E5 nanocarrier aqueous solution with RNA (purchased from Bioengineering (Shanghai) Co., Ltd.) at different mass ratios (9:6, 9:3, 9:1), incubate at room temperature for 30 minutes. Dilute the solution to 1× concentration with 5× loading buffer, add it to the sample well of the gel, add the standard nucleic acid marker to the remaining left and right sample wells, cover the electrophoresis tank, and electrophoresis at 120V for 30 minutes. Use a gel imaging system to develop the agarose gel, and export the image and analyze it using ImageJ software.

[0074] from Figure 6 From the gel hysteresis electrophoresis results of a, it can be seen that the size of naked RNA molecules is slightly larger than 20bp, and the addition of nanocarriers can bind nucleic acid molecules well, preventing them from being separated by the electric field. At the same time, when the mass ratio of RNA to Fe element is 9:6 and 9:3, RNA can be completely bound. When the mass ratio is increased to 9:1, RNA begins to be excessive, and the free RNA is separated by the electric field. Figure 6b shows the grayscale values ​​of each channel counted by ImageJ software. The grayscale values ​​of the second and fifth lanes are 119.04 and 62.45, respectively. It can be roughly estimated that the saturation ratio of RNA and Fe binding is 4.28:1. In subsequent experiments, the binding ratio of nucleic acid and nanocarrier is less than this ratio, which can be approximately considered to have achieved complete loading of nucleic acid.

[0075] Example 3 Study on the oxidation of TMB by hydrogen peroxide catalyzed by nanocarriers

[0076] Prepare a 10mg / mL TMB solution with DMSO, prepare a 0.1M HAc-NaAc buffer with pure water, and adjust the pH to 3.6. Add 200μL pH3.6 HAc-NaAc buffer, 10μL 10mg / mL Fe3O4 nanoparticle aqueous solution, and 10μL 10mg / mL TMB solution dissolved in 10μLDMSO to a 96-well plate at room temperature, and blow evenly to avoid bubbles. Add 10μL 10% H2O2 solution to the above mixed solution and quickly put it into the microplate reader to detect the absorbance change at 650nm within 300s of the reaction system.

[0077] Figure 4 b The results show that Fe3O4@DMSA nanoparticles can catalyze hydrogen peroxide to produce ROS to oxidize TMB. Compared with the blank group, the Fe3O4 nanoparticle group produced a higher amount of oxidized TMB under the same conditions, resulting in an increase in the absorbance value at 650nm, indicating that Fe3O4@DMSA nanoparticles have peroxidase-like activity and can produce ROS under acidic conditions. It can be expected that in the acidic environment containing hydrogen peroxide in cell lysosomes, Fe3O4 nanoparticles can catalyze the production of reactive oxygen species to damage lysosomes, which is conducive to the escape of nucleic acid molecules from lysosomes.

[0078] Example 4 Study on the toxicity of nanocarriers to different cells

[0079] Take Hela, Raw264.7 and A20 cells in the logarithmic growth phase, digest them when the cell density is about 80%, and dilute them to 1×10 5 / mL concentration, inoculated in a 96-well plate at 100μL / well, shake the plate to make the cells evenly distributed, and cultured in a constant temperature incubator at 37℃ and 5% carbon dioxide for 16h. DMEM cell culture medium containing Fe3O4@PEI, Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5, and Fe3O4@PEI@HA-RGD / E5 nanocarriers with final concentrations (in terms of Fe element) of 0, 2, 4, 6, and 8μg / mL was added respectively, and 6 sub-wells were set for each concentration. The plate was placed in a constant temperature incubator at 37℃ and 5% carbon dioxide for 24h. After discarding the culture medium in each well, 10μL CCK8 solution and 90μL DMEM cell culture medium were added to each well. After culturing at 37℃ for 1h, the absorbance at λ=450nm was detected using an ELISA reader.

[0080] Figure 7 a is Raw cell, Figure 7 b is A20 cells, Figure 7 c is Hela cells. The CCK8 method was used to study the effects of five nanocarriers, Fe3O4@PEI, Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5 and Fe3O4@PEI@HA-RGD / E5, on cell viability after co-culture with Hela cells for 24 hours. The results show that the cytotoxicity of the nanocarriers is concentration-dependent, and its cytotoxicity decreases with the modification of surface molecules. At the same time, the toxicity of the nanocarriers to macrophages Raw is lower than that to tumor cells A20.

[0081] Example 5 Study on Cellular Uptake of Nanocarriers

[0082] Prepare 5 mg / mL FITC solution with DMSO solvent, take 10 μL FITC DMSO solution and mix it with Fe3O4 nanocarrier aqueous solution containing 1 mg Fe element, vortex for 30 seconds to mix evenly, place it away from light for 24 hours, and then use ultrafiltration tube with a molecular weight cutoff of 100 kDa to purify and remove free FITC molecules and a small amount of DMSO, repeat 3 times. Take Hela and A20 cells in the logarithmic growth phase, digest them when the cell density is about 80%, and inoculate the cells in confocal dishes, 5×10 per well. 5Cells were placed in a constant temperature incubator at 37°C and 5% carbon dioxide for 24 hours. Nanocarriers modified with different FITC molecules were added to the above six-well plate and confocal dish at a final concentration of 2μg / mL, and placed in a constant temperature incubator at 37°C and 5% carbon dioxide for 6h, 12h and 24h. After the incubation time, the confocal dish was taken out, the supernatant was discarded and rinsed with PBS twice, and then a 1:10 diluted Hoechest33342 dye solution was added according to the instructions. After incubation at 37°C for 20-30min, the supernatant was poured off, and after rinsing with PBS twice, 1mL of solution was added for confocal photography, and confocal images were taken using lasers with excitation wavelengths of 406 and 488nm.

[0083] Figure 8 a is a confocal fluorescence image of Hela cells taking up nanocarriers. Figure 8 b is a statistical histogram of the average fluorescence intensity of the nanocarriers. The results show that the uptake of nanocarriers by Hela cells is time-dependent, and the uptake of nanocarriers is enhanced by the two targeting peptides E5 and RGD.

[0084] Fig. 9 a is the confocal fluorescence image of A20 cells taking up nanocarriers. Fig. 9 b is a statistical histogram of the average fluorescence intensity of the nanocarriers. The results show that the uptake of nanocarriers by A20 cells is time-dependent, and the uptake of nanocarriers is enhanced by the two targeting peptides E5 and RGD, and the enhancement effect is more significant than that of Hela cells.

[0085] Example 6 Effects of external magnetic field, culture medium, carrier / nucleic acid ratio and dosage, and magnetic separation rack enrichment on nucleic acid delivery

[0086] Hela cells in the logarithmic growth phase were taken and digested with trypsin when the cell density was about 80%. The cells were seeded in a 24-well plate with 1×10 cells per well. 5Cells were placed in a constant temperature incubator at 37°C and 5% carbon dioxide for 24 hours. Each culture well was replaced with fresh 500μl DMEM medium (containing 10% FBS) or Opti-MEM serum-reduced culture medium for nucleic acid delivery. After fixing the nucleic acid (RNA) mass to 1μg, the RNA was mixed with 0.5, 0.75, and 1μg of Fe3O4@PEI@HA-RGD / E5 nanocarrier aqueous solution, respectively, incubated at room temperature for 30min, and then added to the well plate and shaken to mix evenly. The control well plate assisted by an external magnetic field was placed above a 400mT magnet for 20min, after which the magnetic plate was removed, and all cells continued to be cultured at 37°C and 5% CO2 for 8 hours to detect the nucleic acid delivery efficiency. After the culture time was reached, the 24-well plate was removed, the supernatant was discarded, and the cells were rinsed twice with PBS, and then trypsin was added to digest the cells. The resulting cells were resuspended with PBS and added to a 1.5mL centrifuge tube for subsequent flow cytometry detection, with an excitation wavelength of 488nm and a detection channel wavelength of 530±30nm. After the test, the remaining cells were enriched using a magnetic separation column, washed twice with PBS, and then the eluted cell samples were subjected to flow cytometry detection.

[0087] Fig.10 The effects of different culture conditions and the presence or absence of an external magnetic field on the efficiency of nucleic acid delivery by nanocarriers are shown in Figure 2. For the nanoparticle nucleic acid delivery carrier of the present invention, the delivery efficiency is less affected by the protein in the culture fluid environment, and may even cause differences in cell state due to changes in the culture environment, which in turn leads to a lower delivery efficiency in the Opti-MEM reduced serum culture medium group than in the ordinary complete culture medium group. Secondly, for the magnetic nanocarriers prepared by the present invention, an external static magnetic field can increase the nucleic acid delivery effect to a certain extent. Fig.10 The pink numbers in the middle represent an external magnetic field, and the gray numbers represent no external magnetic field. The other conditions in the same picture are the same. It is calculated that the efficiency of nucleic acid delivery is improved by 10%-20% when an external magnetic field is added.

[0088] Fig.11 This is the effect of magnetic separation and enrichment on the effectiveness of nanocarrier nucleic acid delivery. From the results, the proportion of successfully delivered cells increased after magnetic separation and enrichment (blue group) compared with before magnetic separation and enrichment (gray group), and all groups could be increased to about 95% through enrichment.

[0089] Example 7 Western Blot Detection of Protein Degradation Ability of Nano-delivery Carrier

[0090] Hela and A20 cells in the logarithmic growth phase were taken and inoculated into six-well plates at a cell density of about 80%, with 5×10 cells per well. 5cells, and cultured them in a constant temperature incubator at 37°C and 5% carbon dioxide for 24 hours. The nanocarriers Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5, and Fe3O4@PEI@HA-RGD / E5 were added to the above six-well plates at a final concentration of 2μg / mL, and cultured in a constant temperature incubator at 37°C and 5% carbon dioxide for 6h, 12h, and 24h. After the culture time was reached, the six-well plate was taken out, the supernatant was discarded, and it was rinsed twice with PBS, and then the cell lysis solution was added, and the cell lysis was performed in an ice bath for 10min, and the liquid was transferred to a 1.5mL centrifuge tube. After the lysis was completed, the centrifuge tube was placed in a centrifuge, centrifuged at 12000g for 5min at 4°C, and the supernatant was taken to store the protein sample at -20°C. Take 5μL of protein sample and use the BCA protein concentration determination kit to determine the protein concentration of the sample. Each protein sample was added with 1 / 4 volume of 5× SDS-PAGE Loading Buffer, denatured at room temperature for 120 min, centrifuged at 12000 rpm for 10 min, and the supernatant was stored at -20°C for subsequent Western Blot experiments.

[0091] Use the WB gel kit according to the instructions to prepare 5% upper gel and 10% lower gel solutions, inject the lower gel into the glass mold, add an appropriate amount of anhydrous ethanol to make the liquid level, wait for the lower gel to solidify, absorb the anhydrous ethanol, inject the upper gel and insert the sample comb, and after the upper gel solidifies, soak the gel and the glass mold in pure water and store in a refrigerator at 4°C. Weigh 3.025g Tris, 18.8g glycine and 1g SDS to prepare 1L Tris-glycine electrophoresis buffer. After loading the gel and glass plate into the electrophoresis instrument, first add electrophoresis solution to the inside until it overflows, then add electrophoresis solution to the outside until it is above the standard line, remove the tooth comb, load 20μg of protein sample into each sample well and add 1×SDS-PAGE Loading Buffer to make up to 10μL, add 5μL of standard protein marker to the sample wells on both ends, use 70V voltage for electrophoresis for 30min until the sample reaches the boundary of the two layers of gel, then switch to 120V voltage for electrophoresis until the sample reaches the bottom of the gel. Weigh 30.3gTris and 144gg glycine to make 1L 10× transfer solution, and make 1× transfer solution with 700mL pure water, 200mL methanol and 100mL 10X transfer solution. Remove the gel from the glass plate, cut off the excess part, immerse the transfer mold in the transfer solution, and activate the PVDF membrane by infiltration with methanol. After laying and clamping in the order of black-sponge-filter paper-film-PVDF membrane-filter paper-sponge-red, transfer to the transfer tank, add 1× transfer solution to the transfer tank mark, and place the transfer tank in an ice water bath. Use 200mA current for transfer for 2h. After the transfer is completed, take out the PVDF membrane, immerse it in 10% skim milk powder and shake it at room temperature for 2h. After completion, rinse it with PBST 3 times, 10min each time. Dilute the primary antibody according to the instructions, add the diluted primary antibody and PVDF membrane to the sealing film to fully contact, place it in a shaker at room temperature for 2h or incubate it in a 4℃ refrigerator overnight. After completion, rinse it with PBST 3 times, 10min each time. Dilute the secondary antibody according to the instructions, add the diluted secondary antibody and PVDF membrane to the sealing film to fully contact, place it in a shaker at room temperature for 1h, and rinse it with PBST 3 times, 10min each time. Mix the ECL luminescent developer according to the instructions, place the PVDF membrane on the imaging table and add drops to cover it, adjust the exposure time and image.

[0092] Fig.12 and Fig.13The changes in CXCR4 protein levels after the nanocarriers acted on Hela cells and A20 cells, respectively, Figure a is the WB result, and Figure b is a statistical chart of the changes in CXCR4 protein relative to GAPDH expression. It can be seen from the results that the nanocarrier Fe3O4@PEI@HA with unmodified polypeptide, the nanocarrier Fe3O4@PEI@HA-RGD modified only with RGD, and the nanocarrier Fe3O4@PEI@HA / E5 modified only with E5 polypeptide, these three nanocarriers caused the CXCR4 protein content of the cells to decrease first and then increase after the action, proving that this downregulation is unstable. After the nanocarrier Fe3O4@PEI@HA-RGD / E5 modified with only E5 and RGD double peptides was incubated with cells, the amount of CXCR4 protein was significantly reduced relative to the control group, and the downregulation of membrane protein CXCR4 remained stable, which was basically the lowest value of the membrane protein CXCR4 content in the four nanocarriers. It is proved that the nanocarrier of the present invention downregulates CXCR4 protein and can exert an effect after acting on different cells.

[0093] Example 8 Fluorescence quantitative PCR detection of the effect of siRNA delivery on CXCR4 mRNA expression in A20 cells

[0094] A20 cells in the logarithmic growth phase were taken and seeded in a six-well plate at a cell density of about 80%, with 2×10 cells per well. 6 cells, and cultured them in a constant temperature incubator at 37°C and 5% carbon dioxide for 6 hours. The Fe3O4@PEI@HA-RGD / E5 nanocarriers were mixed evenly at a mass ratio of Fe: siRNA = 1:1, incubated at 4°C for 30 minutes to fully bind, and then added to six-well plates at a final concentration of 2μg / mL, and cultured in a constant temperature incubator at 37°C and 5% carbon dioxide for 12 hours. After the culture time was reached, the six-well plate was taken out, and the cell suspension was transferred to a centrifuge tube with a pipette, centrifuged at 150g for 5 minutes, washed with PBS 2-3 times, and then 1mL Trizol solution was added. The cells were lysed in an ice bath for 10 minutes, and 200μL of chloroform solution was added to the centrifuge tube. After shaking for 10 seconds to mix evenly, the cells were allowed to stand for 3 minutes, and centrifuged at 12000g at 4°C for 20 minutes. 400μL of the upper aqueous phase liquid was aspirated, 600μL of isopropanol solution was added, and the mixture was mixed by inversion, allowed to stand for 10 minutes, and centrifuged at 12000g at 4°C for 10 minutes. Discard the supernatant, add 1 mL of 75% ethanol solution for washing, centrifuge at 7500 g for 5 min at 4 °C, repeat the washing twice, and discard the upper liquid. After standing and drying the centrifuge tube, add 20 μL of enzyme-free water to dissolve the RNA, and measure the RNA purity and concentration with a micro-UV spectrophotometer. Use the Takara SYBR Green kit to perform reverse transcription and SYBR green fluorescence quantitative PCR on a PCR amplification instrument. Calculate the relative expression of CXCR4 using Actin as an internal reference.

[0095] According to the difference in Ct value of the target CXCR4 gene measured by the instrument relative to the internal reference gene Actin, 2 -△Ct The expression level of the target CXCR4 gene relative to the internal reference gene Actin can be obtained. The expression level of the nanocarrier group is normalized by the expression level of the control group. Fig.14 As shown, after the nanocarrier was loaded with siRNA, the mRNA level of the CXCR4 gene was reduced to 14.8% within 12 hours, which was significantly higher than that of the Control group, proving that the nanocarrier prepared in this article can reduce the mRNA level of the CXCR4 gene in A20 cells by loading and delivering siRNA.

[0096] Example 9 Study on the inhibition of A20 cell migration by nanocarriers via protein degradation and RNA interference

[0097] MS-5 cells (Qingqi (Shanghai) Biotechnology Development Co., Ltd.) were cultured at 5×10 5 The density of each well was inoculated in a 24-well plate in the lower chamber, and the cells were placed in a constant temperature incubator at 37°C and 5% carbon dioxide for 12 hours to form a monolayer of cells. The Fe3O4@PEI@HA-RGD / E5 nanocarriers were mixed evenly at a mass ratio of Fe: siRNA = 1:1, and the empty vector and the vector loaded with nucleic acid were added to the 24-well plate at a final concentration of 2μg / mL, respectively, and incubated with A20 cells for 3 hours. The Fe3O4@PEI@HA-RGD / E5 nanocarriers were mixed evenly at a mass ratio of Fe: siRNA = 1:1, and the empty vector and the vector loaded with nucleic acid were added to the 24-well plate at a final concentration of 2μg / mL, respectively, and incubated with A20 cells for 3 hours. After centrifugation to remove the supernatant, the treated cells were cultured for 8 hours. After counting with a cell counter, the treated A20 cells were counted at 5×10 5 The same volume of suspension of 100 / mL was inoculated into the upper chamber of the Transwell chamber, and the Transwell chamber was combined with a 24-well plate inoculated with MS-5 and cultured in a constant temperature incubator at 37°C and 5% carbon dioxide for 12 hours. The number of A20 cells that migrated to the lower chamber was counted using a cell counter.

[0098] The interaction of the CXCR4-CXCL12 biological axis can trigger the migration of lymphoma cells and is closely related to the metastasis process of malignant lymphoma. Bone marrow stromal cells MS-5 can secrete a large amount of chemokine CXCL12, inducing the migration of lymphoma A20 cells. In this experiment, the Transwell cell migration assay was used to detect the effect of nanocarriers on the migration of A20 cells. The experimental results are shown in Figure 2. Fig.15As shown. The dipeptide-modified nanocarrier Fe3O4@PEI@HA-RGD / E5 can directly reduce the CXCR4 level of A20 cells. Therefore, compared with the control group, the migration ability of A20 cells was significantly reduced after 3 hours of the action of the nanocarrier Fe3O4@PEI@HA-RGD / E5, and after 3 hours of action and removal of the supernatant for continued culture for 8 hours. After the nanocarrier acted for 3 hours and the supernatant was removed and continued to be cultured for 8 hours, the migration ability of A20 was the lowest, only 51.49% of the control group. However, since it takes a certain amount of time for siRNA to take effect, when the nanocarrier acted for 3 hours, whether or not siRNA was loaded had no significant effect on the cell migration ability. Therefore, for the migration ability of A20 cells, the protein-targeted degradation ability of the nanocarrier downregulated the CXCR4 protein level of the cells during the 3-hour culture, and its migration ability was reduced. In the following 8 hours, the loaded siRNA played a role, and by knocking down CXCR4mRNA, it was able to prolong the inhibition time of A20 cell migration and achieve a better auxiliary effect.

[0099] Example 10 Study on Nanocarrier-Induced Macrophage Killing Tumor Cells

[0100] Raw cells in the logarithmic growth phase were taken and digested with trypsin when the cell density was about 80%. The cells were seeded in a six-well plate with 5×10 cells per well. 5 Cells were placed in a constant temperature incubator at 37°C and 5% carbon dioxide for 24 hours. PBS (0.01M, pH7.4), LPS at a final concentration of 1μg / mL, and different nanocarriers of Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5, and Fe3O4@PEI@HA-RGD / E5 at a final concentration of 2μg / mL were added to different wells and placed in a constant temperature incubator at 37°C and 5% carbon dioxide for 12 hours. After the culture time was reached, the six-well plate was removed, the supernatant was discarded, and the cells were rinsed twice with PBS, and then trypsin was added to digest the cells, and the resulting cells were resuspended with PBS. Take 200μL of the cell suspension and place it in a 1mL centrifuge tube, add 2μL of Fc Block blocking solution (Biolegend, Cat. No. 156604) to block the antibody Fc-terminal receptor, and incubate at room temperature in the dark for 5 minutes. Add 1 μg of PE-modified CD86 antibody (Biolegend, Cat. No. 105007), incubate at room temperature in the dark for 25-30 minutes, place the centrifuge tube in a centrifuge, centrifuge at 150g-200g for 5 minutes, discard the supernatant, and resuspend the cells with 1 mL of PBS. Use a flow cytometer for detection, with an excitation wavelength of 488nm and a detection channel wavelength of 574±26nm.

[0101] A20 cells in the logarithmic growth phase were taken and, when the cell density was about 80%, the cells were seeded in six-well plates, with 5×10 5 The cells were placed in a constant temperature incubator at 37°C and 5% carbon dioxide for 6 h. Raw264.7 cells in the logarithmic growth phase were taken and digested with trypsin when the cell density was about 80%. The cells were seeded in a six-well plate with 5×10 cells per well. 5 cells to achieve co-culture of the two cells, and then placed in a constant temperature incubator at 37°C and 5% carbon dioxide for 4 hours. Add different nanocarriers of 1μg / mL LPS, 2μg / mL Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5, and Fe3O4@PEI@HA-RGD / E5 to different wells, respectively, and place in a constant temperature incubator at 37°C and 5% carbon dioxide for 12 hours. Observe the cell growth in each well with an inverted microscope and take photos for record.

[0102] In order to study the ability of nanocarriers to induce the differentiation of macrophages Raw 264.7 into M1 type, CD86, a specific marker of M1 macrophages, was selected as an indicator. APC-modified CD86 antibody was used to detect the difference in CD86 expression levels on the surface of Raw 264.7 cells in different experimental groups by flow cytometry. The experimental results are shown in Figure 2. Fig.16 As shown, the APC fluorescence intensity and side scatter SSC of each cell were used to make a density map, and the Control group was used as the negative control. The positive gate with 95% confidence level of this group was selected for comparison with other groups; the LPS-induced group was the positive control, and its surface CD86 expression level was 92.4% compared with the negative control; the four figures below show the induction of macrophage differentiation by four differently modified nanocarriers. The four nanocarriers, Fe3O4@PEI@HA, Fe3O4@PEI@HA-RGD, Fe3O4@PEI@HA / E5, and Fe3O4@PEI@HA-RGD / E5, can all induce Raw264.7 to differentiate into M1 type, and the CD86 expression level reaches more than 75%. On this basis, the two groups of nanocarriers modified with E5 polypeptide, Fe3O4@PEI@HA / E5 and Fe3O4@PEI@HA-RGD / E5, induced higher expression levels of macrophage CD86, reaching more than 85%.

[0103] By introducing different external influences such as nanocarriers or LPS into the co-cultured A20 cells and macrophage Raw 264.7, the effect of macrophages on tumor cells was observed. The experimental results are as follows Fig.17As shown in a, the Blank group only has A20 cells. Under normal culture conditions, A20 cells can cover the entire well plate. A20 cells are non-adherent cells and have a higher brightness under the microscope field of view. The Control group is a co-culture of A20 cells and macrophages Raw 264.7 without any external influence. The non-adherent A20 cells have a high brightness, while the adherent macrophages have a low brightness. It can be observed that the macrophages clear the tumor cells, and some cell-free spaces appear in the field of view. The two nanocarriers, Fe3O4@PEI@HA and Fe3O4@PEI@HA-RGD / E5, have been proven in previous experiments to be toxic to tumor cells and can promote the M1 polarization of macrophages, thereby enhancing the clearance of tumor cells by macrophages. The LPS group is a positive control, and the effect of M1 polarized macrophages on tumors is observed. Comparing the images of the LPS group and the Fe3O4@PEI@HA-RGD / E5 group, the area of ​​non-A20 cells in the field of view is similar, but the number of macrophages in the field of view is quite different, reflecting that although the proportion of macrophage polarization induced by nanocarriers is not as good as LPS, by weakening tumor cells, it can achieve similar killing and clearance effects with fewer macrophages mobilized. Fig.17 b is to select the threshold through ImageJ software and calculate the ratio of the area of ​​non-A20 cell area to the total area of ​​the picture, so as to quantitatively reflect the difference in the ability to eliminate tumor cells. The results show that the Fe3O4@PEI@HA-RGD / E5 nanocarrier group has the strongest killing effect, which is better than the LPS group.

Claims

1. A magnetic nanocarrier modified with PEI and a targeting peptide, characterized in that: The magnetic nanocarrier uses ferroferric oxide nanoparticles as the internal magnetic core, and uses hyaluronic acid HA, polyethyleneimine PEI, polypeptide E5 and cRGD cyclic peptide as surface modifications. The amino acid sequence of the cRGD cyclic peptide is: Arg-Gly-Asp-Tyr-Lys, and the amino acid sequence of the polypeptide E5 is: GGRSFFLLRRIQGCRFRNTVDD.

2. The method for preparing the PEI and targeting peptide modified magnetic nanocarrier according to claim 1, characterized in that: The steps include: (1) Adding Fe3O4@PEI nanocarrier to RGD-HA aqueous solution under ultrasonic and stirring conditions to obtain Fe3O4@PEI@HA-RGD; (2) Fe3O4@PEI@HA-RGD was coupled with polypeptide E5 using carbodiimide EDC and N-hydroxysuccinimide NHS aqueous solution to prepare Fe3O4@PEI@HA-RGD / E5 nanocarrier.

3. The method for preparing the PEI and targeting peptide modified magnetic nanocarrier according to claim 2, characterized in that, In step (1), the Fe3O4@PEI nanocarrier is obtained by adding Fe3O4@DMSA to a PEI aqueous solution under ultrasonic and stirring conditions. The hydrodynamic size of the Fe3O4@DMSA is 20 to 40 nm, and the surface Zeta potential is -20 to -30 mV.

4. The method for preparing the PEI and targeting peptide modified magnetic nanocarrier according to claim 3, characterized in that: The mass ratio of the Fe3O4@DMSA to PEI is 1:1~1:3, the molecular weight of the PEI is 10000 Da~25000 Da, the ultrasonic power is 80~120 W, the mechanical stirring speed is 500~1000 rpm, and after preparation, it is dialyzed through a dialysis bag with a molecular weight of 100 kD for 24~48 hours to complete purification.

5. The method for preparing the PEI and targeting peptide modified magnetic nanocarrier according to claim 3, characterized in that, The RGD-HA aqueous solution in step (1) is coupled with cRGD and HA by adding EDC and NHS, the molecular weight of HA is 5000, the mass ratio of HA to cRGD is 1:1~2:1, the coupling conditions are shaking at 80~150 rpm at 4°C for 16~24 h, and the cRGD coupling rate is 35%~45%.

6. The method for preparing the PEI and targeting peptide modified magnetic nanocarrier according to claim 3, characterized in that: In step (2), the weight ratio of EDC to NHS is greater than or equal to 2:1, the coupling conditions are shaking at 80 to 150 rpm at 4°C for 16 to 24 hours, and the coupling rate of the E5 polypeptide is 30% to 40%.

7. Use of the PEI and targeting peptide modified magnetic nanocarrier according to claim 1 in preparing drugs for treating tumors.

8. The use according to claim 7, characterized in that: The magnetic nanocarrier is loaded with RNA molecules or other drugs, the mass ratio of the magnetic nanocarrier to the RNA molecule is 1:1.5 to 1:9, and the incubation condition is to place at 4 to 7° C. for 20 to 40 min after mixing evenly.

9. The use according to claim 7, characterized in that: The drug targets the CXCR4 membrane protein of tumor cells to degrade the CXCR4 protein, and combines with the RNA of the delivered nucleic acid to jointly downregulate the CXCR4 protein level of tumor cells to inhibit tumor cell migration.

10. The use according to claim 1, characterized in that: The magnetic nanocarrier kills tumor cells by activating the M1 polarization of macrophages.

Images