Nanometer delivery system capable of responding to metabolism driving of lipid in macrophage

Abstract

The invention discloses a nano delivery system responding to lipid metabolism drive in macrophages, the nano delivery system comprises neutrophils, ferritin nanoparticles and micelles, and the ferritin nanoparticles and the micelles are connected to the surfaces of the neutrophils through sulfydryl; the ferritin nanoparticles are obtained by carrying siRNA (small interfering Ribonucleic Acid) and atrivamib on H subunit apoferritin; and the micelle is prepared from DOPE-PEG2000-SH (Dioctyl Olefin Polyethylene Glycol 2000-SH) and HDL ( According to the nano delivery system disclosed by the invention, diseased region delivery is realized by utilizing inflammation targeting of neutrophil, macrophage targeting at an AS plaque part is realized by utilizing active targeting of HFn, and cytoplasm delivery of siNLRP3, Ava and HDL and excretion of cholesterol are realized, so that a synergistic effect is exerted to effectively inhibit foaming of macrophages.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a nanodelivery system driven by the intracellular lipid metabolism of macrophages. Background Art

[0002] Atherosclerosis (AS) is a disease characterized by chronic inflammation and is the pathological basis of cardiovascular and cerebrovascular diseases. At present, statins for reducing blood lipids, anti-inflammatory drugs, and thrombolytic and anticoagulant drugs are commonly used in the clinical treatment of AS. Although drug treatment plays an important role in the treatment of AS, the current clinical treatment strategies have limitations: weak action specificity, poor lesion area enrichment ability, limited efficacy, and strong side effects. Therefore, developing a delivery vector system targeting the characteristics of the lesion area helps to overcome the limitations in clinical practice.

[0003] Endothelial dysfunction leads to the accumulation of oxidized low density lipoprotein (ox-LDL), thereby inducing inflammation. Monocytes are recruited to the inflammatory area and differentiated into macrophages. The aggregation and activation of macrophages in the AS lesion area are positively correlated with the severity of AS. As "scavengers", macrophages clear lipids and debris in the AS lesion site. When macrophages take up excessive ox-LDL, the balance of intracellular lipid metabolism (uptake, esterification, reverse transport and efflux) is disrupted, and lipids accumulate in macrophages to form lipid droplets, turning them into foam cells. Existing studies have shown that the presence of foam cells is a marker for the occurrence and development of atherosclerosis. When foam cells apoptose, a plaque core is formed, and pro-inflammatory cytokines are released, further recruiting monocytes, thus exacerbating AS.

[0004] The ox-LDL taken up by macrophages is hydrolyzed into free cholesterol (Chol) intracellularly. Chol is esterified under the action of acyl-CoA cholesterol acyltransferase 1 (ACAT-1) and transformed into cholesterol esters, which accumulate in macrophages. Cholesterol esters further enhance the ability of macrophages to take up ox-LDL, promoting the formation of foam cells. Studies have shown that during the development of AS, both the expression and activity of ACAT-1 in macrophages increase significantly. The high expression of ACAT-1 promotes lipid metabolism disorders in macrophages. Avaimibe (Ava) is an ACAT-1 inhibitor that has been clinically studied and developed for the treatment of atherosclerosis, indicating that ACAT-1 can be used as an effective therapeutic target for AS. However, previous studies have shown that inhibiting ACAT-1 alone can lead to a large accumulation of free cholesterol in macrophages, triggering an inflammatory response and exacerbating AS. The accumulation of ox-LDL and cholesterol in macrophages can induce the activation of the NLRP3 inflammasome. The pro-inflammatory signaling pathway mediates the activation of Caspase-1 through the assembly of the NLRP3 inflammasome, producing pro-inflammatory cytokines IL-1β and IL-18, resulting in macrophage apoptosis. The IL-18 inflammatory factor inhibits the efflux of free cholesterol in macrophages, further causing lipid metabolism disorders. At the same time, it enhances the sensitivity and migration ability of macrophages to lipid deposition, inducing macrophage foam formation. Previous studies have shown that the expression of the main components of NLRP3 in AS lesion areas increases significantly. Therefore, while treating with an ACAT-1 inhibitor, it is necessary to promote the efflux of intracellularly accumulated free cholesterol. Studies have shown that the accumulation of cholesterol caused by abnormal lipid metabolism in macrophages can significantly induce the overexpression of ABCA1. High-density lipoprotein (HDL) is an endogenous lipoprotein responsible for lipid metabolism in plasma, mainly composed of phospholipids and apolipoprotein A-I (apo A-I), and has an anti-AS effect.

[0005] In summary, the lipid metabolism of ox-LDL in macrophages is interrelated and inseparable. When treating with therapeutic molecules to inhibit the esterification pathway and the NLRP3 inflammatory pathway, it is necessary to promptly excrete the accumulated cholesterol to effectively inhibit macrophage foam formation. To achieve the above "full-chain" synergistic intervention effect, the present invention designs an effective nano-delivery carrier to co-deliver effective target molecules to macrophages at the inflammatory site, thereby effectively inhibiting macrophage foam formation. Summary of the Invention

[0006] The object of the present invention is to provide a nano-delivery system, including neutrophils, ferritin nanoparticles, and micelles, wherein the ferritin nanoparticles and micelles are connected to the surface of neutrophils through sulfhydryl groups; The ferritin nanoparticles are obtained by encapsulating siRNA and evacetrapib with H-subunit apoferritin; The micelles are made of DOPE-PEG2000-SH and HDL.

[0007] Furthermore, the siRNA is siNLRP3.

[0008] The second object of the present invention is to provide a preparation method of the above-mentioned nano-delivery system, including the following steps: Step 1: Take the H-subunit apoferritin solution, adjust the pH to 2.0, add the siRNA solution, evacetrapib solution and CaCl2 solution, incubate, then adjust the solution pH to 8.0, add the Na2HPO4 solution, and incubate to obtain the ferritin nanoparticles HAS encapsulating siRNA and evacetrapib; Among them, the molar ratio of H-subunit apoferritin to siRNA is 1:8 - 1:20, the mass ratio of H-subunit apoferritin to evacetrapib is 1:6 - 1:20, and the mass ratio of H-subunit apoferritin to CaCl2 is 1:10 - 1:50; Step 2: Use the thin film dispersion method to prepare the micelles DPH from DOPE-PEG2000-SH and HDL; Among them, the mass ratio of DOPE-PEG2000-SH to HDL is 1:4 - 1:16; Step 3: Take the nanoparticles HAS and the micelles DPH and mix them with the DTNB solution respectively to obtain DTNB-activated HAS and DTNB-activated DPH. Mix the two and dilute with the culture medium, and then mix with the neutrophil solution and incubate to obtain the nano-delivery system NH-HAS; Among them, the mass ratio of the nanoparticles HAS to DTNB is 1:0.42, the mass ratio of the micelles DPH to DTNB is 1:0.42, and the dosage ratio of DTNB-activated HAS, DTNB-activated DPH to the neutrophil solution is 2 mg / mL: 2 mg / mL: 2.0×10 6 cells / mL.

[0009] The third object of the present invention is to provide the application of the above-mentioned nano-delivery system in the preparation of drugs for treating atherosclerosis.

[0010] The present invention designs and prepares a nano-delivery system (Neu-DPH-HFn@Ava / siNLRP3, NH-HAS) that can respond to lipid metabolism-driven in macrophages. It uses the inflammatory targeting of neutrophils to achieve delivery to the lesion site, and uses the active targeting of HFn to achieve macrophage targeting at the AS plaque site, realizing the cytoplasmic delivery of siNLRP3, Ava and HDL and the efflux of cholesterol, so as to play a synergistic role in effectively inhibiting macrophage foam cell formation.

[0011] The delivery system has the following beneficial effects: (1) The active targeting endocytosis ability driven by lipid metabolism in macrophages at the inflammatory site Research shows that transferrin receptors 1 (TfR1) are highly expressed in macrophages infiltrating the AS lesion area, and the pro-inflammatory factor IL-1β produced by intracellular disordered lipid metabolism can induce an increase in the expression level of TfR1 in macrophages. H-ferritin (HFn) has a high affinity for the TfR1 receptor and can achieve targeted aggregation of nanoparticles in macrophages at the AS lesion site through receptor-mediated endocytosis.

[0012] Endogenous recombinant HFn has the following advantages as a nano-delivery carrier: (1) It has a unique cavity structure and pH-responsive depolymerization-repolymerization self-assembly performance to achieve the loading and release of target molecules; (2) It has a relatively small particle size (about 12 nm), which can avoid phagocytic clearance by the body's RES system; (3) The H subunit can specifically recognize TfR1 and enter the cell through receptor-mediated endocytosis, showing active cell targeting; (4) The outer surface of the HFn protein has abundant groups such as amino groups and carboxyl groups, which is conducive to functional modification. Therefore, HFn can be used as a potential carrier for targeted delivery of AS therapeutic molecules.

[0013] (2) Cholesterol reverse transport and efflux driven by lipid metabolism in macrophages Inflammation caused by lipid metabolism drives effective therapeutic molecules to target and enter macrophages through TfR1. During the treatment with Ava and siNLRP3, abnormal lipid metabolism will cause the accumulation of free cholesterol in the cells, thereby inducing the high expression of ABCA1. HDL will promote the reverse transport and efflux of cholesterol through the ABCA1 pathway, playing an orderly "full-chain" synergistic intervention role. Description of the drawings

[0014] Figure 1 are the identification results of purified HFn. Among them, A is the electrophoretogram of purified HFn measured by 12% SDS-PAGE (M: Marker; 1: supernatant; 2: 300 mM imidazole eluate); B is the ultraviolet scanning spectrum of purified HFn in the range of 200-400 nm.

[0015] Figure 2 In, A is the particle size distribution diagram of HFn measured by a Malvern particle size analyzer; B is the transmission electron micrograph of HFn after negative staining with 2% phosphotungstic acid (scale bar: 100 nm).

[0016] Figure 3In it, A is the TEM image of HAS nanoparticles under different pH conditions during the preparation by the pH change method (scale bar: 100 nm); B is the particle size distribution diagram of HAS nanoparticles during the formation under different pH conditions measured by DLS; C is the zeta potential of HAS nanoparticles during the formation under different pH conditions measured by DLS.

[0017] Figure 4 In it, A is the detection of HCS nanoparticles with different molar ratios (HFn: siRNA) by agarose gel electrophoresis; B is the encapsulation efficiency of siRNA in HAS nanoparticles prepared with different ratios of HFn to siRNA.

[0018] Figure 5 In it, A is the result of detecting HFn and HAS nanoparticles by Native PAGE; B is the CD spectrogram of HFn and HAC nanoparticles.

[0019] Figure 6 In it, A is the cumulative release diagram of siRNA in pH7.4 and pH5.0 buffers (n = 3); B is the cumulative release diagram of Ava in pH7.4 and pH5.0 buffers (n = 3); C is the particle size result diagram of HCS nanoparticles stored for one week at 4 °C and 25 °C.

[0020] Figure 7 In it, A is the investigation of the connection between nanoparticles and neutrophil nanoparticles by CLSM; B is the determination of the marker proteins (1,2,3-Neu; 4,5,6-NH-HAS) on the surface of neutrophils and NH-HAS by WB; C is the inflammatory migration of neutrophils and NH-HAS nanoparticles.

[0021] Figure 8 In it, A is the distribution of Cy7-siRNA, Cy7-Neu, Cy7-HFn and Cy7-NH-HAS nanoparticles in the arteries of ApoE mice fed with a high-cholesterol diet 6 hours after intravenous injection. - / - B is the semi-quantitative analysis of fluorescence values for Figure A using Image J (* p <0.05, ** p <0.01, *** p <0.001); C is the distribution diagram of Cy7-siRNA, Cy7-Neu, Cy7-HFn and Cy7-NH-HAS nanoparticle delivery systems in various tissues and organs (heart, liver, spleen, lung, kidney); D is the semi-quantitative analysis of fluorescence for Figure C using Image J.

[0022] Figure 9 In it, A is for ApoE fed with a high-cholesterol diet after treatment with each preparation. - / -Expression of proteins in the NLRP3 inflammation-related pathway in mouse aorta; B was the semi-quantitative analysis of NLRP3, ASC, Cleaved-caspase1, and p-p65 proteins in Figure A using Image J (compared with Control: ## p <0.05; compared with Model: ** p <0.01).

[0023] Figure 10 Among them, A was the H&E staining photo of the aorta of atherosclerotic mice; B was the quantitative analysis of the ratio of plaque area to total aortic area in Figure C using Image J (** p <0.01, ## p <0.01); C-G were the contents of pro-inflammatory related factors IL-1β, IL-18, TNF-α, MCP-1, and ox-LDL in the aorta (* p <0.05, ** p <0.01, # p <0.05). Detailed implementation manners

[0024] The preferred implementation manners of the present invention will be described in detail below in conjunction with the embodiments. It should be understood that the following embodiments are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0025] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0026] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified. Example 1

[0027] 1. Expression and purification of H-subunit apoferritin (H-apoferritin, HFn) The preparation of HFn was based on the FTH gene coding sequence (NCBI Reference Sequence: NM_002032.2). The CCATGGCT and GCGGCCGC sequences were added to the 5' end and 3' end of the gene coding sequence respectively to introduce NcoI and NotI restriction enzyme recognition sites. Subsequently, this sequence was optimized to ensure its correct expression in the Escherichia coli system, and the final obtained gene sequence had a length of 568 bp. The synthesized sequence was double digested with NcoI and NotI enzymes, and the plasmid vector pET-30a(+) was subjected to the same double digestion treatment. Under the action of T4 DNA ligase, the two digested products were ligated overnight at 4 °C, and finally the pET-30a(+) / FTH recombinant plasmid was obtained. This recombinant plasmid was constructed by Nanjing Genscript Corporation. Next, the construction and screening of FTH positive engineering bacteria were carried out, and the specific process can refer to the "Preparation of recombinant apoferritin by genetic engineering recombinant expression technology" described in Patent CN 110075087 A.

[0028] The bacterial cell supernatant and 300 mM imidazole eluate were analyzed by 12% SDS-PAGE electrophoresis. The results are as Figure 1 shown in A. The results showed that an obvious protein band appeared at 29 kDa, and its molecular weight was basically consistent with the theoretical molecular weight. Next, the ultraviolet characteristic absorption peak of HFn was investigated using an ultraviolet spectrophotometer, and the results are as Figure 1 shown in B. In the range of 200 - 400 nm, a characteristic peak appeared at 280 nm, indicating that there was a large amount of protein in the purified solution. The above results proved that the HFn vector was successfully prepared by genetic engineering method.

[0029] The particle size and potential of HFn protein were measured by DLS. The results showed that the particle size of HFn protein was 16.89 ± 2.12 nm, the PDI was 0.21 ± 0.052, and the zeta potential was -8.12 ± 1.28 mV. The transmission electron microscope results of the HFn nanocage vector are as Figure 2 shown in B. It can be seen from the figure that HFn presented a regular spherical structure with a uniform particle size of about 15 nm. This was consistent with the results measured by the above Malvern laser particle size analyzer, indicating that the recombinant apoferritin nanocage was successfully expressed in the Escherichia coli expression system.

[0030] 2. Preparation and characterization of HFn@Ava / Cap / siNLRP3 (HAS) nanoparticles The pH change method was used to load avaimibe and siNLRP3 (Sangon Biotech). Take 220 μL of the HFn solution at 8 mg / mL, and add 0.1 M HCl solution dropwise to adjust the pH of the solution to 2.0. Then, add 40 μL of the siRNA solution at 8.75 nmol / L, 20 μL of Ava at 5 mg / mL, and 40 μL of the 20 mM CaCl2 solution. After mixing evenly, incubate for 20 min at 25 °C and 200 rpm in a thermostatic mixer. Dropwise add 0.1 M NaOH solution to adjust the pH to 8.0. Add 80 μL of the 10 mM Na2HPO4 solution, mix evenly, and incubate for 2 h at 25 °C and 200 rpm in a thermostatic mixer. Add 400 μL of the RNase A solution at 3 mg / mL and incubate at 37 °C for 30 min to remove the unreacted siNLRP3. Next, ultrafiltration was carried out using a 100KD ultrafiltration tube to remove the unencapsulated Ava.

[0031] During the preparation of HAS nanoparticles, the depolymerization and repolymerization of HFn occurred, and the particle size would change with depolymerization-repolymerization. Therefore, in this invention, DLS was used to investigate the changes in the particle size and zeta potential of HAS nanoparticles under different pH conditions during the preparation process ( Figure 3 B and C in). At the same time, transmission electron microscopy (TEM) was used to investigate the morphology of the nanoparticles under different pH conditions ( Figure 3 A in). It was observed by transmission electron microscopy that when the pH of the solution was adjusted to 2.0, HFn depolymerized from the cage structure into irregular nanoparticles and aggregated, with a significant increase in particle size. The measured particle size was 188.78 ± 2.82 nm, the PDI was 0.16 ± 0.028, and at pH 2.0, its zeta potential was 18.98 ± 1.31 mV, carrying a positive charge. When the pH of the solution was adjusted to 5.0, the particle size of the nanoparticles decreased, and the subunits began to repolymerize and recombine. The nanoparticles had a spherical structure, with a particle size of 86.26 ± 6.42 nm, the PDI was 0.26 ± 0.011, and at this time, the potential was still positive, and the zeta potential was 4.57 ± 1.33 mV. When the pH of the solution was adjusted to 6.0, flocculent precipitates appeared in the solution, and the particle size increased greatly, which might be because the isoelectric point of the HFn protein was reached, resulting in precipitation. As the pH continued to increase, the flocculent precipitates redissolved, and the particle size of the formed nanoparticles decreased greatly. When the pH of the solution was adjusted to 8.0, the HFn protein repolymerized into a complete cage structure, and the particle size of the HAS nanoparticles was 20.53 ± 0.86 nm, indicating the successful repolymerization of HFn, and the zeta potential was -10.79 ± 2.71 mV.

[0032] Next, the encapsulated Ava and siNLRP3 were characterized. First, the encapsulation of siRNA (siNLRP3) was characterized by agarose gel electrophoresis. As Figure 4 shown in A, as the molar ratio of HFn / siRNA increased, the fluorescence intensity of the free siRNA below gradually decreased, indicating that most of the siRNA was encapsulated by HFn and retained in the loading well. The encapsulation efficiency of HAS nanoparticles with different HFn / siRNA molar ratios was quantitatively determined by fluorescence spectrophotometry. The results are as Figure 4 shown in B, among which the HAS nanoparticles prepared at a ratio of 1:10 had the highest encapsulation efficiency for siRNA, and the encapsulation efficiency results were consistent with the Figure 4 qualitative results in A. In native polyacrylamide gel electrophoresis (Native PAGE), protein macromolecules can maintain their intact state and are separated according to the size of the protein molecular weight, the shape of the protein, and the charge carried. Native PAGE electrophoresis was used to investigate whether the configuration of HFn in the prepared HAS nanoparticles changed. The results are as Figure 5 shown in A, HAS had the same band and position as HFn, indicating that the configuration of HFn did not change during the preparation of HAS nanoparticles. Circular dichroism spectroscopy is a special absorption spectroscopy, and the secondary structure of biological macromolecules can be obtained by measuring the CD spectrum of protein biological macromolecules. As can be seen from Figure 5 B, both HFn and HAS nanoparticles showed negative peaks near 208 nm and 221 nm, and positive peaks near 190 nm, showing the characteristic of the classical α-helix conformation. The CD spectral results of HFn and HAS nanoparticles were basically indistinguishable, indicating that the preparation of HAS nanoparticles by the pH change method did not cause a change in the secondary structure of HFn protein.

[0033] Due to the pH depolymerization and recombination characteristics of HFn, its subunits depolymerize under pH 5.0 conditions, and the nanoparticles expand, which is beneficial to the release of the encapsulated drug. Therefore, the in vitro release of siRNA and Ava under pH 5.0 and pH 7.4 conditions was investigated, and the results are as Figure 6 shown in A and B in 6. Under physiological conditions, the release amounts of the encapsulated Ava and siRNA were both very small, indicating that they were stable under physiological conditions and no drug leakage occurred. Under pH 5.0 conditions, the release of siRNA and Ava was significantly accelerated, and the cumulative release amounts both reached more than 80% within 24 h. This indicates that under the acidic conditions of lysosomes, the release of siRNA and Ava encapsulated by HFn can be promoted. Finally, the particle size of HAS nanoparticles stored at 4 °C and room temperature was measured by DLS. The results showed that within 7 days of storage at 4 °C and room temperature, the particle size of HAS nanoparticles did not change, indicating that the prepared HAS nanoparticles were relatively stable.

[0034] 3. Preparation and Characterization of DOPE-PEG@HDL (DPH) Micelles Weigh 20 mg of DOPE-PEG2000-SH and 1.5 mg of HDL into a 15 mL round-bottom flask, dissolve them in methanol, and then sonicate for 1 min to completely dissolve. After removing the organic solvent under reduced pressure using a rotary evaporator, a drug-polymer thin film is formed. Place the flask under a high vacuum pump (<0.2 mbar) overnight to thoroughly dry the film and remove residual methanol. Then rehydrate the obtained dry film with 2 mL of PBS buffer saline. Sonicate the round-bottom flask containing DPH micelles for 5 minutes, then place it in an incubator at 50 °C for 1 hour, then cool to room temperature, and then pass through a 0.22 μm nylon syringe filter. Ultrafilter through a 10KDa ultrafiltration tube to remove unencapsulated HDL.

[0035] 4. Extraction and Characterization of Neutrophils (Neu) Extract neutrophils according to the literature method. Dissolve 3 g of mercaptoacetate in 100 mL of purified water to prepare a 3% mercaptoacetate solution, sterilize it under the conditions of 121 °C and 40 min, and store it at 4 °C. Take a mouse, intraperitoneally inject 2 mL of the above solution, and sacrifice it by decapitation after 5 h. Then soak it in 75% ethanol solution for 5 min and take it out, and place it on a sterile operating table. Again, intraperitoneally inject 3 mL of pre-cooled PBS, and massage the mouse's abdomen for 6 min at the same time. Make a small incision in the abdomen, and use a sterile syringe to deeply penetrate the abdominal cavity from the incision to extract the peritoneal lavage fluid, and use pre-cooled PBS to lavage the abdominal cavity until the extracted lavage fluid becomes a colorless solution. Centrifuge the collected liquid at 1000 r / min for 10 min. Discard the supernatant, add red blood cell lysate to resuspend the cells and let it stand for 10 min. Centrifuge again at 1000 r / min for 10 min, and resuspend the obtained cells in DMEM medium and place them in an incubator for static culture at 37 °C and 5% CO2 for 30 min. Take the upper culture solution and centrifuge it at 1000 r / min for 5 min to obtain suspended neutrophils.

[0036] 5. Preparation and Characterization of NH-HAS Nanoparticles Preparation of DTNB solution: Weigh 0.0396 g of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), place it in a 10 mL brown volumetric flask, add 5 mL of 10 mM Na2HPO4 solution (pH 7.0), sonicate to dissolve and then make up the volume to 10 mL, and store it at 4 °C for later use.

[0037] Mix the nanoparticles with the DNTB solution according to the weight ratios of DPH:DNTB = 1:0.42 and HAS:DNTB = 1:0.42, respectively, and incubate for 2 h at room temperature. Then, ultrafilter using an ultrafiltration tube with a molecular weight cut-off of 8000 Da to remove the unlinked DNTB. Mix the DNTB-linked DPH and HAS in a weight ratio of 1:1. After that, dilute the DNTB-activated nanoparticle mixed solution (with a HAS concentration of 2 mg / mL and a DPH concentration of 2 mg / mL) with 1640 medium. Take 1 mL of the nanoparticle solution and incubate it with 1 mL of the neutrophil solution containing 2.0×10 6 cells / mL at room temperature for 30 min to obtain NH-HAS nanoparticles.

[0038] Prepare a cross-linking reaction solution (NaHCO3, Na2CO3, NaCl), and disperse HAS with the cross-linking reaction solution. Dissolve FITC in an appropriate amount of DMSO solution, add the FITC solution to the above HAS cross-linking reaction solution to label HAS and obtain FITC-HAS. Connect FITC-HAS and Dil-DPH to the surface of Neu by the above method, and observe the surface connection situation using CLSM.

[0039] Resuscitate RAW264.7 cells, and after culturing, seed them in the lower chamber of a Transwell in a 24-well plate (the pore size of the filter membrane is 8 μm), and incubate overnight at 37 °C and 5% CO2. When the confluence of RAW cells reaches 70 - 80%, expose the cells to fresh serum-free 1640 medium containing 20 ng / mL ox-LDL, and select the RAW cells exposed to fresh serum-free medium as the control. After co-incubating for 4 h, add 1 mL of the NH-HAS nanoparticles (1×10 5 cells / mL) suspended in fresh serum-free 1640 medium to the upper chamber of the Transwell chamber. Incubate in an incubator at 37 °C and 5% CO2 for 6 h. Then, take out the upper chamber of the Transwell chamber, gently wipe off the un-migrated Neu cells above the filter membrane with a cotton swab, and stain the cells that have migrated to the lower surface of the membrane with 0.1% crystal violet solution for 10 min, and then observe and take pictures under an optical microscope. After imaging, elute the crystal violet on the chamber membrane with 33% acetic acid solution, and measure the OD value of each group at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0040] DTNB can be linked to sulfhydryl groups. Therefore, DTNB was first linked to HAS nanoparticles and DPH micelles, and then co-incubated with neutrophils, so that HAS and DPH were bonded to the surface of neutrophils via -S-S- bonds. HAS and DPH nanoparticles were labeled with FITC, and FITC was used as a fluorescent probe to indicate their connection with neutrophils. As shown in Figure 7 A, a green fluorescence appeared in the outer circle of the nanoparticles, indicating that the nanoparticles could be successfully linked to the surface of neutrophils. Neutrophils have inflammatory tropism, and their inflammatory tropism depends on the surface characteristic protein CXCR2. Therefore, WB was used to investigate the characteristic proteins on the surface of neutrophils and NH-HAS, and the results are shown in Figure 7 B. The results showed that the connection of HAS and DPH did not affect the expression of the characteristic proteins on the surface of neutrophils. Finally, the inflammatory tropism ability of neutrophils was investigated using Transwell. The results are shown in Figure 7 C. The connection of HAS and DPH did not affect their inflammatory tropism ability.

[0041] 6. In vivo targeting ApoE- / - female mice fed a high-fat diet for 12 weeks were selected to establish an atherosclerotic mouse model. Cy7-siRNA (Sangon Biotech), Cy7-Neu (the extracted neutrophils were co-incubated with Cy7 dye), Cy7-HFn (the liquid of HFn was exchanged using an ultrafiltration tube to 0.1 M sodium bicarbonate at pH 8.5, and Cy7-NHS solution was added in a ratio of HFn:Cy7-NHS = 1:6, and the reaction was shaken in the dark for 2 h, and the unreacted Cy7-NHS was removed by ultrafiltration), and Cy7-NH-HAS nanoparticles (the liquid of HFn was exchanged using an ultrafiltration tube to 0.1 M sodium bicarbonate at pH 8.5, and Cy7-NHS solution was added in a ratio of HFn:Cy7-NHS = 1:6, and the reaction was shaken in the dark for 2 h, and the unreacted Cy7-NHS was removed by ultrafiltration) (dosage: Cy7 1 mg / kg) were administered via the tail vein. After 8 h, the mice were dissected, and PBS perfusion was used to remove the Cy7 not bound to the tissue. The aorta was isolated and the fluorescence intensity at the plaque site in the aorta was qualitatively and quantitatively analyzed again using a small animal in vivo optical imager.

[0042] Free siRNA has a very short half-life in vivo and is easily degraded by nucleases in the body, so it is very easily metabolized and cleared by the system. The neutrophil-based delivery system designed in the present invention has two characteristics in vivo: (1) Neutrophils have inflammatory tropism and can tend to the lesion site; (2) HFn has macrophage targeting at the plaque site. In order to investigate the NH-HAS delivery system in ApoE fed a high-cholesterol diet - / -Active targeting of plaque sites in mice. In this invention, Cy7-siRNA was used as a negative control, and the distribution of Cy7-siRNA in the arteries of mice was evaluated by using small animal in vivo imaging technology. As Figure 8 shown in A and B of Figure 8 , there was no fluorescence in the arteries of the free Cy7-siRNA group, indicating that after the free Cy7-siRNA entered the body, it was quickly cleared, metabolized or degraded, and did not have the property of actively targeting arterial plaque sites. In the Cy7-Neu group, partial fluorescence appeared in the arterial site, which may be due to the inflammatory targeting effect of neutrophils. The inflammatory lesions in the arterial site caused neutrophils to chemotax to the inflammatory site, thus achieving targeting of the lesion site. In the Cy7-HFn group, obvious fluorescence was observed, indicating that HFn could actively target arterial plaques. Finally, enhanced fluorescence intensity was observed in the NH-HAS group, indicating that the binding of Neu and HFn enabled more delivery systems to accumulate in the arterial site, thus achieving better therapeutic effects. In addition, as Figure 8 shown in C and D of Figure 8 , the fluorescence distribution maps of major organs showed that most of the nanoparticles were metabolized through the liver and kidneys, and obvious fluorescence appeared in the liver and kidneys. Among them, the fluorescence intensity in the liver of the Cy7-Neu group was significantly higher than that of the Cy7-HFn and Cy7-NH-HAS groups, and the fluorescence intensity in the kidneys of the Cy7-NH-HAS group was significantly lower than that of the Cy7-Neu group and the Cy7-HFn group, which also indirectly indicated that the accumulation of the delivery system in non-target organs decreased, thus increasing the number of nanoparticles retained in the arterial site. In summary, the prepared NH-HAS delivery system has good targeting to atherosclerotic plaques.

[0043] 7. In vivo pharmacodynamics Refer to the above method steps to prepare nanoparticles HFn@Cap without encapsulating Ava and siNLRP3, and then connect it with Neu through DTNB to obtain an empty carrier (Neu-HFn@Cap).

[0044] Refer to the above method steps to prepare nanoparticles HFn@Ava / Cap / siNLRP3 encapsulating Ava and siNLRP3, and then connect it with Neu through DTNB to obtain N-HAS (Neu-HFn@Ava / Cap / siNLRP3).

[0045] Refer to the above method steps to prepare micelles DPH, and then connect it with Neu through DTNB to obtain NH (Neu@HDL).

[0046] Female ApoE- / - mice were randomly divided into the following 8 groups: (1) Control, (2) Model, (3) siNLRP3, (4) HDL, (5) empty vector (Neu-HFn@Cap), (6) N-HAS (Neu-HFn@Ava / Cap / siNLRP3), (7) NH (Neu@HDL), (8) NH-HAS (Neu@HDL-HFn@Ava / Cap / siNLRP3). The mice were fed a high-fat diet for 18 consecutive weeks. Starting from week 7, the mice in each group were intravenously injected with nanoparticles of different groups. Different preparations were administered weekly (3 mg / kg Ava; 1.0 mg / kg siNLRP3, HDL: 1 mg / kg) for 2 months (once every 3 days). The mice were sacrificed at week 18, serum was collected, and the aortas of the mice were isolated.

[0047] Next, a pharmacodynamic evaluation was carried out in ApoE mice fed a high-cholesterol diet. - / - First, WB was used to determine the protein expression levels of the NLRP3 inflammatory-related pathway in the aorta after treatment with different preparations. The results are as Figure 9 shown. The results showed that the protein expression level of NLRP3 was significantly increased in the model group, while the treatment with free siNLRP3, HDL, empty vector, and NH could not change the protein expression level of NLRP3. After treatment with N-HAS and NH-HAS, the protein expression level of NLRP3 was significantly decreased, indicating that siNLRP3 can effectively enter cells and reach the action site to play a gene interference role with the assistance of the delivery system. At the same time, the protein expression levels of related proteins such as ASC and Caspase1 were measured, and it can be seen from the results that siNLRP3 can effectively inhibit the NLRP3 / ASC / caspase-1 pathway and play an anti-inflammatory role.

[0048] The aorta was frozen-sectioned and stained with H&E. The results are as Figure 10 shown in A. Image J was used to quantify the plaque area. Figure 10 As shown in B, the proportion of collagen fibers in the NH-HAS group was significantly decreased, and the plaque area was significantly decreased, showing a better effect in the treatment of atherosclerosis. At the same time, after treatment with the NH-HAS group, the expression levels of pro-inflammatory factors IL-1β, IL-18, TNF-α, and inflammatory chemokine MCP-1 in the artery were significantly decreased, showing a highly significant difference compared with the model group. The above results confirmed the good effect of the NH-HAS delivery system in the treatment of atherosclerosis.

[0049] In summary, the NH-HAS delivery system can effectively deliver HDL, Ava, and siNLRP3, play a synergistic role, and effectively treat atherosclerosis.

Claims

1. A nano-delivery system, characterized in that, It includes neutrophils, ferritin nanoparticles and micelles, and the ferritin nanoparticles and micelles are connected to the surface of neutrophils through thiol groups; The ferritin nanoparticles are obtained by encapsulating siRNA and evacetrapib with H-subunit apoferritin; The micelles are made of DOPE-PEG2000-SH and HDL.

2. The nano-delivery system according to claim 1, characterized in that, The siRNA is siNLRP3.

3. The preparation method of the nano-delivery system according to claim 1, characterized in that, It includes the following steps: Step 1: Take the H-subunit apoferritin solution, adjust the pH to 2.0, add the siRNA solution, evacetrapib solution and CaCl2 solution, incubate, then adjust the solution pH to 8.0, add the Na2HPO4 solution, and incubate to obtain the ferritin nanoparticles HAS encapsulating siRNA and evacetrapib; Step 2: Use the thin film dispersion method to prepare the micelles DPH from DOPE-PEG2000-SH and HDL; Step 3: Respectively take the nanoparticles HAS and the micelles DPH and mix them with the DTNB solution to obtain DTNB-activated HAS and DTNB-activated DPH. After mixing the two, dilute them with the culture medium, and then mix them with the neutrophil solution and incubate to obtain the nano-delivery system NH-HAS.

4. The preparation method according to claim 3, characterized in that, In Step 1, the molar ratio of H-subunit apoferritin to siRNA is 1:8 - 1:20, the mass ratio of H-subunit apoferritin to evacetrapib is 1:6 - 1:20, and the mass ratio of H-subunit apoferritin to CaCl2 is 1:10 - 1:

50.

5. The preparation method according to claim 3, characterized in that In Step 2, the mass ratio of DOPE-PEG2000-SH to HDL is 1:4 - 1:

16.

6. The preparation method according to claim 3, characterized in that, In Step 3, the mass ratio of nanoparticle HAS to DTNB is 1:0.42, the mass ratio of micelle DPH to DTNB is 1:0.42, and the dosage ratio of DTNB-activated HAS, DTNB-activated DPH to the neutrophil solution is 2 mg / mL: 2 mg / mL: 2.0×10 6 cells / mL.

7. Use of the nano-delivery system according to claim 1 in the preparation of a therapeutic drug for atherosclerosis.

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