Multifunctional bionic nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a multifunctional bionic nano-particle as well as a preparation method and application thereof. The multifunctional bionic nano-particle comprises a PBAE / pDNA nano-composite, a macrophage membrane coated on the PBAE / pDNA nano-composite, and an IL-7R targeting peptide anchored on the macrophage membrane. The multifunctional bionic nano-particle provided by the invention is prepared by the following steps: firstly, synthesizing a biodegradable poly-beta-amino ester polymer by a two-step method, and then carrying out electrostatic compounding on the biodegradable poly-beta-amino ester polymer and ETS1 therapeutic plasmid DNA to form a PBAE / pDNA nano-core; then, the nano core is coated with a macrophage membrane, and bionic nano particles are constructed; finally, the surface of a macrophage membrane is modified with IL-7R targeting peptide (ITP) through a lipid fusion technology, and a final product PBAE / pDNA (at) ITP-MM is prepared.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceuticals, in particular to a multifunctional biomimetic nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Multiple sclerosis (MS) as the primary chronic inflammatory disease of the central nervous system poses a significant threat to the neurological function of young people and is one of the main causes of non-traumatic neurological dysfunction, affecting about 2.5 million patients worldwide. After decades of interdisciplinary in-depth research, the scientific community has initially revealed the pathogenesis of multiple sclerosis, and it is clear that T cells play a key role in triggering the process of autoimmune demyelination. The blood-brain barrier (BBB), a complex structure composed of endothelial cells, astrocytes, pericytes, etc., constitutes a physical barrier between the central nervous system and the external environment, which precisely regulates molecular transport and effectively blocks the entry of immune cells, thereby maintaining the homeostatic balance of the nervous system. It is worth noting that at the onset of multiple sclerosis, the damage to the blood-brain barrier serves as a gateway for pathogenic T lymphocytes to penetrate into the central nervous system, which in turn triggers a chain reaction of nerve damage and demyelination, ultimately leading to progressive neurological dysfunction in patients. Currently, commonly used immunomodulatory drugs (such as interferon beta, teriflunomide, etc.) can delay disease progression to some extent, but have obvious limitations: on the one hand, most drugs are difficult to penetrate the intact or damaged BBB, resulting in insufficient drug concentration in the central nervous system, which cannot effectively inhibit local immune inflammatory response; on the other hand, existing drugs target a single pathological link (such as broad-spectrum inhibition of T cell activation), which cannot simultaneously regulate the repair of BBB, inhibit the infiltration of pathogenic T cells, and improve nerve damage, and long-term use can easily cause immune suppression-related side effects. Therefore, simultaneous regulation of pathological activities of T cells and endothelial cells is considered a potential strategy to control immune infiltration in the central nervous system and suppress the progression of multiple sclerosis. Our previous research results reveal the close relationship between endothelial to mesenchymal transition (EndMT) and the progression of multiple sclerosis, and point out the key role of transcription factor ETS1 in maintaining endothelial cell phenotype and homeostasis.

[0003] Therefore, the development of a new drug delivery system that can precisely target key sites of MS pathology and achieve multi-mechanism synergistic therapy has become a core direction to break through the current treatment bottleneck. SUMMARY

[0004] The main purpose of the present application is to provide a multifunctional biomimetic nanoparticle and its preparation method and application, aiming to provide a nanomedicine delivery system, which realizes the effect of synchronous inhibition of endothelial mesenchymal transition and pathogenic differentiation of T cells by loading ETS1 expression plasmid, and comprehensively inhibits the immune infiltration of the nervous system through this multi-target strategy, so as to improve the treatment effect of multiple sclerosis.

[0005] In order to achieve the above-mentioned purpose, the present application provides a multifunctional biomimetic nanoparticle, which comprises: a PBAE / pDNA nanocomposite, a macrophage membrane coated on the PBAE / pDNA nanocomposite, and an IL-7R targeting peptide anchored on the macrophage membrane.

[0006] The present application also provides a preparation method of the multifunctional biomimetic nanoparticle as described above, comprising the following steps: S1, mixing a base monomer 1,4-butanediol diacrylate and a side chain monomer 4-amino-1-butanol, stirring, and reacting to obtain a polymer; S2, dissolving the polymer in anhydrous tetrahydrofuran to obtain a polymer solution, dissolving an amino-containing capping agent 1-(3-aminopropyl)-4-methylpiperazine in DMSO to obtain a capping agent solution, mixing the polymer solution and the capping agent solution, and performing a capping reaction to obtain a crude product; S3, precipitating the crude product in diethyl ether, washing, and vacuum drying to obtain a PBAE polymer; S4, mixing the PBAE polymer with a sodium acetate buffer to obtain a PBAE polymer solution, mixing pDNA with a sodium acetate buffer to obtain a pDNA solution, mixing the PBAE polymer solution and the pDNA solution, and incubating to obtain a PBAE / pDNA nanocomposite; S5, washing RAW 264.7 cells, low-osmotic lysis, centrifugation, washing and resuspending the precipitate to obtain purified macrophage membranes; S6, mixing the purified macrophage membranes with the PBAE / pDNA nanocomposite, ultrasonic treatment, and then using a polycarbonate membrane for extrusion filtration to obtain MNP nanoparticles; S7, dissolving the IL-7R targeting peptide with a hydrophobic tail in a DMSO-containing aqueous solution to obtain a targeting peptide solution, then adding the MNP nanoparticles, mixing, incubating, ultrafiltrating to obtain a multifunctional biomimetic nanoparticle.

[0007] The multifunctional bionic nanoparticles (IMNP) are formed by using PBAE polymer as a carrier material, loading therapeutic plasmid ETS1 DNA (pDNA), and forming an outer layer camouflage by wrapping the macrophage membrane, so as to form a composite nanostructure with excellent biocompatibility and targeting property. By wrapping the macrophage membrane, the present application not only solves the problem that the traditional gene delivery carrier is easily removed by the immune system, but also significantly improves the enrichment efficiency of the nanoparticles in the target tissue and enhances the curative effect of gene therapy.

[0008] Preferably, the particle size of the multifunctional bionic nanoparticles is between 50 and 200 nanometers, so as to optimize the circulation time and tissue permeability thereof in the organism.

[0009] Preferably, in step S1, the molar ratio of 1,4-butanediol diacrylate to 4-amino-1-butanol is (1-1.2):1. The reaction temperature is 85-95 ℃, the reaction time is 23-25 h, and the reaction is carried out under light shielding conditions.

[0010] Preferably, in step S2, the concentration of the polymer in the polymer solution is 90-110 mg / mL; and the concentration of 1-(3-aminopropyl)-4-methylpiperazine in the end-capping agent solution is 0.4-0.6 M.

[0011] Preferably, in step S3, the vacuum drying time is 36-48 h. In step S4, the mass ratio of the PBAE polymer to pDNA is (3-500):1, for example, the mass ratio of the PBAE polymer to pDNA is 3:1, 5:1, 10:1, 50:1, 100:1 or 500:1.

[0012] Preferably, in step S4, the PBAE polymer solution and the pDNA solution are mixed in different weight ratios of 3:1, 5:1, 10:1, 50:1, 100:1 or 500:1, and then incubated at room temperature to obtain the PBAE / pDNA nanocomposite.

[0013] Preferably, step S5 specifically comprises the following steps: The RAW 264.7 cells are collected in a centrifuge tube, washed with cold 1xPBS, resuspended with a cell membrane extraction reagent, then incubated in an ice bath for 10-15 min, and then subjected to low osmotic lysis by repeating freezing in liquid nitrogen and thawing at room temperature for 2-4 times, and then centrifuged to remove the cell nuclei and intact cells, and then the supernatant is centrifuged to obtain the cell membrane fragment precipitate, which is resuspended with ddH2O and then centrifuged and washed to obtain the purified macrophage membrane.

[0014] Preferably, in step S6, the weight ratio of the purified macrophage membrane to the PBAE / pDNA nanocomplex is (0.9-1.1):1.

[0015] Preferably, in step S7, the concentration of the targeting peptide solution is 90-110 μg / mL, and the incubation time is 1.5-2.5 h.

[0016] Preferably, the concentration of the targeting peptide solution is 100 μg / mL.

[0017] The application also provides a use of the multifunctional bionic nanoparticle or the multifunctional bionic nanoparticle prepared by the method as described above in the preparation of a drug for treating a chronic inflammatory disease such as multiple sclerosis.

[0018] The multifunctional bionic nanoparticle can precisely introduce the ETS1 gene into the endothelial cells and infiltrated T lymphocytes in the central nervous system at the lesion site through a dual-targeting delivery mechanism, so as to achieve synergistic, efficient and safe gene expression regulation and achieve the treatment purpose.

[0019] Preferably, the drug is a dual-targeting endothelial cell and T cell multifunctional bionic nanoparticle drug.

[0020] Compared with the prior art, the application has the following beneficial effects: (1) The multifunctional bionic nanoparticle provided by the application is first synthesized by a two-step method to obtain a biodegradable poly-β-amino ester (PBAE) polymer, and then the PBAE polymer is electrostatically compounded with an ETS1 therapeutic plasmid DNA (pDNA) to form a PBAE / pDNA (NP) nanocore; then the nanocore is coated with a macrophage membrane to construct a bionic nanoparticle (MNP), so as to significantly improve the in-vivo circulation stability and natural targeting ability of the bionic nanoparticle to the inflammatory site; finally, an IL-7R targeting peptide (ITP) is modified on the surface of the macrophage membrane by a lipid fusion technology to obtain the final product PBAE / pDNA@ITP-MM (IMNP). The multifunctional bionic nanoparticle utilizes the characteristics that the endothelial cells and T cells co-express IL-7R under an inflammatory environment, and realizes the synchronous targeting (dual targeting) of the two types of key pathological cells through a single targeting head (ITP). The IMNP system not only can efficiently deliver the ETS1 gene to the endothelial cells and T cells at the lesion site, effectively inhibit endothelial mesenchymal transition (EndMT) and regulate T cell function, but also can play a therapeutic role by competitively inhibiting the IL-7 signaling pathway, and is particularly suitable for the treatment of chronic inflammatory diseases such as multiple sclerosis.

[0021] (2) The drug for treating multiple sclerosis chronic inflammatory diseases provided by the application adopts multifunctional biomimetic nanoparticles, breaks through the limitation of single target treatment, and realizes synergistic systemic treatment: existing therapies usually only target a single link in the pathological process of nerve inflammation, such as simply inhibiting the activity of immune cells or strengthening the blood vessel barrier, and it is difficult to effectively block the vicious cycle of mutual aggravation of each link in the disease process. The application proposes a new strategy of "double target point synergistic intervention". By designing a single nano delivery system, it can simultaneously act on the two core cell targets in the pathogenesis mechanism of pathogenic T lymphocytes and blood brain barrier endothelial cells. This strategy adjusts the pathogenic differentiation of T cells from the source on the one hand, and directly enhances the barrier function of endothelial cells and inhibits the infiltration of immune cells into the central nervous system on the other hand. This synchronous intervention on two key pathological links realizes the systemic treatment of chronic inflammatory diseases such as multiple sclerosis, and is expected to produce a synergistically enhanced therapeutic effect.

[0022] (3) The drug for treating multiple sclerosis chronic inflammatory diseases provided by the application realizes precise active targeting: the "double targeting" ability of the application is not derived from the mechanical combination of two independent targeting elements, but is based on the clever use of disease-specific microenvironment, that is, it is found that activated T cells and endothelial cells at the lesion site will jointly overexpress IL-7R under inflammatory conditions. Accordingly, the application introduces a targeting peptide capable of specifically recognizing IL-7R, and uses the same target to realize precise recognition and combination of two different target cells. This targeting strategy based on a common receptor is a deep exploration and application of the biological mechanism of the disease, and compared with simply relying on the inflammatory chemotaxis effect of the carrier (such as the macrophage membrane), it has stronger initiative and targeting specificity.

[0023] (4) The drug for treating multiple sclerosis chronic inflammatory diseases provided by the application adopts a safer and more controllable gene therapy mode, and guides the positive remodeling of body function: the application delivers plasmid DNA encoding ETS1 transcription factor, aiming to "up-regulate" or "restore" the protective mechanism existing in the body. The expression of ETS1 can stabilize the endothelial cell barrier and inhibit the pathogenic differentiation of T cells, which is an "additive" treatment that guides the "remodeling" of the body's own function in a beneficial direction. The strategy is more gentle and controllable, avoiding the potential risks of permanent genomic changes, and is especially suitable for chronic diseases such as multiple sclerosis that need long-term management, and the safety is significantly improved.

[0024] (5) The drug for treating chronic inflammatory diseases of multiple sclerosis provided by this invention has both high efficacy and biocompatibility: The multifunctional biomimetic nanomedicine (PBAE / pDNA@ITP-MM, abbreviated as IMNP) constructed in this invention ingeniously combines the advantages of multiple delivery strategies: its macrophage membrane coating endows the nanoparticles with excellent accumulation ability at inflammatory sites and inherent biocompatibility; while the surface-modified IL-7R targeting peptide provides active and precise cell targeting capability. In vitro and in vivo experiments show that this system can efficiently deliver the therapeutic gene (ETS1 plasmid) to target cells, thereby ensuring therapeutic efficacy while minimizing non-specific interactions with non-target tissues, demonstrating good biocompatibility and clinical translation potential. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The 1H NMR spectrum of the PBAE polymer provided by this invention.

[0027] Figure 2 Fourier transform infrared spectrum of PBAE polymer provided by the present invention.

[0028] Figure 3 The gel permeation chromatography characterization diagram provided by this invention.

[0029] Figure 4 Agarose gel retardation assays of pDNA and PBAE / pDNA nanoassemblies at various mass ratios provided by this invention.

[0030] Figure 5 Agarose gel retardation assays of NP, MNP, and IMNP provided by this invention.

[0031] Figure 6 The protein spectra of macrophage cytoplasm (MC), macrophage membrane (MM), NP, MNP and IMNP provided for this invention.

[0032] Figure 7 Microscopic morphology images of NP (A), MNP (B) and IMNP (C) observed by transmission electron microscopy for the present invention; scale bar represents 100 nm.

[0033] Figure 8Figure for stability determination of pDNA and IMNP provided by the present application in serum-containing medium.

[0034] Figure 9 Figure for expression of EndMT-related proteins (ZO-1, Occludin, Vimentin, and αSMA) and ETS1 detected by Western blotting provided by the present application; (A) is a result figure of electrophoresis bands for expression of EndMT-related proteins (ZO-1, Occludin, Vimentin, and αSMA) detected by Western blotting; (B)-(F) are respectively quantitative statistical result figures of ZO1, Occludin, Vimentin, ETS1, and αSMA; data are expressed as mean ± standard error (n=4).

[0035] Figure 10 Figure for expression of ZO-1, Occludin, Vimentin, and αSMA of mouse brain microvascular endothelial cells after EndMT induction and different treatments (control group, IMNP empty vector group, or IMNP treatment group) provided by the present application; (A) is a figure for observation of immunofluorescence staining of expression amount of ZO-1, Occludin, Vimentin, and αSMA of mouse brain microvascular endothelial cells after EndMT induction and different treatments (control group, IMNP empty vector group, or IMNP treatment group); (B)-(E) are respectively fluorescence intensity quantitative columnar charts of ZO1, Occludin, Vimentin, and αSMA; the scale bar represents 50 μm, and data are expressed as mean ± standard error (n=4).

[0036] Figure 11 Figure for determination result of transendothelial electrical resistance value of primary mouse brain microvascular endothelial cells after EndMT induction and different treatments (control group, IMNP empty vector group, or IMNP treatment group) provided by the present application; (A) is a schematic diagram of blood-brain barrier model; (B) is a columnar statistical chart of transendothelial electrical resistance value of different treatment groups; the scale bar represents 50 μm, and data are expressed as mean ± standard error (n=4).

[0037] Figure 12 Figure for dynamic change of neurological deficit scores within 28 days of disease course of experimental autoimmune encephalomyelitis model mice after intervention with normal saline, IMNP-EV, or IMNP provided by the present application (n=5).

[0038] Figure 13 Figure for representative LK fast blue staining sections of lumbar spinal cords of mice in each group (A) and quantitative analysis of demyelination area (B) provided by the present application; data are expressed as mean ± standard error (n=5).

[0039] Figure 14 Figure 28 provides representative hematoxylin-eosin-stained sections of lumbar spinal cord of mice in each group at day 28 (A) and the quantification of inflammatory lesion area (B); data are presented as mean ± SEM (n=5).

[0040] The purposes, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0041] In order to make the purposes, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products which can be purchased in the market. In addition, the technical solutions in each embodiment can be combined with each other, but it should be considered that the combination of the technical solutions does not exist and is not within the protection scope of the present application when the combination of the technical solutions is contradictory or unachievable by the ordinary skilled in the art. Based on the embodiments in the present application, all other embodiments obtained by the ordinary skilled in the art without making creative efforts are within the protection scope of the present application.

[0042] The technical solutions of the present application will be further described in detail below in combination with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present application and do not limit the present application.

[0043] Material description in the following embodiments: ETS1 overexpression plasmid (pDNA): purchased from Shanghai Dianjun Biotechnology Co., Ltd., pCDH-CMV-mEts1-EF1a-CopGFP-T2A-Puro plasmid.

[0044] IL-7R targeting peptide with hydrophobic tail: DSPE-PEG-ITP, the core sequence of the targeting peptide is ASACPPH, purchased from Jier Biochemical Co., Ltd.

[0045] Example 1 Preparation method of multifunctional biomimetic nanoparticles 1. Preparation of PBAE: S1, the base monomer 1,4-butanediol diacrylate and the side chain monomer 4-amino-1-butanol are mixed in a molar ratio of 1.1:1 (acrylate group: amino), and the polymerization is carried out by Michael addition reaction under the conditions of 90 ℃, light shielding and magnetic stirring for 24 h, to obtain the polymer; S2, the polymer was dissolved in anhydrous tetrahydrofuran at a concentration of 100 mg / mL to obtain a polymer solution, and an amino-containing capping agent 1-(3-aminopropyl)-4-methylpiperazine was dissolved in DMSO at a concentration of 0.5 M to obtain a capping agent solution, the polymer solution and the capping agent solution were mixed, and the capping reaction was carried out at room temperature under the condition of a shaking table to obtain a crude product; S3, the crude product was precipitated in diethyl ether to remove unreacted monomers, and after repeated washing for 3 times and vacuum drying for 40 h, the final PBAE polymer, i.e., PBAE, was obtained.

[0046] The reaction equation is as follows: .

[0047] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the PBAE polymer is shown in FIG. 1; Figure 2 The Fourier infrared spectrum of the PBAE polymer is shown in FIG. 2, and the Fourier infrared spectrum characterizes the absorption peaks specific to the PBAE polymer; Figure 3 The gel permeation chromatography characterization diagram is shown in FIG. 3, and the number average molecular weight and weight average molecular weight of the polymer and the polydispersity coefficient were determined, the PDI of the polymer molecule was 1.12, the number average molecular weight was 11.75 kDa, and the weight average molecular weight was 13.13 kDa.

[0048] 2. Preparation of PBAE / pDNA nanocomplexes: The PBAE polymer solution and the ETS1 overexpression plasmid (pDNA) solution were respectively prepared with 25 mM pH 5.5 sodium acetate buffer, and then the PBAE polymer solution and the pDNA solution were mixed in different weight ratios of 1:1, 3:1, 5:1, 10:1, 50:1, 100:1 and 500:1, and incubated at room temperature for 30 min to obtain PBAE / pDNA nanocomplexes (NP).

[0049] Figure 4 The agarose gel retardation assay diagrams of pDNA and PBAE / pDNA nanocomplexes at various mass ratios are shown in FIG. 4, and the results are shown in FIG. 5. Figure 4 As can be seen from the results of FIG. 5, when the mass ratio of PBAE / pDNA is 1:1, 3:1, 5:1 and 10:1, the band of pDNA can still be seen (only the brightness is weaker), which indicates that at this time the wrapping of PBAE on pDNA is not sufficient, and part of the unbound pDNA can move out of the band; when the mass ratio is increased to 10:1 or more (50:1, 100:1 and 500:1), the band of pDNA completely disappears, which indicates that PBAE has fully combined (wrapped) pDNA, so that pDNA cannot move in the gel (i.e., complete retardation is achieved).

[0050] 3. Extraction of macrophage membrane: 1 x 10 8 RAW 264.7 cells were collected in 15 mL centrifuge tubes, washed twice with cold 1 x PBS, resuspended with cell membrane extraction reagent, then incubated in ice bath for 10-15 min, and then repeated 3 times of low permeability lysis by liquid nitrogen freezing- room temperature thawing, then centrifuged to remove the cell nucleus and intact cells, and then the supernatant was centrifuged to obtain the cell membrane fragment precipitate, which was resuspended with ddH2O and then centrifuged and washed to obtain the purified macrophage membrane.

[0051] 4. Preparation of MNP nanoparticles: The purified macrophage membrane and the PBAE / pDNA nanocomplex were ultrasonically treated at a weight ratio of 1:1 for 5 min, and then extrusion filtered using polycarbonate membranes with pore sizes of 400 nm, 200 nm and 100 nm in sequence to obtain MNP nanoparticles.

[0052] 5. Preparation of multifunctional biomimetic nanoparticles: The IL-7R targeting peptide (DSPE-PEG-ITP) with a hydrophobic tail was dissolved in an aqueous solution containing 20% DMSO at a final concentration of 100 μg / mL to obtain a targeting peptide solution, 100 μL of which was mixed with MNP nanoparticles (containing 3 x 10 7 macrophage membranes derived from cells) to obtain a mixture, which was incubated at 37 ℃ on a shaker for 2 hours to allow the IL-7R targeting peptide to be anchored on the cell membrane through lipid fusion; then unbound DSPE-PEG-ITP was removed by ultrafiltration, and finally the modified membrane material was combined with the NP nanocomplex to obtain IMNP nanoparticles, i.e. multifunctional biomimetic nanoparticles.

[0053] Figure 5 The results of the agarose gel retardation assay of NP, MNP and IMNP, in which the mass ratio of PBAE polymer to pDNA was 50:1, Figure 5 indicate that under this mass ratio, PBAE polymer and pDNA can be effectively combined, and the processes of macrophage membrane wrapping (MNP) and IL-7R targeting peptide modification (IMNP) do not destroy the combination of PBAE and pDNA, and pDNA is still stably wrapped in the nanoparticles.

[0054] Figure 6 The results of the protein spectrum of macrophage cytoplasm (MC), macrophage membrane (MM), NP, MNP and IMNP, Figure 6 prove that the macrophage membrane is successfully wrapped on the surface of NP, and the modification of IL-7R targeting peptide does not destroy the membrane structure of the macrophage membrane, and the biomimetic function of the membrane material is retained.

[0055] Figure 7 Figure 16 shows the micro-morphology of NPs, MNPs and IMNPs observed by transmission electron microscopy, with a scale bar representing 100 nm.

[0056] Example 2 Stability test of multifunctional biomimetic nanoparticles (1) 200 μL of mouse serum was taken and 5 μg of free plasmid DNA (pDNA) and a corresponding amount of IMNP nanoparticles were added, respectively, and mixed, and then incubated at 37°C.

[0057] (2) The corresponding samples were taken at 0 h, 3 h, 6 h and 9 h, respectively, and placed in a 80°C incubator for 5 minutes to terminate the serum activity.

[0058] (3) 40 μL of each sample was mixed with an equal volume of loading buffer, and 15 μL of the mixture was loaded into a sample well containing a 0.8% agarose gel (1xTAE buffer) with GelRed.

[0059] (4) Electrophoresis was performed at a constant voltage of 100 V for 25 minutes, and then the bands were observed using a BioSpectrum chemiluminescence imaging system.

[0060] The results are shown in Figure 17. Figure 8 As can be seen from the results of the figure, IMNP degrades more slowly than pDNA in serum, and IMNP encapsulating pDNA has better stability.

[0061] Example 3 Efficacy of multifunctional biomimetic nanoparticles prepared in Example 1 of the application for treating multiple sclerosis 1. Cell level experiment 1.1 Experimental materials Cells: primary brain microvascular endothelial cells extracted from C57BL / 6 mice.

[0062] Main reagents: recombinant human TGF-β1 was purchased from MCE, USA; multifunctional biomimetic nanoparticles (IMNP) and its empty carrier (IMNP-EV) were prepared by the laboratory according to the method of Example 1; phosphate buffered saline (PBS), DMEM high glucose medium, fetal bovine serum and 0.25% trypsin and other cell culture related reagents were purchased from Thermo Fisher Scientific, USA.

[0063] Main antibodies: the primary antibodies used in the experiment, including anti-αSMA, anti-Vimentin, anti-ZO-1, anti-Occludin and anti-ETS1, and the corresponding fluorescently labeled secondary antibodies, were purchased from Abeam, UK and CST, USA, respectively.

[0064] Main instruments: the main instruments used in the experiment include transendothelial electrical resistance meter, real-time fluorescent quantitative PCR instrument, Western blot system and confocal microscope.

[0065] 1.2 Experimental Methods 1.2.1 Experimental Grouping and Treatment To accurately evaluate the therapeutic potential of IMNP and its mechanism of action, we established an in vitro endothelial-mesenchymal transition (EndMT) model by stimulating primary brain microvascular endothelial cells with TGF-β1 and set up the following experimental groups: Control group (Normal + Ctrl): No special treatment.

[0066] TGF-β model group (EndMT + Ctrl): Only TGF-β1 (20 ng / mL) was added to induce EndMT.

[0067] IMNP-EV group (EndMT + IMNP-EV): While TGF-β1 was stimulated, empty vector nanoparticles (IMNP-EV) carrying no ETS1 gene were added.

[0068] IMNP treatment group (EndMT + IMNP): While TGF-β1 was stimulated, complete multifunctional biomimetic nanoparticles (IMNP) carrying ETS1 gene were added.

[0069] After the corresponding treatment, cells in each group were collected at the designated time points for subsequent detection.

[0070] 1.2.2 Detection Indicators and Methods Gene and protein expression detection: Real-time fluorescent quantitative PCR and Western blotting were used to quantitatively analyze the mRNA and protein expression levels of mesenchymal markers (αSMA, Vimentin), endothelial junction proteins (ZO-1, Occludin), and ETS1.

[0071] Immunofluorescence staining: The localization and expression of the above indicators in cells were observed by confocal microscopy.

[0072] Blood-brain barrier integrity assessment: The transendothelial electrical resistance values of cells in each group were continuously monitored using a transendothelial electrical resistance meter to assess endothelial barrier function.

[0073] 1.3 Experimental Results 1.3.1 IMNP Inhibits TGF-β-induced EndMT by Upregulating ETS1 As shown in Figure 9 , Figure 9(A): Western blotting results of EndMT-related proteins (ZO-1, Occludin, Vimentin, aSMA) expression; (B)-(F): Quantitative results of ZO1, Occludin, Vimentin, ETS1, aSMA expression, respectively; this figure is used to evaluate the effect of IMNP on reversing EndMT. Phosphate buffer treatment group as negative control, IMNP empty vector group as empty vector control. The protein expression level was quantified by band gray value, and GAPDH was used as internal reference for standardization. The data are expressed as mean ± standard error (n = 4). Compared with the control group, TGF-β1 treatment significantly up-regulated the expression of mesenchymal markers aSMA and Vimentin, while down-regulated the expression of endothelial junction proteins ZO-1 and Occludin. Notably, TGF-β1 treatment also inhibited the expression of ETS1, which confirmed the successful construction of the EndMT model.

[0074] In the intervention experiment, compared with the TGF-β model group and the IMNP-EV group, the IMNP treatment group can significantly reverse the above-mentioned phenotype changes, which is manifested as: the expression of mesenchymal markers is reduced, the expression of endothelial junction proteins is restored, and the expression of ETS1 is significantly up-regulated.

[0075] As Figure 10 shown, Figure 10 (A): Immunofluorescence staining observation of ZO-1, Occludin, Vimentin and aSMA expression in mouse brain microvascular endothelial cells after EndMT induction and different treatments (control group, IMNP empty vector group or IMNP treatment group); Figure 10 (B)-(E): Fluorescence intensity quantitative column chart of ZO1, Occludin, Vimentin, aSMA, respectively. Immunofluorescence staining results further confirmed this finding, which directly showed that IMNP treatment effectively inhibited the TGF-β-induced EndMT process, while the IMNP-EV group did not show obvious inhibitory effect. These results collectively indicate that the inhibitory effect of IMNP on EndMT is mainly due to the ETS1 gene it carries, rather than the nano-carrier itself, revealing the core role of ETS1 in regulating the EndMT process.

[0076] 1.3.2 IMNP improves blood brain barrier integrity To explore the protective effect of IMNP on blood brain barrier function under inflammatory conditions, we detected the transendothelial electrical resistance value. As Figure 11As shown, (A): is a schematic diagram of blood brain barrier model; (B): is a columnar statistical chart of transendothelial electrical resistance value of different treatment groups. The results show that under the stimulation of TGF-β1, the IMNP treatment group can effectively maintain a higher TEER value, which is significantly better than the TGF-β model group and the IMNP-EV group. This indicates that IMNP can significantly improve the endothelial barrier function damage induced by inflammatory factors, and help to maintain the integrity of the blood brain barrier.

[0077] 2. Animal level experiment (1) Establishment of MS animal model 1.1 Experimental materials C57BL / 6 male mice (8 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. Heat-inactivated Mycobacterium tuberculosis H-37Ra, Freund's complete adjuvant and pertussis toxin were purchased from Sigma Company, USA.

[0078] 1.2 Experimental method 1.2.1 Experimental grouping To evaluate the therapeutic effect of multifunctional biomimetic nanoparticles (IMNP), this study used experimental autoimmune encephalomyelitis (EAE) model mice, which were randomly divided into the following three groups: model control group (EAE+Ctrl): physiological saline was given after EAE induction. Empty vector control group (EAE+IMNP-EV): empty vector nanoparticles (IMNP-EV) without carrying ETS1 gene were given after EAE induction. This particle retains the ITP functional membrane structure to maintain its IL-7R targeting ability, which is used as a control to exclude non-specific effects. IMNP treatment group (EAE+IMNP): complete IMNP carrying ETS1 gene was given for treatment after EAE induction.

[0079] 1.2.2 EAE model induction First, prepare complete Freund's adjuvant (CFA) containing 5 mg / ml heat-inactivated Mycobacterium tuberculosis H-37Ra. MOG 35-55 After dissolving the polypeptide in physiological saline, mix it with an equal volume of CFA and emulsify it thoroughly to prepare a MOG 35-55 / CFA emulsion with a concentration of 1.5 mg / ml. After anesthesia by isoflurane inhalation (flow rate: 1 L / min), subcutaneous injection was performed at 4 points on the ventral and dorsal sides of each mouse, with 50 μl of emulsion injected at each point. Subsequently, on the day of immunization (day 0) and day 2, pertussis toxin (200 ng per mouse, dissolved in 200 μl PBS) was injected intraperitoneally.

[0080] 1.3 Neurological function score and animal ethics Blind neurological deficit score was performed daily after immunization. The scoring criteria were as follows: 0, no abnormality; 0.5, weakness of the distal tail with mild movement abnormality; 1, complete weakness of the tail; 1.5, tail paralysis with weakness of the hind limbs; 2, partial paralysis of the unilateral hind limbs; 2.5, partial paralysis of the bilateral hind limbs; 3, complete paralysis of the bilateral hind limbs; 3.5, complete hind limb paralysis with partial paralysis of the unilateral forelimb; 4, complete hind limb paralysis with partial paralysis of the bilateral forelimb; 4.5, complete paralysis of the four limbs; 5, moribund or death. The study strictly abided by the animal ethics standards. If the mouse neurological score reached 4.0 and lasted more than 48 hours, humane euthanasia was performed, and its final score was recorded as 5.0 in subsequent data statistics.

[0081] 1.4 Histopathological analysis The spinal cord tissues of mice were taken for LFB staining to assess the degree of demyelination and H&E staining to observe the overall morphology of the tissues and inflammatory cell infiltration.

[0082] (2) Therapeutic effect of multifunctional biomimetic nanoparticles prepared in Example 1 on MS animal models 2.1 Neurological deficit score results As shown in Figure 12 , the model control group (EAE+Ctrl) mice began to develop the disease on average on the 9th day after immunization, and the neurological deficit score continued to rise, reaching a peak on the 18th day. Compared with the model control group, the disease progression of the empty vector control group (EAE+IMNP-EV) and the IMNP treatment group (EAE+IMNP) was inhibited to a certain extent, which was manifested as delayed onset, a more gentle score rising curve, and a later peak of symptoms than the model control group. Among them, the improvement effect of the IMNP treatment group was the most significant.

[0083] 2.2 Histopathological results Myelin integrity analysis: As shown in Figure 13 , the LFB staining results showed that the spinal cord tissue of the model control group (EAE+Ctrl) had significant demyelination. The empty vector control group (EAE+IMNP-EV) only had a small amount of demyelination, while the myelin structure of the IMNP treatment group (EAE+IMNP) was basically intact, and no obvious demyelination was observed.

[0084] Inflammatory infiltration analysis: As shown in Figure 14 , the H&E staining results showed that there were a large number of inflammatory cell infiltrations in the spinal cord of the model control group (EAE+Ctrl). The inflammatory infiltration of the empty vector control group (EAE+IMNP-EV) was lighter, while the immune cell infiltration of the IMNP treatment group (EAE+IMNP) was significantly reduced.

[0085] 2.3 Conclusion The above results show that the multifunctional biomimetic nanoparticles (IMNP) prepared by the present application can effectively alleviate the disease severity of EAE model mice, and its mechanism of action includes reducing myelin damage, promoting myelin repair, and significantly inhibiting inflammatory cell infiltration in the central nervous system. Notably, compared with the model control group, the empty vector control group (EAE+IMNP-EV) showed a mild therapeutic effect, confirming that the IL-7R blockade mediated by the ITP peptide on the surface has an independent contribution to the therapeutic effect. However, the therapeutic effect of the IMNP treatment group is significantly better than that of the model control group and the empty vector control group. In summary, the therapeutic effect of IMNP is not due to a single mechanism, but rather the result of the synergistic effect of ITP peptide-mediated IL-7R targeting / inhibition on its surface and ETS1 gene delivery in its core. The combination of these two strategies produces the best therapeutic efficacy.

[0086] The above merely describes the preferred embodiments of the present application, and is not intended to limit the patent scope of the present application. Various modifications and changes can be made to the present application by those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the patent protection scope of the present application.

Claims

1. A multifunctional biomimetic nanoparticle, characterized in that, The multifunctional biomimetic nanoparticles include: a PBAE / pDNA nanocomposite, a macrophage membrane coated on the PBAE / pDNA nanocomposite, and an IL-7R targeting peptide anchored on the macrophage membrane.

2. A method for preparing multifunctional biomimetic nanoparticles as described in claim 1, characterized in that, Includes the following steps: S1. Mix the basic monomer 1,4-butanediol diacrylate with the side-chain monomer 4-amino-1-butanol, stir, and react to obtain the polymer. S2. Dissolve the polymer in anhydrous tetrahydrofuran to obtain a polymer solution. Simultaneously, dissolve the amino-containing capping agent 1-(3-aminopropyl)-4-methylpiperazine in DMSO to obtain a capping agent solution. Mix the polymer solution and the capping agent solution to carry out a capping reaction and obtain a crude product. S3. The crude product is precipitated in diethyl ether, washed, and vacuum dried to obtain PBAE polymer; S4. Mix PBAE polymer with sodium acetate buffer to obtain PBAE polymer solution, mix pDNA with sodium acetate buffer to obtain pDNA solution, mix PBAE polymer solution and pDNA solution, incubate to obtain PBAE / pDNA nanocomposite. S5. Wash RAW 264.7 cells, lyse them under hypotonic conditions, centrifuge, wash and resuspend the precipitate to obtain purified macrophage membranes. S6. The purified macrophage membrane was mixed with the PBAE / pDNA nanocomposite, sonicated, and then extruded and filtered using a polycarbonate membrane to obtain MNP nanoparticles. S7. Dissolve the IL-7R targeting peptide with a hydrophobic tail in an aqueous solution containing DMSO to obtain a targeting peptide solution. Then add MNP nanoparticles, mix well, incubate, and ultrafilter to obtain multifunctional biomimetic nanoparticles.

3. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of 1,4-butanediol diacrylate to 4-amino-1-butanol is (1~1.2):1; The reaction is carried out at a temperature of 85~95 ℃ for 23~25 h, and under light-protected conditions.

4. The preparation method according to claim 2, characterized in that, In step S2, the concentration of the polymer in the polymer solution is 90~110 mg / mL; the concentration of 1-(3-aminopropyl)-4-methylpiperazine in the capping agent solution is 0.4~0.6 M.

5. The preparation method according to claim 2, characterized in that, In step S3, the vacuum drying time is 36-48 hours; In step S4, the mass ratio of the PBAE polymer to pDNA is (3~500):

1.

6. The preparation method according to claim 2, characterized in that, Step S5 specifically includes the following steps: RAW 264.7 cells were collected in centrifuge tubes, washed with cold 1×PBS, resuspended in cell membrane extraction reagent, and then incubated on ice for 10–15 min. After repeated 2–4 cycles of liquid nitrogen freezing-room temperature thawing, the cells were lysed under hypotonic conditions. The cells were then centrifuged to remove the nuclei and intact cells. The supernatant was centrifuged to obtain cell membrane fragments. The precipitate was resuspended in ddH2O, centrifuged, and washed to obtain purified macrophage cell membranes.

7. The preparation method according to claim 2, characterized in that, In step S6, the weight ratio of the purified macrophage membrane to the PBAE / pDNA nanocomplex is (0.9~1.1):

1.

8. The preparation method according to claim 2, characterized in that, In step S7, the concentration of the targeting peptide solution is 90~110 μg / mL, and the incubation time is 1.5~2.5 h.

9. The use of a multifunctional biomimetic nanoparticle as described in claim 1 or a multifunctional biomimetic nanoparticle prepared by the method described in any one of claims 2-8 in the preparation of a medicament for treating chronic inflammatory diseases.

10. The application according to claim 9, characterized in that, The drug is a multifunctional biomimetic nanomedicine that targets both endothelial cells and T cells.

11. The application according to claim 9, characterized in that, The chronic inflammatory disease mentioned is multiple sclerosis.

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