Multifunctional biomimetic nanoparticles, methods of making and using the same

Abstract

This invention discloses a multifunctional biomimetic nanoparticle, its preparation method, and its applications. The multifunctional biomimetic nanoparticle 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. The multifunctional biomimetic nanoparticle provided by this invention first synthesizes a biodegradable poly-β-amino ester polymer via a two-step method, then electrostatically combines it with ETS1 therapeutic plasmid DNA to form a PBAE / pDNA nanocore; subsequently, this nanocore is coated with a macrophage membrane to construct the biomimetic nanoparticle; finally, the IL-7R targeting peptide (ITP) is modified on the surface of the macrophage membrane using lipid fusion technology to obtain the final product PBAE / pDNA@ITP-MM.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a multifunctional biomimetic nanoparticle, its preparation method, and its application. Background Technology

[0002] Multiple sclerosis (MS), the leading 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 approximately 2.5 million patients worldwide. After decades of in-depth interdisciplinary research, the scientific community has preliminarily revealed the pathogenesis of MS, clarifying the central role of T cells in triggering autoimmune demyelination. The blood-brain barrier (BBB), a complex structure composed of endothelial cells, astrocytes, pericytes, and other cells, constitutes a physical barrier between the central nervous system and the external environment. It precisely regulates molecular transport, effectively blocking the entry of immune cells, thereby maintaining the homeostasis of the nervous system. Notably, in the early stages of MS, damage to the BBB becomes a gateway for pathogenic T lymphocytes to infiltrate the central nervous system. This series of events triggers a chain reaction of nerve damage and demyelination, ultimately leading to progressive neurological dysfunction in patients. Currently used immunomodulatory drugs (such as interferon-β and teriflunomide) can delay disease progression to some extent, but they have significant limitations: on the one hand, most drugs cannot penetrate intact or damaged BBBs, resulting in insufficient drug concentrations in the central nervous system and failing to effectively inhibit local immune inflammatory responses; on the other hand, existing drugs mostly target single pathological processes (such as broad-spectrum inhibition of T cell activation), failing to simultaneously regulate multiple targets such as BBB repair, inhibition of pathogenic T cell infiltration, and improvement of nerve damage, and long-term use is prone to causing immunosuppressive side effects. Therefore, synchronous regulation of the pathological activities of T cells and endothelial cells is considered a potential strategy for controlling central nervous system immune infiltration and curbing the progression of multiple sclerosis. Our previous research revealed the close link between endothelial-mesenchymal transition (EndMT) and the progression of multiple sclerosis, and pointed out the key role of transcription factor ETS1 in maintaining endothelial cell phenotype and homeostasis.

[0003] Therefore, developing novel drug delivery systems that can precisely target key pathological sites in MS and achieve synergistic treatment through multiple mechanisms has become a core direction for breaking through current treatment bottlenecks. Summary of the Invention

[0004] The main objective of this invention is to propose a multifunctional biomimetic nanoparticle, its preparation method, and its application. The aim is to provide a nanodrug delivery system that, by loading an ETS1 expression plasmid, simultaneously inhibits endothelial-mesenchymal transition and pathogenic differentiation of T cells. Through this multi-target strategy, it comprehensively inhibits immune infiltration of the nervous system, thereby improving the therapeutic effect of multiple sclerosis.

[0005] To achieve the above objectives, the present invention proposes 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] This invention also proposes a method for preparing the multifunctional biomimetic nanoparticles as described above, comprising the following steps:

[0007] 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.

[0008] 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.

[0009] S3. The crude product is precipitated in diethyl ether, washed, and vacuum dried to obtain PBAE polymer;

[0010] 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.

[0011] S5. Wash RAW 264.7 cells, lyse them under hypotonic conditions, centrifuge, wash and resuspend the precipitate to obtain purified macrophage membranes.

[0012] 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.

[0013] 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.

[0014] The described multifunctional biomimetic nanoparticles (IMNPs) use PBAE polymer as a carrier material, load therapeutic plasmid ETS1 DNA (pDNA), and form an outer camouflage by encapsulating macrophage membranes, resulting in a composite nanostructure with excellent biocompatibility and targeting capabilities. Through macrophage membrane encapsulation, this invention not only solves the problem of traditional gene delivery vectors being easily cleared by the immune system, but also significantly improves the enrichment efficiency of nanoparticles in target tissues, enhancing the efficacy of gene therapy.

[0015] Preferably, the particle size of the multifunctional biomimetic nanoparticles is between 50 and 200 nanometers to optimize their circulation time and tissue permeability in living organisms.

[0016] Preferably, in step S1, the molar ratio of 1,4-butanediol diacrylate to 4-amino-1-butanol is (1~1.2):1;

[0017] The reaction is carried out at a temperature of 85-95 °C for 23-25 ​​h, and under light-protected conditions.

[0018] 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 capping agent solution is 0.4~0.6 M.

[0019] Preferably, in step S3, the vacuum drying time is 36-48 hours;

[0020] In step S4, the mass ratio of the PBAE polymer to the pDNA is (3~500):1, for example, the mass ratio of the PBAE polymer to the pDNA is 3:1, 5:1, 10:1, 50:1, 100:1, or 500:1.

[0021] Preferably, in step S4, the PBAE polymer solution and the pDNA solution are mixed at different weight ratios of 3:1, 5:1, 10:1, 50:1, 100:1, and 500:1, and incubated at room temperature to obtain the PBAE / pDNA nanocomposite.

[0022] Preferably, step S5 specifically includes the following steps:

[0023] 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.

[0024] Preferably, in step S6, the weight ratio of the purified macrophage membrane to the PBAE / pDNA nanocomposite is (0.9~1.1):1.

[0025] 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.

[0026] Preferably, the concentration of the targeted peptide solution is 100 μg / mL.

[0027] The present invention also proposes the application of the multifunctional biomimetic nanoparticles as described above or the multifunctional biomimetic nanoparticles prepared by the method described above in the preparation of drugs for treating chronic inflammatory diseases such as multiple sclerosis.

[0028] The multifunctional biomimetic nanoparticles can precisely deliver the ETS1 gene to the central nervous system endothelial cells and infiltrating T lymphocytes at the lesion site through a dual-target delivery mechanism, thereby achieving synergistic, efficient and safe gene expression regulation to achieve therapeutic goals.

[0029] Preferably, the drug is a multifunctional biomimetic nanomedicine that targets both endothelial cells and T cells.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) The multifunctional biomimetic nanoparticles provided by this invention first synthesize biodegradable poly-β-amino ester (PBAE) polymers through a two-step method, and then combine them with ETS1 therapeutic plasmid DNA (pDNA) through electrostatic recombination to form a PBAE / pDNA (NP) nanocore; subsequently, this nanocore is coated with a macrophage membrane to construct biomimetic nanoparticles (MNPs) to significantly enhance their in vivo circulation stability and natural targeting ability to inflammatory sites; finally, IL-7R targeting peptide (ITP) is modified on the surface of macrophage membranes using lipid fusion technology to obtain the final product PBAE / pDNA@ITP-MM (IMNP). This multifunctional biomimetic nanoparticle utilizes the characteristic of endothelial cells and T cells co-expressing IL-7R under inflammatory conditions to achieve simultaneous targeting (dual targeting) of these two key pathological cell types through a single target (ITP). This IMNP system can not only efficiently deliver the ETS1 gene to endothelial cells and T cells at the lesion site, effectively inhibit endothelial-mesenchymal transition (EndMT) and regulate T cell function, but also exert a therapeutic effect by competitively inhibiting the IL-7 signaling pathway, making it particularly suitable for the treatment of chronic inflammatory diseases such as multiple sclerosis.

[0032] (2) The drug for treating chronic inflammatory diseases such as multiple sclerosis provided by this invention uses multifunctional biomimetic nanoparticles, breaking through the limitations of single-target therapy and achieving synergistic systemic treatment: Existing therapies usually target only a single link in the neuroinflammatory pathological process, such as simply inhibiting immune cell activity or strengthening the vascular barrier, which is difficult to effectively block the vicious cycle of mutual aggravation of various links in the disease process. This invention proposes a new strategy of "dual-target synergistic intervention". By designing a single nanodelivery system, it can act simultaneously on the two core cellular targets in the pathogenesis mechanism: pathogenic T lymphocytes and blood-brain barrier endothelial cells. This strategy regulates 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 simultaneous intervention on two key pathological links achieves systemic treatment for chronic inflammatory diseases such as multiple sclerosis, and is expected to produce synergistic and enhanced therapeutic effects.

[0033] (3) The drug for treating chronic inflammatory diseases of multiple sclerosis provided by this invention achieves precise active targeting: The "dual-targeting" capability of this invention does not stem from the mechanical combination of two independent targeting elements, but is based on the ingenious utilization of the disease-specific microenvironment, namely, the discovery that in an inflammatory state, activated T cells and endothelial cells at the lesion site both highly express IL-7R. Accordingly, this invention introduces a targeting peptide that can specifically recognize IL-7R, thereby achieving precise recognition and binding to two different types of target cells using the same target. This targeting strategy based on a shared receptor is a deep exploration and application of the biological mechanisms of the disease, and compared with the inflammatory chemotactic effect that relies solely on carriers (such as macrophage membranes), it has stronger initiative and targeting specificity.

[0034] (4) The drug for treating multiple sclerosis, a chronic inflammatory disease, provided by this invention employs a safer and more controllable gene therapy modality, guiding positive functional remodeling of the body: This invention aims to "upregulate" or "restore" the body's own protective mechanisms by delivering plasmid DNA encoding the ETS1 transcription factor. The expression of ETS1 can simultaneously stabilize the endothelial cell barrier and inhibit pathogenic T cell differentiation, which is an "additive" treatment that guides the body's own functions to "remodel" in a beneficial direction. This strategy is more mild and controllable, avoiding the potential risks of permanent genomic alterations, and is particularly suitable for chronic diseases such as multiple sclerosis that require long-term management, with significantly improved safety.

[0035] (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

[0036] 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.

[0037] Figure 1 The 1H NMR spectrum of the PBAE polymer provided by this invention.

[0038] Figure 2 Fourier transform infrared spectrum of PBAE polymer provided by the present invention.

[0039] Figure 3 The gel permeation chromatography characterization diagram provided by this invention.

[0040] Figure 4 Agarose gel retardation assays of pDNA and PBAE / pDNA nanoassemblies at various mass ratios provided by this invention.

[0041] Figure 5 Agarose gel retardation assays of NP, MNP, and IMNP provided by this invention.

[0042] Figure 6 The protein spectra of macrophage cytoplasm (MC), macrophage membrane (MM), NP, MNP and IMNP provided for this invention.

[0043] 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.

[0044] Figure 8This is a graph showing the stability of pDNA and IMNP provided by the present invention in serum-containing culture medium.

[0045] Figure 9 The following diagrams are provided for the present invention: (A): Electrophoretic bands of the proteins involved in endothelial-mesenchymal transition (ZO-1, Occludin, Vimentin, αSMA) and ETS1 detected by Western blotting; (B) to (F): Statistical results of the quantitative expression of ZO1, Occludin, Vimentin, ETS1 and αSMA, respectively; Data are expressed as mean ± standard error (n = 4).

[0046] Figure 10 Figure 1 shows the expression of ZO-1, Occludin, Vimentin, and αSMA in mouse brain microvascular endothelial cells induced by EndMT according to the present invention after different treatments (control group, IMNP empty vector group, or IMNP treatment group); (A): Immunofluorescence staining observation of the expression levels of ZO-1, Occludin, Vimentin, and αSMA in mouse brain microvascular endothelial cells induced by EndMT after different treatments (control group, IMNP empty vector group, or IMNP treatment group); (B)~(E): Quantitative fluorescence intensity bars for ZO1, Occludin, Vimentin, and αSMA, respectively; the scale bar represents 50 μm, and the data are expressed as mean ± standard error (n = 4).

[0047] Figure 11 The following figures show the transendothelial resistance values ​​of primary mouse brain microvascular endothelial cells induced by EndMT according to the present invention after different treatments (control group, IMNP empty vector group or IMNP treatment group); (A): schematic diagram of blood-brain barrier model; (B): bar chart of transendothelial resistance values ​​of different treatment groups; scale bar represents 50 μm, and data are expressed as mean ± standard error (n = 4).

[0048] Figure 12 Dynamic changes in neurological deficit scores during the 28-day course of the experimental autoimmune encephalomyelitis model mice provided by this invention after intervention with saline, IMNP-EV or IMNP (n=5).

[0049] Figure 13 Representative Locke's Fast Blue stained sections (A) and quantitative analysis diagram (B) of the myelin sheath loss area of ​​each group of mouse lumbar spinal cord provided by the present invention; data are expressed as mean ± standard error (n=5).

[0050] Figure 14 The data provided for this invention are representative hematoxylin-eosin stained sections (A) of the lumbar spinal cord of each group of mice on day 28 and quantitative analysis diagram (B) of the area of ​​inflammatory lesions; the data are expressed as mean ± standard error (n=5).

[0051] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0054] Material descriptions in the following embodiments:

[0055] ETS1 overexpression plasmid (pDNA): purchased from Shanghai Dianjun Biotechnology Co., Ltd., pCDH-CMV-mEts1-EF1a-CopGFP-T2A-Puro plasmid.

[0056] The IL-7R targeting peptide with a hydrophobic tail, DSPE-PEG-ITP, with the core sequence of the targeting peptide being ASACPPH, was purchased from Jier Biochemical Co., Ltd.

[0057] Example 1: Preparation method of multifunctional biomimetic nanoparticles

[0058] 1. Preparation of PBAE:

[0059] S1. The basic monomer 1,4-butanediol diacrylate and the side chain monomer 4-amino-1-butanol were mixed in a molar ratio of 1.1:1 (acrylate group: amino group) and reacted at 90 °C under light-proof and magnetic stirring conditions for 24 h. Polymerization was carried out through Michael addition reaction to obtain the polymer.

[0060] S2. The polymer was dissolved in anhydrous tetrahydrofuran at a concentration of 100 mg / mL to obtain a polymer solution. At the same time, the 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 and on a shaker to obtain the crude product.

[0061] S3. The crude product is precipitated in diethyl ether to remove unreacted monomers. After repeated washing three times and vacuum drying for 40 h, the final PBAE polymer, namely PBAE, is obtained.

[0062] The reaction equation is shown below:

[0063] .

[0064] Figure 1 The 1H NMR spectrum of the PBAE polymer is shown. Figure 2 The image shows the Fourier transform infrared spectrum of the PBAE polymer. Fourier transform infrared spectroscopy characterizes the unique absorption peaks of the PBAE polymer. Figure 3 The image shows the characterization chromatogram by gel permeation chromatography. The number-average molecular weight, weight-average molecular weight, and polydispersity index (PDI) of the polymer 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.

[0065] 2. Preparation of PBAE / pDNA nanocomposites:

[0066] The PBAE polymer solution and the ETS1 overexpression plasmid (pDNA) solution were prepared separately with 25 mM sodium acetate buffer at pH 5.5. Then, the PBAE polymer solution and the pDNA solution were mixed at 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 the PBAE / pDNA nanocomplex (NP).

[0067] Figure 4 Agarose gel retardation assays of pDNA and PBAE / pDNA nanoassemblies at various mass ratios are presented. Figure 4The results showed that when the mass ratio of PBAE / pDNA was 1:1, 3:1, 5:1, and 10:1, the pDNA band could still be seen (although the brightness was weaker), indicating that the PBAE did not fully encapsulate the pDNA at this time, and some unbound pDNA could migrate out of the band. When the mass ratio increased to more than 10:1 (50:1, 100:1, 500:1), the pDNA band disappeared completely, indicating that the PBAE had fully bound (encapsulated) the pDNA, preventing the pDNA from migrating in the gel (i.e., achieving complete blockade).

[0068] 3. Extraction of macrophage cell membranes:

[0069] 1×10 8 RAW 264.7 cells were collected in 15 mL centrifuge tubes, washed twice with cold 1×PBS, resuspended in cell membrane extraction reagent, and then incubated on ice for 10–15 min. After repeated freezing-thawing at room temperature in liquid nitrogen three times, the cells were lysed using hypotonic lysis. 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.

[0070] 4. Preparation of MNP nanoparticles:

[0071] The purified macrophage membrane was sonicated with PBAE / pDNA nanocomposite at a weight ratio of 1:1 for 5 min, and then extruded and filtered using polycarbonate membranes with pore sizes of 400 nm, 200 nm and 100 nm, respectively, to obtain MNP nanoparticles.

[0072] 5. Preparation of multifunctional biomimetic nanoparticles:

[0073] The IL-7R targeting peptide (DSPE-PEG-ITP) with a hydrophobic tail was dissolved in an aqueous solution containing 20% ​​DMSO to a final concentration of 100 μg / mL to obtain the targeting peptide solution. 100 μL of this solution was then added to MNP nanoparticles (the nanoparticles contain 3 × 10⁻⁶ MNP nanoparticles). 7 The macrophage membranes derived from individual cells were mixed and incubated at 37 °C on a shaker for 2 hours to allow the IL-7R targeting peptide to be anchored to the cell membrane via lipid fusion. Unbound DSPE-PEG-ITP was then removed by ultrafiltration. Finally, the modified membrane material was combined with the NP nanocomposite to obtain IMNP nanoparticles, i.e., multifunctional biomimetic nanoparticles.

[0074] Figure 5 The images show agarose gel retardation assays of NP, MNP, and IMNP, where the mass ratio of PBAE polymer to pDNA is 50:1. Figure 5The results show that at this mass ratio, PBAE polymer can effectively bind to pDNA, and the macrophage membrane encapsulation (MNP) and IL-7R targeting peptide modification (IMNP) processes do not disrupt the binding of PBAE to pDNA, and the pDNA remains stably encapsulated in the nanoparticles.

[0075] Figure 6 The protein profiles are of macrophage cytoplasm (MC), macrophage membrane (MM), NP, MNP, and IMNP. Figure 6 The results demonstrate that the macrophage membrane was successfully coated onto the NP surface, and the modification of the IL-7R targeting peptide did not damage the macrophage membrane structure, thus preserving the biomimetic function of the membrane material.

[0076] Figure 7 Microscopic morphology images of NP, MNP and IMNP observed by transmission electron microscopy, with the scale bar representing 100 nm.

[0077] Example 2: Stability Test of Multifunctional Biomimetic Nanoparticles

[0078] (1) Take 200 μL of mouse serum, add 5 μg of free plasmid DNA (pDNA) and the corresponding amount of IMNP nanoparticles, mix well and incubate at 37°C.

[0079] (2) Take out the corresponding samples at 0 h, 3 h, 6 h and 9 h respectively, and heat them in an 80℃ constant temperature oven for 5 minutes to terminate serum activity.

[0080] (3) Take 40 μL of each sample and mix it with an equal volume of loading buffer. Take 15 μL of the mixture and load it into the sample well of a 0.8% agarose gel containing GelRed (prepared with 1×TAE buffer).

[0081] (4) Electrophoresis was performed at a constant voltage of 100 V for 25 minutes, and the bands were then observed using the BioSpectrum chemiluminescence imaging system.

[0082] The results are as follows Figure 8 As shown in the figure, the results indicate that IMNPs degrade more slowly in serum than pDNA, and IMNPs encapsulating pDNA exhibit better stability.

[0083] Example 3: The efficacy of the multifunctional biomimetic nanoparticles prepared in Example 1 of this invention in the treatment of multiple sclerosis.

[0084] 1. Cell-level experiments

[0085] 1.1 Experimental Materials

[0086] Cells: Primary brain microvascular endothelial cells extracted from C57BL / 6 mice.

[0087] Main reagents: Recombinant human TGF-β1 was purchased from MCE Corporation, USA; Multifunctional biomimetic nanoparticles (IMNP) and their empty vector (IMNP-EV) were prepared in our laboratory according to the method in Example 1; Cell culture-related reagents such as phosphate-buffered saline (PBS), DMEM high glucose medium, fetal bovine serum and 0.25% trypsin were all purchased from Thermo Fisher Scientific, USA.

[0088] Main antibodies: The primary antibodies used in the experiment, including anti-αSMA, anti-Vimentin, anti-ZO-1, anti-Occludin and anti-ETS1, as well as the corresponding fluorescently labeled secondary antibodies, were purchased from Abcam (UK) and CST (USA), respectively.

[0089] Main instruments: The main instruments used in the experiment include transendothelial resistance meter, real-time quantitative PCR instrument, Western blot system and confocal microscope.

[0090] 1.2 Experimental Methods

[0091] 1.2.1 Experimental Grouping and Processing

[0092] To accurately evaluate the therapeutic potential and mechanism of action of IMNP, 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:

[0093] Control group (Normal+Ctrl): No special treatment was given.

[0094] TGF-β model group (EndMT+Ctrl): EndMT was induced by adding only TGF-β1 (20 ng / mL).

[0095] IMNP-EV group (EndMT+IMNP-EV): During TGF-β1 stimulation, empty vector nanoparticles (IMNP-EV) without carrying the ETS1 gene were added.

[0096] IMNP treatment group (EndMT+IMNP): TGF-β1 stimulation was combined with the addition of complete multifunctional biomimetic nanoparticles (IMNP) carrying the ETS1 gene.

[0097] Cells from each group were collected at designated time points after appropriate treatment for subsequent testing.

[0098] 1.2.2 Detection Indicators and Methods

[0099] Gene and protein expression detection: Real-time quantitative PCR and Western blotting were used to quantitatively analyze the mRNA and protein expression levels of mesenchymal markers (αSMA, Vimentin), endothelial connexin (ZO-1, Occludin), and ETS1.

[0100] Immunofluorescence staining: The localization and expression of the above indicators in cells were observed using confocal microscopy.

[0101] Blood-brain barrier integrity assessment: Transendothelial resistance values ​​of cells in each group were continuously monitored using a transendothelial resistance meter to assess endothelial barrier function.

[0102] 1.3 Experimental Results

[0103] 1.3.1 IMNP inhibits TGF-β-induced EndMT by upregulating ETS1

[0104] like Figure 9 As shown, Figure 9 (A) Electrophoretic bands showing the expression of endothelial-mesenchymal transition (EMT) related proteins (ZO-1, Occludin, Vimentin, αSMA) detected by Western blotting; (B)-(F) Quantitative statistical results of ZO1, Occludin, Vimentin, ETS1, and αSMA, respectively; this figure was used to evaluate the effect of IMNP in reversing EndMT. The phosphate-buffered saline (PFS) group served as a negative control, and the IMNP empty vector group served as an empty vector control. Protein expression levels were quantified by band gray values ​​and standardized using GAPDH as an internal reference. Data are expressed as mean ± standard error (n = 4). Compared with the control group, TGF-β1 treatment significantly upregulated the expression of mesenchymal markers αSMA and Vimentin, while downregulating the expression of endothelial connexins ZO-1 and Occludin. Notably, TGF-β1 treatment also inhibited ETS1 expression, confirming the successful construction of the EndMT model.

[0105] In the intervention experiment, compared with the TGF-β model group and the IMNP-EV group, the IMNP treatment group could significantly reverse the above phenotypic changes, specifically: decreased expression of mesenchymal markers, restored expression of endothelial connexins, and significantly upregulated expression of ETS1.

[0106] like Figure 10 As shown, Figure 10 (A): Immunofluorescence staining observation of the expression levels of ZO-1, Occludin, Vimentin and αSMA in mouse brain microvascular endothelial cells induced by EndMT after different treatments (control group, IMNP empty vector group or IMNP treatment group). Figure 10 (B)–(E): Quantitative fluorescence intensity bar charts for ZO1, Occludin, Vimentin, and αSMA, respectively. Immunofluorescence staining further confirmed this finding, visually demonstrating that IMNP treatment effectively inhibited the TGF-β-induced EndMT process, while the IMNP-EV group did not show a significant inhibitory effect. These results collectively indicate that the inhibitory effect of IMNP on EndMT is mainly attributed to the ETS1 gene it carries, rather than the nanocarrier itself, revealing the core role of ETS1 in regulating the EndMT process.

[0107] 1.3.2 IMNP improves blood-brain barrier integrity

[0108] To investigate the protective effect of IMNP on blood-brain barrier function under inflammatory conditions, we measured transendothelial resistance values. For example... Figure 11 As shown, (A) is a schematic diagram of the blood-brain barrier model; (B) is a bar chart of transendothelial resistance values ​​in different treatment groups. The results showed that under TGF-β1 stimulation, the IMNP treatment group effectively maintained a higher TEER value, significantly superior to the TGF-β model group and the IMNP-EV group. This indicates that IMNP can significantly improve endothelial barrier function damage induced by inflammatory factors and help maintain the integrity of the blood-brain barrier.

[0109] 2. Animal-level experiments

[0110] (1) Establishment of MS animal model

[0111] 1.1 Experimental Materials

[0112] 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 all purchased from Sigma-Aldrich, USA.

[0113] 1.2 Experimental Methods

[0114] 1.2.1 Experimental Grouping

[0115] To evaluate the therapeutic effect of multifunctional biomimetic nanoparticles (IMNPs), this study used experimental autoimmune encephalomyelitis (EAE) mouse models and randomly divided them into three groups: Model control group (EAE+Ctrl): mice were given saline after EAE induction. Empty vector control group (EAE+IMNP-EV): mice were given empty vector nanoparticles (IMNP-EV) without the ETS1 gene after EAE induction. These nanoparticles retained the ITP functionalized membrane structure to maintain their IL-7R targeting ability and served as a control to exclude nonspecific effects. IMNP treatment group (EAE+IMNP): mice were treated with intact IMNPs carrying the ETS1 gene after EAE induction.

[0116] 1.2.2 EAE Model Induction

[0117] First, prepare a complete Freund's adjuvant (CFA) containing 5 mg / ml of heat-inactivated Mycobacterium tuberculosis H-37Ra. Then, prepare MOG... 35-55 The peptide was dissolved in physiological saline, mixed with an equal volume of CFA, and thoroughly emulsified to prepare MOG with a concentration of 1.5 mg / ml. 35-55 / CFA emulsion. After anesthesia by inhalation of isoflurane (flow rate: 1 L / min), 50 μl of the emulsion was injected subcutaneously at four points on the ventral and dorsal sides of each mouse. Subsequently, on the day of immunization (day 0) and day 2, pertussis toxin (200 ng / mouse, dissolved in 200 μl PBS) was injected intraperitoneally.

[0118] 1.3 Neurological Function Scoring and Animal Ethics

[0119] Following immunization, mice underwent daily double-blind neurological deficit assessments. The scoring criteria were as follows: 0 points, no abnormalities; 0.5 points, distal tail weakness with mild motor abnormalities; 1 point, complete tail weakness; 1.5 points, tail paralysis with hind limb weakness; 2 points, partial paralysis of one hind limb; 2.5 points, partial paralysis of both hind limbs; 3 points, complete paralysis of both hind limbs; 3.5 points, complete hind limb paralysis with partial paralysis of one forelimb; 4 points, complete hind limb paralysis with partial paralysis of both forelimbs; 4.5 points, complete paralysis of all four limbs; 5 points, near death or deceased. This study strictly adhered to animal ethics guidelines. Mice with a neurological function score of 4.0 or higher for more than 48 hours were euthanized, and their final score was recorded as 5.0 in subsequent data analysis.

[0120] 1.4 Histopathological Analysis

[0121] Mouse spinal cord tissue was collected and subjected to Laugh blue (LFB) staining to assess the degree of myelin loss, and hematoxylin and eosin (H&E) staining to observe the overall morphology of the tissue and inflammatory cell infiltration.

[0122] (2) Therapeutic effect of the multifunctional biomimetic nanoparticles prepared in Example 1 on MS animal models

[0123] 2.1 Neurological deficit score results

[0124] like Figure 12 As shown, mice in the model control group (EAE+Ctrl) began to develop symptoms on average on day 9 post-immunization, with neurological deficit scores steadily increasing, peaking on day 18. Compared to the model control group, disease progression was inhibited to some extent in both the empty vector control group (EAE+IMNP-EV) and the IMNP treatment group (EAE+IMNP), specifically manifested as delayed onset, a flatter score increase curve, and a later peak symptom appearance compared to the model control group. The IMNP treatment group showed the most significant improvement.

[0125] 2.2 Histopathological Results

[0126] Myelin sheath integrity analysis: such as Figure 13 As shown, LFB staining results revealed significant demyelination in the spinal cord tissue of the model control group (EAE+Ctrl). The empty vector control group (EAE+IMNP-EV) showed only minor demyelination, while the myelin structure of the IMNP treatment group (EAE+IMNP) remained largely intact, with no obvious demyelinating lesions observed.

[0127] Analysis of inflammatory infiltration: such as Figure 14 As shown, H&E staining results revealed extensive inflammatory cell infiltration in the spinal cord of the model control group (EAE+Ctrl). The degree of inflammatory infiltration was milder in the empty vector control group (EAE+IMNP-EV), while the degree of immune cell infiltration was significantly reduced in the IMNP treatment group (EAE+IMNP).

[0128] 2.3 Conclusion

[0129] The above results demonstrate that the multifunctional biomimetic nanoparticles (IMNPs) prepared in this invention can effectively alleviate the severity of disease in EAE model mice. Their 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) exhibited a milder therapeutic effect, confirming that its surface ITP peptide-mediated IL-7R blockade has an independent contribution to the therapeutic effect. However, the efficacy of the IMNP treatment group was significantly better than that of the model control group and the empty vector control group. In summary, the therapeutic effect of IMNPs does not stem from a single mechanism, but rather is the result of the synergistic effect of its surface ITP peptide-mediated IL-7R targeting / inhibition and its core-delivered ETS1 gene. The combination of these two strategies produces optimal therapeutic efficacy.

[0130] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. The application of a multifunctional biomimetic nanoparticle in the preparation of a drug for treating chronic inflammatory diseases, wherein the drug is a multifunctional biomimetic nanodrug that targets both endothelial cells and T cells, and the chronic inflammatory disease is multiple sclerosis; The multifunctional biomimetic nanoparticles include: PBAE / pDNA nanocomposite, macrophage membrane coated on PBAE / pDNA nanocomposite, and IL-7R targeting peptide anchored on macrophage membrane; The IL-7R targeting peptide can bind to IL-7R on the surface of blood-brain barrier endothelial cells and T cells, mediating the passage of nanoparticles across the blood-brain barrier and protecting the integrity of the blood-brain barrier. The core sequence of the IL-7R targeting peptide is ASACPPH, and the pDNA is an ETS1 overexpression plasmid.

2. The application of the multifunctional biomimetic nanoparticles according to claim 1 in the preparation of drugs for treating chronic inflammatory diseases, characterized in that, The preparation method of the multifunctional biomimetic nanoparticles 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 application of the multifunctional biomimetic nanoparticles according to claim 2 in the preparation of drugs for treating chronic inflammatory diseases, 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 application of the multifunctional biomimetic nanoparticles according to claim 2 in the preparation of drugs for treating chronic inflammatory diseases, 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 application of the multifunctional biomimetic nanoparticles according to claim 2 in the preparation of drugs for treating chronic inflammatory diseases, 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 application of the multifunctional biomimetic nanoparticles according to claim 2 in the preparation of drugs for treating chronic inflammatory diseases, 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 application of the multifunctional biomimetic nanoparticles according to claim 2 in the preparation of drugs for treating chronic inflammatory diseases, characterized in that, In step S6, the weight ratio of the purified macrophage membrane to the PBAE / pDNA nanocomposite is (0.9~1.1):

1.

8. The application of the multifunctional biomimetic nanoparticles according to claim 2 in the preparation of drugs for treating chronic inflammatory diseases, 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.

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