An engineered nano-vesicle of artemisia annua targeting macrophages, and a preparation method and application thereof
By modifying the surface of Artemisia annua nanovesicles with DSPE-PEG-CRV targeting peptides, engineered nanovesicles targeting M1 macrophages were prepared, solving the targeting and immunogenicity problems of Artemisia annua nanovesicles in the treatment of rheumatoid arthritis. This enabled precise delivery and metabolic reprogramming of M1 macrophages, significantly improving disease symptoms.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANJIN HOSPITAL
- Filing Date
- 2026-06-11
- Publication Date
- 2026-07-14
AI Technical Summary
Existing natural Artemisia annua nanovesicles lack lesion targeting, resulting in low effective concentrations in the treatment of rheumatoid arthritis, making it difficult to target M1 macrophages, and existing targeting methods have the problem of high immunogenicity risk.
By modifying the surface of natural Artemisia annua nanovesicles with DSPE-PEG-CRV targeting peptides, engineered Artemisia annua nanovesicles with a particle size of 100-200 nm and a negative charge were prepared, achieving specific binding and targeted delivery to M1 macrophages.
It achieves precise targeting of M1 macrophages, inhibits their inflammatory polarization and metabolism, reduces CD86 expression, promotes CD206 expression, interrupts NAD+ metabolism, reduces glucose uptake and lactic acid secretion, and significantly improves rheumatoid arthritis symptoms.
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Figure CN122376772A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically an engineered Artemisia annua nanovesicle targeting macrophages, its preparation method, and its application. Background Technology
[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis, cartilage, and bone destruction. Its pathogenesis is complex, but the core pathological element of RA is currently recognized as the abnormal polarization and metabolic remodeling of macrophages in the synovial microenvironment. Specifically, in the hypoxic, hyperinflammatory synovial microenvironment of RA, infiltrating macrophages undergo abnormal polarization toward the M1 type. These M1 macrophages are highly dependent on aerobic glycolysis (Warburg effect) for energy, consuming large amounts of glucose and secreting lactate and pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). This not only directly drives the abnormal proliferation of synovial fibroblasts but also leads to bone erosion through osteoclast activation. Therefore, targeted intervention of the abnormal energy metabolism of M1 macrophages, particularly blocking the rate-limiting step in their glycolysis pathway—NAD+ salvage synthesis—has become a highly promising new strategy for RA treatment.
[0003] In recent years, plant-derived nanovesicles (PDNVs) have attracted much attention due to their naturally low immunogenicity, excellent biocompatibility, and inherent pharmacological activity. Artemisia annua, a traditional Chinese medicine for anti-inflammatory purposes, contains nanovesicles (AELNs) extracted from it, which are rich in anti-inflammatory active substances (such as artemisinin and its derivatives, and specific miRNAs), and have potential therapeutic value for rheumatoid arthritis (RA).
[0004] However, existing natural Artemisia annua nanovesicles have a fatal flaw: a lack of lesion targeting. After systemic administration, unmodified AELNs are easily captured and cleared by the reticuloendothelial system (mononuclear-macrophage system) of organs such as the liver and spleen, resulting in extremely low effective concentrations reaching the synovium of RA-affected joints, severely limiting their in vivo anti-inflammatory efficacy. How to endow AELNs with the ability to target M1 macrophages at RA lesion sites, while simultaneously utilizing their own active ingredients to regulate the metabolic reprogramming of macrophages, is a pressing technical challenge that needs to be addressed in this field.
[0005] While there have been attempts to target macrophages using artificially synthesized liposomes or exosomes in existing technologies, these usually involve complex chemical synthesis or engineering modifications, which pose a high risk of immunogenicity. Furthermore, there are few reports of modifying peptides targeting M1 macrophages onto the surface of Artemisia annua natural nanovesicles and further exploring their synergistic effects in RA treatment through metabolic reprogramming.
[0006] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention
[0007] The purpose of this invention is to provide an engineered Artemisia annua nanovesicle that targets macrophages, its preparation method, and its application, in order to solve the problems in the prior art.
[0008] To achieve the above objectives, the present invention provides the following technical solution: an engineered Artemisia annua nanovesicle targeting macrophages, wherein the engineered Artemisia annua nanovesicle includes natural nanovesicles derived from Artemisia annua, and a targeting polypeptide anchored to the surface of the natural nanovesicles via lipids; the targeting polypeptide can specifically bind to M1 macrophages.
[0009] Preferably, the lipid anchor is DSPE-PEG, and the targeting peptide is attached to the surface of the nanovesicles via a DSPE-PEG-CRV copolymer.
[0010] Preferably, the engineered Artemisia annua nanovesicles have a particle size of 100-200 nm and a negative Zeta potential.
[0011] This invention also provides a method for preparing engineered Artemisia annua nanovesicles targeting macrophages as described above, comprising the following steps:
[0012] S1. Natural nanovesicles extracted from Artemisia annua;
[0013] S2. Synthesize DSPE-PEG-targeting polypeptide copolymer, wherein the targeting polypeptide specifically binds to M1 macrophages;
[0014] S3. The natural nanovesicles obtained in step S1 are mixed and incubated with the DSPE-PEG-targeting polypeptide copolymer obtained in step S2, so that the DSPE-PEG-targeting polypeptide is modified onto the surface of the natural nanovesicles by lipid insertion. After purification, the product is obtained.
[0015] Preferably, the extraction in step S1 includes: mixing fresh Artemisia annua with PBS buffer and juicing, filtering through gauze, centrifuging at low speed and then at high speed, and collecting the precipitate to obtain natural nanovesicles.
[0016] Preferably, the low-speed centrifugation includes centrifugation at 400g for 10 min and centrifugation at 10000g for 30 min; the ultracentrifugation is centrifugation at 150000g for 120 min.
[0017] Preferably, the mass ratio of the natural nanovesicles to the DSPE-PEG-targeting polypeptide copolymer in step S3 is 10:1; the incubation conditions are co-incubation at 37°C for 2 hours.
[0018] The present invention also provides the use of engineered Artemisia annua nanovesicles targeting macrophages as described above in the preparation of medicaments for treating rheumatoid arthritis.
[0019] Preferably, the treatment of rheumatoid arthritis includes: inhibiting inflammatory polarization of synovial M1 macrophages, reducing the expression of the M1 marker CD86, and increasing the expression of the M2 marker CD206.
[0020] Preferably, the treatment of rheumatoid arthritis further includes: reducing the NAD+ / NADH ratio in M1 macrophages, inhibiting glycolytic metabolism in M1 macrophages, and reducing glucose uptake and lactic acid secretion.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] 1. This invention modifies natural Artemisia annua vesicles with CRV peptides using a simple lipid intercalation method, a process that is stable and convenient. The engineered vesicles specifically recognize and accumulate in M1 macrophages in the synovium of rheumatoid arthritis, overcoming the shortcomings of traditional plant vesicles, such as lack of targeting and dispersed drug efficacy.
[0023] 2. This invention is the first to combine targeted delivery of plant nanovesicles with the theory of "NAD+ metabolic reprogramming". It not only exerts anti-inflammatory effects through the artemisinin active substances carried by the vesicles, but also cuts off the "main power source" of NAD+ required for glycolysis of pathogenic macrophages from the metabolic source, achieving a therapeutic depth of 1+1>2.
[0024] 3. Artemisia annua extract has been proven safe for human use over a long period. Engineered delivery systems using it as a chassis organelle exhibit lower immunogenicity and better humoral stability compared to synthetic liposomes, demonstrating significant clinical translational potential in the treatment of autoimmune diseases. Attached Figure Description
[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.
[0026] Figure 1 The images show the morphological structure of CRV-AELNs under transmission electron microscopy (TEM) and the particle size distribution of NTA.
[0027] Figure 2 A schematic diagram showing the results of the hemolysis assay for the biosafety of CRV-AELNs;
[0028] Figure 3 This is a schematic diagram of the in vivo targeting experiment results of CRV-AELNs;
[0029] Figure 4 This image is a fluorescence image of macrophages specifically targeting and phagocytizing nanovesicles before and after CRV modification using laser confocal microscopy. Detailed Implementation
[0030] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0031] Example 1: Preparation and purification of engineered Artemisia annua nanovesicles (CRV-AELNs)
[0032] Step 1. Extraction of natural Artemisia annua vesicles (AELNs): Fresh Artemisia annua was washed with PBS buffer to remove impurities, then mixed with PBS at a ratio of 1g:2ml and juiced in a high-speed blender. The resulting homogenate was filtered through multi-layer gauze. The filtrate was placed in centrifuge tubes and centrifuged at 400g and 10000g for 10min and 30min respectively at 4℃ to remove large cell debris and precipitate, retaining the supernatant. The supernatant was placed in an ultracentrifuge and centrifuged at 150000g for 120min at 4℃. The supernatant was discarded, and the precipitate was resuspended in pre-cooled PBS buffer to obtain purified high-concentration AELNs.
[0033] Step 2. Synthesis of the peptide-liposome (DSPE-PEG-CRV): DSPE-PEG2000-NHS and a CRV peptide targeting M1 macrophages were dissolved in anhydrous dimethylformamide (DMF) at a molar ratio of 1:1.2. A suitable amount of triethylamine was added as a catalyst, and the reaction was carried out at room temperature in the dark for 24 hours. After dialyzing and freeze-drying, the DSPE-PEG-CRV polymer was obtained.
[0034] Step 3. Biomimetic engineering modification of vesicle surface (lipid insertion method): The AELNs extracted in Step 1 and the DSPE-PEG-CRV synthesized in Step 2 were mixed in PBS at a mass ratio of 10:1 and incubated on a constant temperature shaker at 37°C for 2 hours. Due to hydrophobic interactions, the lipid tails of DSPE spontaneously inserted into the lipid bilayer of the nanovesicles. After the reaction, unbound free peptides were removed by centrifugation and washing through an ultrafiltration tube (100kDa MWCO) to finally obtain CRV-modified engineered Artemisia annua nanovesicles (CRV-AELNs), which were aliquoted and stored at -80°C for later use.
[0035] Example 2: Characterization and Targeting Validation of Engineered Vesicles
[0036] 1. Transmission Electron Microscopy (TEM) and Particle Size Detection (NTA): The prepared CRV-AELNs suspension was dropped onto a copper grid, negatively stained with 2% phosphotungstic acid, and observed under a transmission electron microscope. The results are as follows: Figure 1 As shown, CRV-AELNs exhibit a typical intact lipid bilayer vesicle structure. Particle size and zeta potential were detected using NTA. The results showed that the average particle size of CRV-AELNs was 130 nm, and the zeta potential was negative, indicating that peptide modification did not disrupt the physicochemical stability of the vesicles.
[0037] 2. In vitro macrophage targeted phagocytosis assay: AELNs and CRV-AELNs were labeled with PKH67 fluorescent dye. RAW264.7 cells were stimulated to M1 type with LPS (100 ng / mL) + IFN-γ (20 ng / mL), and then incubated with equal amounts of the two fluorescently labeled vesicles for 4 hours. Confocal microscopy ( Figure 4 Flow cytometry results showed that the intracellular fluorescence intensity of the CRV-AELNs group was significantly higher than that of the unmodified AELNs group, demonstrating that the CRV peptide significantly enhanced the precise delivery of vesicles to inflammatory macrophages.
[0038] Example 3: CRV-AELNs drive macrophage NAD+ metabolic reprogramming and phenotypic reversal (in vitro efficacy)
[0039] 1. Cell culture and standard intervention model: RAW264.7 cells were seeded into culture plates and divided into three groups after adhesion: M0 group (blank culture medium), M1 group (100ng / ml LPS + 20ng / ml IFN-γ), and treatment group (LPS + IFN-γ, and 10μg / ml CRV-AELNs were added and incubated for 24 hours).
[0040] 2. Flow cytometry detection of macrophage polarization phenotype: 24 h after intervention, macrophages were blocked on ice using anti-mouse CD16 / 32 antibody (Fc Block), followed by CD86 and CD206 fluorescent surface staining. Flow cytometry results showed that after the addition of CRV-AELNs, the positivity rate of the M1 marker CD86 decreased sharply, while the positivity rate of the M2 marker CD206 increased significantly, confirming the phenotype reversal effect of vesicles and indicating that CRV-AELNs can effectively inhibit M1 polarization and promote the conversion to the M2 phenotype.
[0041] 3. NAD+ metabolism blockade and glycolysis inhibition assay: After the intervention, cells from each group were extracted.
[0042] The intracellular NAD+ / NADH ratio was detected by chemiluminescence immunoassay. The ratio in the CRV-AELNs group was found to be significantly lower than that in the M1 group, which proves that the mechanism of targeted deprivation of NAD+ is established.
[0043] Cell supernatants were collected, and D-glucose uptake was determined using the GOD-POD colorimetric method. Extracellular L-lactic acid secretion was determined using an enzymatic colorimetric method. The results showed that CRV-AELNs precisely disrupted the macrophage's "glycolysis pipeline," significantly reducing glucose consumption and the emission of pathogenic acidic waste (lactic acid).
[0044] Example 4: The in vivo therapeutic effect of CRV-AELNs on rheumatoid arthritis
[0045] 1. Construction and treatment of a mouse model of collagen-induced arthritis (CIA): DBA / 1J mice were selected, and a CIA rheumatoid arthritis model was established by intradermal injection of an emulsion of bovine type II collagen and complete Freund's adjuvant at multiple points in the tail root. Mice were randomly divided into the Blank group, the CIA model group, the AELNs group (unmodified vesicles injected via tail vein, dose based on protein), and the CRV-AELNs group. The mice were administered the drug twice weekly via tail vein for 4 weeks, and clinical joint redness and swelling scores were assessed periodically.
[0046] 2. Micro-CT Bone Destruction Scan: Mice were sacrificed, and the ankle / knee joints of the hind limbs were harvested for Micro-CT scanning and 3D reconstruction. Bone volume fraction (BV / TV) and the number of trabeculae were analyzed. The results showed that the joint space in the CIA model group was extremely narrow, and the cortical bone showed severe osteolytic destruction; while the bone erosion in the CRV-AELNs treatment group was significantly protected, the joint space was normal, and the integrity of the bone microstructure was far superior to that of the non-targeted AELNs group.
[0047] 3. Histopathological evaluation of synovial tissue: Mice joint tissue was decalcified and sectioned for H&E staining and Safranin O-Fast Green staining. Results confirmed that CRV-AELNs effectively inhibited the infiltration of inflammatory cells into the synovium, protected articular cartilage from erosion, and demonstrated excellent in vivo targeted anti-RA efficacy.
[0048] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.
Claims
1. An engineered Artemisia annua nanovesicle targeting macrophages, characterized in that: The engineered Artemisia annua nanovesicles include natural nanovesicles derived from Artemisia annua, and a targeting peptide linked to the surface of the natural nanovesicles via lipid anchoring; the targeting peptide can specifically bind to M1 macrophages.
2. The engineered Artemisia annua nanovesicles targeting macrophages according to claim 1, characterized in that: The lipid is anchored as DSPE-PEG, and the targeting peptide is attached to the surface of the nanovesicles via a DSPE-PEG-CRV copolymer.
3. An engineered Artemisia annua nanovesicle targeting macrophages according to any one of claims 1 or 2, characterized in that: The engineered Artemisia annua nanovesicles have a particle size of 100-200 nm and a negative Zeta potential.
4. A method for preparing engineered Artemisia annua nanovesicles targeting macrophages as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Natural nanovesicles extracted from Artemisia annua; S2. Synthesize DSPE-PEG-targeting polypeptide copolymer, wherein the targeting polypeptide specifically binds to M1 macrophages; S3. The natural nanovesicles obtained in step S1 are mixed and incubated with the DSPE-PEG-targeting polypeptide copolymer obtained in step S2, so that the DSPE-PEG-targeting polypeptide is modified onto the surface of the natural nanovesicles by lipid insertion. After purification, the product is obtained.
5. The preparation method according to claim 4, characterized in that: The extraction described in step S1 includes: mixing fresh Artemisia annua with PBS buffer and juicing, filtering the mixture through gauze, centrifuging at low speed and then at high speed, and collecting the precipitate to obtain natural nanovesicles.
6. The preparation method according to claim 5, characterized in that: The low-speed centrifugation includes centrifugation at 400g for 10 min and centrifugation at 10000g for 30 min; the ultra-speed centrifugation is centrifugation at 150000g for 120 min.
7. The preparation method according to claim 4, characterized in that: In step S3, the mass ratio of the natural nanovesicles to the DSPE-PEG-targeting polypeptide copolymer is 10:1; the incubation conditions are 37°C for 2 hours.
8. The use of engineered Artemisia annua nanovesicles targeting macrophages according to any one of claims 1-3 in the preparation of a medicament for treating rheumatoid arthritis.
9. The application according to claim 8, characterized in that, The treatment for rheumatoid arthritis includes: inhibiting inflammatory polarization of synovial M1 macrophages, reducing the expression of the M1 marker CD86, and increasing the expression of the M2 marker CD206.
10. The application according to claim 8, characterized in that, The treatment of rheumatoid arthritis also includes: reducing the NAD+ / NADH ratio in M1 macrophages, inhibiting glycolytic metabolism in M1 macrophages, and reducing glucose uptake and lactic acid secretion.