Paeonol-loaded plant nanovesicles and application thereof in preparation of medicine for regulating rheumatoid arthritis

Abstract

The application discloses a paeonol-loaded plant nanovesicle and application thereof in preparation of a medicine for regulating rheumatoid arthritis; the plant nanovesicle takes gardenia-derived exosome as a carrier, loads paeonol through an ultrasonic method, and is modified with a cyclic peptide RGD and a CD47 mimetic peptide on the surface, so that the plant nanovesicle can not only avoid being removed by macrophages, but also can penetrate a synovial vascular barrier and a dense extracellular matrix and be accurately delivered to an inflammatory synovial tissue, the delivery system effectively improves the stability and bioavailability of paeonol, can down-regulate a MALAT1 related signal path, inhibit abnormal activation of ERK / MAPK, and significantly reduce the expression of inflammatory factors such as IL-2, IL-6 and TNF-alpha, thereby relieving synovial inflammation of rheumatoid arthritis; the application has high targeting, long circulation, low immunogenicity and multi-dosage form adaptability, provides a novel delivery strategy of a safe and efficient active ingredient of traditional Chinese medicine, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomedicine preparation and application, and particularly relates to application of paeoniflorin exosomes in preparation of a nanomedicine for relieving symptoms of rheumatoid arthritis, especially synovitis, based on MALAT1 regulation of the ERK / MAPK pathway. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovitis, and its clinical manifestations include joint swelling, pain, morning stiffness, and limited mobility, which can gradually lead to joint destruction and loss of function. Although modern medicine has proposed various pathogenesis theories, including genetic predisposition, immune inflammatory response, cytokine storm, and abnormal proliferation of synoviocytes, the pathogenesis has not been fully elucidated. Currently, commonly used chemical drugs or biological agents can relieve symptoms, but they have limitations such as high side effects, high recurrence rate, and high drug cost.

[0003] Traditional Chinese medicine has a long history of regulating RA. In traditional Chinese medicine, RA is classified as "Bi syndrome", which is caused by wind, cold, dampness, and heat evil blocking collaterals and poor blood circulation. Baishao and Zhizi are classic medicinal combinations for treating Bi syndrome. Baishao has the effects of nourishing blood, soothing liver, relieving pain, and Zhizi has the effects of clearing heat, purging fire, and detoxifying blood. Modern pharmacological studies have shown that paeoniflorin is the main active ingredient of Baishao, which has anti-inflammatory, immunoregulatory, and antioxidant effects. The active ingredients in Zhizi also play a role in anti-inflammatory and immune regulation. From the theory of traditional Chinese medicine combination to modern mechanism research, it suggests that the two have potential synergistic effects in regulating synovial inflammation, which is the core pathological link of RA.

[0004] In recent years, the development of nanomedicine delivery systems has provided new ideas for the efficient use of active ingredients of traditional Chinese medicine. Plant-derived exosomes have attracted widespread attention due to their natural biocompatibility, low immunogenicity, and cross-barrier transport capacity. In particular, plant nanovesicles derived from Zhizi are not only widely available and safe and reliable, but also can be used as natural nanocarriers to encapsulate and protect unstable small molecule drugs, improving their in vivo stability and targeted delivery efficiency.

[0005] However, the existing technology still has the following problems in using paeoniflorin to treat rheumatoid arthritis:

[0006] (1) Paeoniflorin has high water solubility and fast in vivo metabolism, resulting in low bioavailability;

[0007] (2) Paeoniflorin is difficult to efficiently cross the synovial vascular barrier and deliver to the diseased synovial tissue;

[0008] (3) The traditional drug delivery method has defects such as unstable drug release, insufficient targeting and fast immune clearance.

[0009] Therefore, the present application plans to propose a new method by research, through the construction of plant source nanovesicles loaded with paeoniflorin (PF), as a very promising nanocomposite drug, to regulate the inflammatory pathway of fibroblast-like synoviocytes intervened by MALAT1, so as to improve the therapeutic effect of paeoniflorin in rheumatoid arthritis synovial inflammation. SUMMARY

[0010] Based on the above problems, the present application proposes a new idea: using gardenia source plant nanovesicles as carriers, loading paeoniflorin, and realizing long circulation, immune escape and efficient delivery of synovial lesions through surface modification of target peptides (such as cRGD and CD47 mimic peptides), so as to improve the synovitis of RA typical pathological state, and achieve the purpose of reducing IL-2, IL-6, TNF-α and other inflammatory factors.

[0011] To achieve the above purpose, the present application adopts the following technical solutions:

[0012] A plant nanovesicle loaded with paeoniflorin, comprising paeoniflorin and gardenia source plant nanovesicles, the surface of the plant nanovesicle being modified with cyclic peptide RGD and CD47 mimic peptide.

[0013] Preferably, the plant nanovesicle loaded with paeoniflorin is prepared by the following method:

[0014] 1) Plant nanovesicle extraction and purification: take gardenia plants, soak them in 2-(N-morpholine) ethanesulfonic acid (MES) buffer at 4℃ for 3-8h, and perform 1-3 times of vacuum extraction, each time for 5-10s, with an interval of 30-60s between adjacent two times; discard the supernatant, collect the wet sample, remove the tissue fragments and cell nuclei by low-speed separation, and then perform 400xg, 2000xg and 10000xg centrifugation in sequence, and then filter through 0.22μm; the obtained supernatant is subjected to ultrahigh-speed centrifugation combined with density gradient separation, the conditions being 100000xg, 4℃, 1.5-2h, the enriched layer is collected, and then washed with buffer and purified by ultrafiltration;

[0015] Adopting the technical scheme, the buffer and pH: selecting MES pH 6.0 (20 mM) + 150 mM NaCl, keeping close to acidic / neutral environment, being conducive to the stability of gardenia components and paeoniflorin, and reducing membrane damage caused by plant endogenous acid-base at the same time; operating at 4°C, inhibiting nuclease / protease activity and protecting membrane structure; less frequent and longer interval gentle vacuuming, avoiding instantaneous stress on cell walls and membranes and thus avoiding rupture, reducing content pollution; pre-centrifugation ladder (400g→2,000g→10,000g) and 0.22μm filtration: gradually removing large particles / cell fragments, reducing the burden of co-sedimentation during ultracentrifugation, and reducing mechanical damage to exosome membrane integrity; adopting a separation method that increases purity while maintaining vesicle integrity; using low-shear ultrafiltration instead of repeated high-speed centrifugation to protect membrane morphology, improve freeze stability, and avoid storage at -20°C (easily forming ice crystals and causing damage);

[0016] 2) Paeoniflorin loading: taking the purified plant nanovesicles, mixing with a paeoniflorin methanol solution, and ultrasonating under an ice water bath; after ultrasonation, recovering in a 37°C constant temperature incubator for 1h;

[0017] 3) Purification: centrifuging at 4°C, 120000xg for 65-75min, discarding the supernatant, and removing free paeoniflorin;

[0018] 4) Washing: resuspending with buffer and centrifuging again; taking the precipitate and resuspending with an appropriate amount of buffer to obtain plant nanovesicles loaded with paeoniflorin;

[0019] 5) Surface modification: modifying DSPE-PEG2000-c(RGDfK) and DSPE-PEG2000-CD47 mimic peptides on the surface of exosomes by membrane insertion to obtain plant exosomes Exo-PF loaded with paeoniflorin and modified with cRGD and CD47 mimic peptides, i.e. plant nanovesicles Exo-PF loaded with paeoniflorin.

[0020] Adopting the technical scheme, the surface modification structure can reduce phagocytosis by macrophages and prolong circulation time in the systemic circulation, and the cRGD ligand can bind to integrin receptors on the endothelial cells of newly formed blood vessels of inflammatory synovial tissue, improving targeting.

[0021] Preferably, the mixing ratio of plant nanovesicles and PF solution, in terms of concentration, is 0.5-2:1, and the gardenia plant includes flowers, leaves, rhizomes, and fruits.

[0022] Preferably, the ultrasonation conditions in step 2) are 20% amplitude, 5s on, 5s off, 1min cycle, a total of 6 cycles, and a cycle interval of 2min.

[0023] Preferably, in step 4), the centrifugation is at 4°C, 120000xg for 65-75min.

[0024] Preferably, in step 5), the surface modification adopts a DSPE-PEG2000 lipid anchor structure, wherein cRGD is a cyclic peptide c(RGDfK), CD47 mimic is a short peptide fragment of a SIRPα binding site, and the modification ratio is: DSPE-PEG2000-OMe accounts for 70-85 mol%, DSPE-PEG2000-c(RGDfK) accounts for 5-10 mol%, and DSPE-PEG2000-CD47 mimic accounts for 1-3 mol%.

[0025] Preferably, in step 1), the buffer condition is 20 mM, containing 150 mM NaCl, and pH=6.0.

[0026] Preferably, the average particle size of the nanovesicle is 80-160 nm, and the Zeta potential is-10-+10 mV; the drug loading amount of paeoniflorin is 2-12%.

[0027] The application further discloses a use of the plant nanovesicle loaded with paeoniflorin in preparation of a medicine for regulating rheumatoid arthritis, and based on MALAT1 regulation of the ERK / MAPK signal pathway, the expression of IL-2, IL-6 and TNF-α is reduced.

[0028] Preferably, the preparation of the medicine can be in any one of the following forms: the plant nanovesicle loaded with paeoniflorin is used as an additive; the plant nanovesicle loaded with paeoniflorin is used as an additive of an efficacy component; an injection type; an oral type; a patch type; the plant nanovesicle loaded with paeoniflorin is loaded and gelled to prepare an enhanced patch.

[0029] The application has the following beneficial effects:

[0030] 1) improving drug stability and bioavailability: paeoniflorin, as the main active ingredient of radix paeoniae alba, is easy to be metabolized and inactivated in the body, and the application effectively avoids the rapid degradation and inactivation of paeoniflorin in the body fluid environment through the loading effect of the gardenia source plant nanovesicle, thereby significantly improving the stability and utilization rate of the medicine;

[0031] 2) enhancing targeting and delivery efficiency: through surface modification of the cyclic peptide RGD and the CD47 mimic peptide of the exosome, on one hand, active targeting of the inflammatory synovial tissue neovascular endothelial cells can be realized, and on the other hand, the phagocytosis of macrophages is reduced, thereby ensuring the long circulation of the exosome and directional delivery to the synovial membrane site;

[0032] 3) improving cross-barrier transportation and tissue penetration: the gardenia source exosome has a natural membrane fusion and cross-barrier transportation capacity, and combined with surface targeting peptide modification, the exosome can efficiently penetrate the synovial vascular barrier and the dense extracellular matrix, thereby realizing effective delivery of paeoniflorin to the synovial inflammation lesion;

[0033] 4) Anti-inflammatory and immune regulation: The paeoniflorin-loaded gardenia source nanovesicle can inhibit the abnormal activation of the ERK / MAPK signaling pathway by down-regulating the MALAT1 level, and reduce the expression of inflammatory factors such as IL-2, IL-6 and TNF-alpha, thereby relieving the typical symptoms of rheumatoid arthritis synovial inflammation.

[0034] In summary, the present application combines the dual advantages of traditional Chinese medicine active ingredients and plant exosome nanocarriers, improves the treatment effect of paeoniflorin, and provides a new treatment strategy for rheumatoid arthritis that is safe, efficient and has strong targeting. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is a schematic diagram of the morphology of Exo and Exo-PF under transmission electron microscope;

[0036] Figure 2 It is a schematic diagram of the particle size change of Exo-PF at 4 and 37℃;

[0037] Figure 3 It is a schematic diagram of the uptake of Exo-PF by MH7A cells;

[0038] Figure 4 It is a TUNEL detection of apoptosis;

[0039] Figure 5 It is a schematic diagram of cell migration ability detection (200x);

[0040] Figure 6 It is a schematic diagram of ELISA detection of inflammatory factors IL-2, IL-6 and TNF-alpha content;

[0041] Figure 7 It is a qRT-PCR detection of key factors of ERK / MAPK pathway;

[0042] Figure 8 It is a qRT-PCR detection of apoptosis factor expression. DETAILED DESCRIPTION

[0043] The present application will be further described below by specific examples. In order to make the inventive purpose, technical scheme and beneficial technical effects of the present application clearer, the present application will be further described in detail below in combination with examples. It should be understood that the examples described in the specification are only for the purpose of explaining the present application, and are not intended to limit the present application.

[0044] Unless otherwise stated, all adhesive films and reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further treatment, and the instruments used in the examples are commercially available.

[0045] Example 1:

[0046] 1) Material and buffer: Take 50 g of gardenia flower / leaf mixture fresh weight according to the laboratory batch, prepare MES buffer, 20 mM MES, 150 mM NaCl, pH = 6.0, pre-cooled to 4°C, add broad-spectrum protease inhibitors and 0.5 mM DTT, and place on ice;

[0047] 2) Exosome extraction and purification: immerse the gardenia material in the MES buffer at 4°C for 4-6 h, and perform 1 negative pressure (vacuum for 5-10 s, back pressure for 30-60 s) to reduce tissue damage.

[0048] Discard the soaking supernatant, collect the wet sample and quickly freeze it at -80°C, then warm it to 4°C for half-melting; remove large particles by continuous centrifugation: 400 x g, 10 min→2,000 x g, 20 min→10,000 x g, 30 min (all at 4°C). The supernatant is removed and filtered through a 0.22 μm filter.

[0049] Place the pretreated liquid on top of the iodixanol density gradient (10% / 20% / 40%), use a swing-bucket rotor to perform ultracentrifugation at 100,000 x g, 4°C, for about 1.5-2 h, and collect the exosome enrichment layer (density about 1.08-1.18 g / mL).

[0050] Concentrate by 100 kDa ultrafiltration, and wash twice in buffer, and finally resuspend to the desired volume.

[0051] 3) Paeoniflorin loading: prepare an exosome suspension with a concentration of 400 mg / L (protein mass) and a PF methanol solution (200 mg / L). Mix the two at a ratio of 2:1 (PF:Exo by concentration), perform probe sonication in an ice water bath: 20% amplitude, 5 s on / 5 s off, 1 min per cycle, a total of 6 cycles, with a 2 min interval between cycles (the entire sonication process is in an ice water bath). After sonication, place in a 37°C constant temperature incubator for 1 h.

[0052] 4) Free drug removal and purification: centrifuge the mixture at 4°C, 120,000 x g, for 70 min, discard the supernatant, resuspend with PBS and centrifuge again for 70 min, finally resuspend in buffer to obtain Exo-PF, and further remove free small molecules by SEC.

[0053] 5) Surface modification (membrane insertion): mix loaded Exo-PF with DSPE-PEG2000-OMe, DSPE-PEG2000-c(RGDfK), DSPE-PEG2000-CD47-mimic (molar ratio: OMe 75 mol%, cRGD 7 mol%, CD47 2 mol%) at 37 °C for 30-60 min, allowing DSPE chains to insert into the exosome membrane, followed by ultrafiltration to remove free lipid anchor molecules.

[0054] Example 2:

[0055] Variable compared to Example 1: PF:Exo mixing ratio taken as 1:1 (concentration basis); ultrasound parameters same as Example 1 (20% amplitude, 6 cycles); centrifugation time taken as 65 min;

[0056] PF:Exo = 1:1 mixed ultrasound loading -> 37 °C recovery for 1 h; 4 °C, 120,000 x g, 65 min centrifugation to remove free PF; remaining steps consistent, not elaborated.

[0057] Characterization and results: according to the document drug loading curve, LC ~ 1.93% at 1:1 ratio (see Table 3), particle size and Zeta are within the range of nanoscale exosomes, and the stability is excellent.

[0058] Example 3:

[0059] Variable: PF:Exo = 0.5:1; ultrasound parameters halved or changed to passive incubation; centrifugation taken as 75 min; surface modification molar ratio cRGD 5 mol%, CD47 1 mol%; step summary:

[0060] Extraction and purification of Exo (same as Example 1);

[0061] Mix PF aqueous phase or methanol dilution (≤1% v / v methanol) with Exo at 0.5:1 in an ice bath and passively incubate for 4 h (avoid strong ultrasound to ensure the retention activity of PF in the patch / oral preparation); or use ultrasound: 20% amplitude, 3 cycles;

[0062] 4 °C, 120,000 x g, 75 min centrifugation to remove free PF; washing -> surface modification;

[0063] Characterization: LC ~ 1.93% (see control table), particle size ~ 110 nm or so, stable at 4 °C for 7 d; suitable for preparing patch / oral dosage forms (low immunostimulation).

[0064] Example 4:

[0065] The preparation method is the same as that of Example 1, but the centrifugation time is set to 65 min / 70 min / 75 min for three groups, respectively, and other conditions remain unchanged. The residual free PF, drug loading capacity LC and particle size stability of the three groups of Exo-P are compared, and the data are recorded to determine the optimal centrifugation time.

[0066] Example 5: PF:Exo ratio gradient

[0067] System comparison of PF:Exo = 0.5:1, 1:1, 2:1 three groups (corresponding to Table 3), other processes are the same as Example 1.

[0068] Example 6: Surface modification molar ratio variation

[0069] On the basis of Example 1, three modification formulas are set respectively:

[0070] A: OMe 85% / cRGD 10% / CD47 1%;

[0071] B (Example 1): OMe 75% / cRGD 7% / CD47 2%;

[0072] C: OMe 70% / cRGD 10% / CD47 3%.

[0073] The differences of the three groups in macrophage phagocytosis (RAW264.7) and MH7A targeted delivery efficiency (uptake amount, inflammatory factor inhibition) are compared to balance the circulation time and targeting.

[0074] Example 7: Multi-formulation dosage form

[0075] Oral preparation: Exo-PF in Example 3 is formulated into a capsule or sustained-release tablet form for oral administration. The excipients are limited to inactive excipients necessary for the dosage form, such as microcrystalline cellulose, hydroxypropyl methylcellulose as molding and disintegrating agents, etc. The physicochemical properties of Exo-PF in the formulation are maintained within the range of particle size 100 nm, Zeta−10~+10 mV, drug loading capacity 8%, and the temperature is strictly controlled during preparation and drying steps (≤25°C drying or low-temperature freeze-drying and adding 1% w / v trehalose as a cryoprotectant) to protect the structure of PF and exosomes;

[0076] Injection, Exo-PF is formulated into a sterile solution for injection, only using buffer and isotonic excipients necessary for injection dosage form, such as 20mM MES, 150mM NaCl, pH=6.0, or injection PBS adjusted to isotonic, preparation and filtration (0.22μm sterile filtration) are operated under cold chain conditions (4°C) throughout the process. The final product can be stored in cold storage for short-term storage or in aliquots-frozen (−80°C) for long-term storage and protected with trehalose.

[0077] Gel enhanced patch, Exo-PF is embedded in a degradable matrix to make a microneedle patch or a gel patch system, the excipients are only biocompatible polymers necessary for the formation of the preparation, such as sodium hyaluronate, gelatin methacryl derivative or controllable dissolving polymer and supporting materials for microneedle substrate, the microneedle structure can be hyaluronic acid-based microneedle or gelatin / PLGA composite microneedle, the arrangement density and needle length are designed according to the penetration depth, typically 200-600 μm needle length range.

[0078] Characterization and experiment

[0079] 1) Characterization of Exo-PF

[0080] DLS determination of particle size and zeta potential: Exo-PF and Exo-coumarin6 suspensions were diluted with ultrapure water, respectively, and after equilibration at room temperature, the nanoparticle size analyzer was used to detect the particle size and potential of exosomes, repeated 3 times.

[0081] Observation of morphology and size: Take the exosome suspension on the copper mesh, 2 min standing, 1% phosphotungstic acid solution restaining 5 min, dry and take pictures with transmission electron microscope.

[0082] 2) PF quality determination of Exo-PF

[0083] Chromatographic conditions: Prepare 100µg / ml (HPLC grade) PF and dilute it to the appropriate concentration as the working standard solution, 5mg is dissolved in 50ml of methanol, the mobile phase is methanol and water (80:20 v / v), and the retention time of the compound is reported as 6 minutes. Chromatographic separation was performed using a YMC-triart C18 column (4.6mm x 250mm, 5μm), and the detection wavelength was 285nm.

[0084] Sample preparation: accurately weigh 5mg of PF reference substance into a 5mL volumetric flask, and dissolve with methanol. Filter with a microporous membrane to obtain the stock solution. Preparation of test solution: take Exo-PF and Exo-coumarin6 suspensions 100μL, centrifuge at 120000 x g for 70 min at 4℃, remove the supernatant, add an equal volume of methanol to break the emulsion, centrifuge at 12000 x g for 10 min at 4℃, and take the supernatant.

[0085] Specificity determination: accurately pipette 10μL of PF reference solution, Exo-PF and Exo-coumarin 6 solution after emulsion breaking, and inject according to the above chromatographic conditions for determination.

[0086] Drug loading determination: Prepare PF solutions with mass concentrations of 4, 8, 16, 32, and 64 mg / L, respectively, to draw a standard curve; take 100 μL of Exo-PF solution, inject it according to the chromatographic conditions to determine the mass of PF, and calculate the drug loading (LC). LC = W1 / W2 x 100%, wherein W1 is the total mass of encapsulated PF; and W2 is the total mass of protein in Exo-PF.

[0087] Stability observation: Place Exo-PF in 4°C and 37°C environments, respectively, and use DLS to determine the change in particle size, for a continuous determination of 7 days.

[0088] MH7A cell uptake: Stain Exo-PF according to the PKH67 kit instructions, centrifuge at 120,000 x g for 70 min at 4°C, resuspend, centrifuge again, resuspend in an appropriate amount of PBS, and obtain the PKH67-labeled Exo-PF. Seed MH7A cells in a 96-well plate at 5 x 10 3

[0089] Apoptosis detection using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL): After dehydration, mix the sample with 50 μL of TdT + 450 μL of fluorescein-labeled dUTP solution, and incubate in a 37°C humidified box for 2 hours. Drop DAB developing solution on the dried glass slide, and then wash to stop the reaction. Immediately after taking a photograph, rinse with running water, and then apply hematoxylin or methyl green. After dehydration with alcohol, xylene, and neutral resin, observe the apoptotic cells under an optical microscope.

[0090] Migration experiment: Collect MH7A, inoculate the upper chamber with serum-free cell suspension at 5 x 10 4

[0091] Use the corresponding enzyme-linked immunosorbent assay kit (ELISA) to determine the expression levels of IL-2, IL-6, and TNF-α. Add the diluent in a 96-well plate, and incubate at 37°C for 2 hours; add 100 μL of biotin-labeled antibody to each well, and then add 100 μL of HRP-labeled avidin. After washing, add 90 μL of TMB reaction substrate to each well. Finally, after incubation in the dark for a period of time, add 50 μL of stop buffer to each well. Measure the absorbance at 450 nm wavelength using a microplate reader.​​

[0092] qRT-PCR: The purity and concentration of RNA were determined using a NanoDrop 2000 spectrophotometer. Total RNA was converted into cDNA according to the instructions of the PrimeScript RT Kit and gDNA Eraser. RT-PCR was performed using the Premix Ex Taq™ II Kit. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference, and the results were calculated according to 2 -ΔΔCt Formulas. The primers of MALAT1, ERK1 / 2, JNK1 / 2, p38 and GAPDH were synthesized (Table 1 qRT-PCR primer sequences).

[0093]

[0094] Statistical methods: Graphpad Prism 10.5 was used for statistical analysis. The measurement data was expressed as mean ± standard deviation, the difference between two groups was evaluated by t test, the measurement data between multiple groups was compared by one-way analysis of variance (ANOVA), P<0.05 indicated statistical significance.

[0095] 3) Experimental materials and reagents: MH7A cells, PF control, purified gardenia exosomes (Exo), Exo-PF (prepared according to reference example 1), si-MALAT1 and si-NC (control siRNA), Lipofectamine 3000 or equivalent transfection reagent, ELISA kit (IL-2 / IL-6 / TNF-α), qRT-PCR reagent, TUNEL kit, etc.

[0096] Grouping design (3 replicate holes or more per group, experimental repetition ≥3 times):

[0097] 1. Blank control group (Control, untreated)

[0098] 2. si-NC group (transfection of negative control) - mechanism control

[0099] 3. si-MALAT1 group (only knock down MALAT1, used to verify its function)

[0100] 4. PF group (free paeoniflorin treatment)

[0101] 5. Exo group (empty exosome treatment)

[0102] 6. Exo-PF group (exosome loaded with paeoniflorin treatment)

[0103] 7. PF+si-MALAT1 group (transfect si-MALAT1 first, then treat with free PF, to test whether the effect of PF depends on MALAT1)

[0104] 8. Exo-PF+si-MALAT1 group (transfect si-MALAT1 first, then treat with Exo-PF, to test whether the effect of Exo-PF still depends on MALAT1) Note: si-MALAT1 in groups 7 and 8 is still only a mechanism verification tool to knock down MALAT1.

[0105] Operation step points:

[0106] 1. siRNA transfection: according to the kit instructions, transfect si-MALAT1 (or si-NC) into MH7A using Lipofectamine, and change the culture medium 24 h after transfection; the transfection efficiency / knockdown effect is detected by qRT-PCR to confirm MALAT1 expression at 24-48 h (knockdown rate ≥ 60% is qualified).

[0107] 2. Drug treatment: after transfection confirmation, treat the cells for 24-48 h at the specified dose: PF (concentration according to the effective concentration range in vitro in Example 1, for example 10 μg / mL); Exo-PF is administered at the same PF content (or administered according to the exosome protein mass / particle number standard). Exo and Exo-PF are administered with the same volume of buffer / equal carrier amount as controls

[0108] 3. Detection index (sampled for determination 24-48 h after treatment):

[0109] qRT-PCR: detection of MALAT1 expression and ERK, JNK, p38 (MAPK family) mRNA levels;

[0110] ELISA: determination of IL-2, IL-6, TNF-α content in culture supernatant;

[0111] Migration ability: wound healing / Transwell migration experiment;

[0112] Apoptosis: TUNEL staining and Caspase-3 / 9, Bax / Bcl-2 mRNA (or protein) detection;

[0113] Cell proliferation: CCK-8 or EdU experiment.

[0114] The relevant parameters of the example are shown in the following table:

[0115]

[0116] Note: Table 2 (DLS / Zeta): Particle size and potential data corresponding to Example 1 (comparison between Exo and Exo-PF).

[0117]

[0118] Note: Table 3 (Drug Loading - PF:Exo Gradient): LC values ​​(12.03%, 10.11%, 1.93%) at three points corresponding to Examples 1, 2, and 3 (PF:Exo = 2:1 / 1:1 / 1:2).

[0119]

[0120] Note: Table 4 shows the data on residual free PF, drug loading LC and particle size stability of Exo-P in Example 4.

[0121]

[0122] Note: Table 5 corresponds to Example 6.

[0123] Analysis and Conclusion:

[0124] Figure 1 (TEM): Used to demonstrate the integrity of exosome membranes in the examples (checked after the extraction / purification step);

[0125] Figure 2 (Particle size dynamics): Stability testing in the corresponding examples (7-day observations at 4°C and 37°C);

[0126] Figure 3 (Cellular uptake) Figure 4 (TUNEL) Figure 5 (migrate), Figure 6 (ELISA), Figures 7–8 (qRT-PCR / apoptosis factor): corresponding to the functional verification results of Examples 1, 2, and 8, showing that Exo-PF (especially Exo-PF+si-MALAT1) is superior to free PF in inhibiting inflammation and promoting apoptosis;

[0127] Appendix Figure 5 Results showed that cell proliferation and migration were significantly inhibited in the Exo-PF+si-MALAT1 group compared with the control group. The cell proliferation and migration rates in the Exo-PF+si-MALAT1 group were significantly lower than those in the PF+si-MALAT1 group (see attached). Figure 5Compared with the control group, the Exo-PF+si-MALAT1 group significantly increased cell apoptosis. Compared with the si-MALAT1 group, the cell apoptosis rate of this group and the PF+si-MALAT1 group increased significantly. Compared with PF+si-MALAT1, the Exo-PF+si-MALAT1 group significantly increased cell apoptosis (P<0.01).

[0128] Appendix Figure 6 The results show that: PF treatment can reduce the expression of IL-2, IL-6 and TNF-α, while MALAT1 knockout can increase their expression (P<0.01 or 0.05). Compared with PF+si-MALAT1, Exo-PF+si-MALAT1 group significantly reduced the expression of the above inflammatory factors.

[0129] Appendix Figure 7 The results show that: after MALAT1 knockout, the mRNA levels of ERK, JNK and p38 increase, but after PF and Exo-PF treatment, they decrease significantly. Compared with the PF treatment group, the mRNA levels of ERK, JNK and p38 significantly decrease after Exo-PF treatment (P<0.05).

[0130] Appendix Figure 8 The results show that: compared with the control group, the mRNA expression of caspase3, caspase9 and bax significantly decreases after MALAT1 knockout (P<0.05), and the mRNA expression of bcl2 increases (P<0.01). Compared with the si-MALAT1 group, it significantly increases after PF and Exo-PF treatment (P<0.01). Compared with the PF treatment group, the mRNA expression of caspase3, caspase9 and bax significantly increases after Exo-PF treatment (P<0.01), and the mRNA expression of bcl2 significantly decreases (P<0.01).

[0131] From the above, it can be seen that plant nanovesicles loaded with paeoniflorin can be used in various dosage forms. Low-load Exo-PF can be used as a plaster or oral supplement for chronic and long-term administration. Oral preparations can avoid the first-pass effect of the liver and can be delivered systemically. Injection into the blood does not cause immune mechanisms. Microneedle plasters can be long-acting and effective. All three preparation forms focus on the fact that the exosome can break through the special physiological structure barrier of the synovial membrane and effectively administer drugs.

[0132] The DLS particle size in Table 2 suggests that it belongs to the exosome particle size definition range, and the Zeta potential suggests good stability. There is no signal for Exo at the same time point, indicating that Exo has no interference with the determination of PF and good specificity.

[0133] The drug loading ratio of Exo-PF in Table 3 is optimized, and the Exo carrier and PF ratio of 2:1 tends to be saturated.

[0134] In Table 4, the residual free PF, drug loading capacity LC and particle size stability of Exo-P: 65 min: The centrifugation time is too short, the residual free PF is relatively high, although the particle size change is small, but the free substance removal is insufficient; 70 min (median): The residual free PF is significantly reduced and the LC reaches the peak, the initial particle size and PDI are optimal, and Day 7 shows good stability at 4°C and 37°C - the comprehensive performance is the best; 75 min: The residual free PF is similar to that of 70 min, but the particle size and PDI are observed to increase, and the aggregation at 37°C is obvious, suggesting that long-time ultracentrifugation may have a negative impact on the integrity / dispersibility of the vesicles - slight damage or aggregation promotion, resulting in a decrease in apparent LC and poor stability; under the process conditions and detection methods, 100,000 x g centrifugation for 70 min at 4°C is the best choice for the removal of equilibrium free drug, drug loading capacity and vesicle particle size / stability.

[0135] Table 5 phagocytosis rate (RAW264.7) and MH7A targeted delivery efficiency: Group C (higher cRGD and higher CD47-mimic) shows the lowest macrophage phagocytosis rate in this test range, that is, the least removal, the highest MH7A uptake and the most obvious inhibition of inflammatory factors, indicating that under this molar ratio, both the circulation time / phagocytosis can be prolonged and the synoviocyte localization and drug efficacy can be enhanced; Group A (high PEG shielding, low CD47) can reduce part of the phagocytosis, but the strong PEG shielding inhibits the exposure of the targeting ligand, resulting in weak MH7A uptake and inflammatory inhibition effect; Group B is a compromise / baseline scheme, showing a good balance: moderate phagocytosis rate, higher targeted uptake and better inflammatory inhibition, suitable for scenarios that pursue a balance between circulation time and targeting.

[0136] In summary, the data of the test results provided by the present application show that in MH7A cells, MALAT1 can inhibit the expression of ERK / MAPK pathway and inflammatory factors, PF can enhance the inhibition of synovial inflammation by MALAT1, and by constructing Exo-PF, the regulation of PF on MALAT1 can be enhanced, promoting the apoptosis of fibroblast-like synoviocytes, inhibiting their proliferation and migration, and delaying the progression of inflammation.

[0137] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A plant nanovesicle loaded with paeoniflorin, characterized in that, Plant nanovesicles containing paeoniflorin and gardenia extract, wherein the surface of the plant nanovesicles is modified with cyclic peptide RGD and CD47 mimic peptide. The loading capacity of paeoniflorin is 2-12%; The plant nanovesicles were prepared by the following method: 1) Extraction and purification of plant nanovesicles: Gardenia plants were soaked in 2-(N-morpholino)ethanesulfonic acid (MES) buffer at 4℃ for 3-8 hours, and vacuumed 1-3 times, each time for 5-10 seconds, with an interval of 30-60 seconds between adjacent vacuuming. The supernatant was discarded, and the moist sample was collected. After removing tissue fragments and cell nuclei by low-speed separation, the sample was centrifuged at 400×g, 2000×g and 10000×g in sequence, and then filtered through 0.22μm. The obtained supernatant was centrifuged at ultra-high speed combined with density gradient separation at 100000×g, 4℃, for 1.5-2 hours. The enriched layer was collected, washed with buffer, and purified by ultrafiltration. 2) Paeoniflorin loading: Purified plant nanovesicles were mixed with paeoniflorin methanol solution and sonicated in an ice-water bath; after sonication, the mixture was incubated at 37°C for 1 hour. 3) Purification: Centrifuge at 4℃ and 120000×g for 65-75 min, discard the supernatant, and remove free paeoniflorin; 4) Washing: Wash and resuspend with buffer solution, and centrifuge again; take the precipitate and resuspend with an appropriate amount of buffer solution to obtain plant nanovesicles loaded with paeoniflorin; 5) Surface modification: DSPE-PEG2000-c(RGDfK) and DSPE-PEG2000-CD47 mimic peptides were modified onto the surface of exosomes by membrane insertion to obtain plant exosomes Exo-PF loaded with paeoniflorin and modified with cRGD and CD47 mimic peptides, which is the plant nanovesicles Exo-PF loaded with paeoniflorin. The surface modification employs a DSPE-PEG2000 lipid anchor structure, wherein cRGD is a cyclic peptide c(RGDfK), and the CD47 mimic peptide is a short peptide fragment at the SIRPα binding site. The modification ratio is as follows: DSPE-PEG2000-OMe accounts for 70-85 mol%, DSPE-PEG2000-c(RGDfK) accounts for 5-10 mol%, and DSPE-PEG2000-CD47 mimic accounts for 1-3 mol%.

2. The plant nanovesicles loaded with paeoniflorin according to claim 1, characterized in that, The mixing ratio of plant nanovesicles to PF solution, in terms of concentration, is 0.5~2:

1.

3. The plant nanovesicles loaded with paeoniflorin according to claim 1, characterized in that, Step 2) The ultrasound conditions are 20% amplitude, 5s on, 5s off, 1min cycle, for a total of 6 cycles, with a 2min interval between cycles.

4. The plant nanovesicles loaded with paeoniflorin according to claim 1, characterized in that, In step 4), centrifuge at 4℃ and 120000×g for 65-75 minutes.

5. The plant nanovesicles loaded with paeoniflorin according to claim 1, characterized in that, In step 1), the buffer conditions are 20 mM, containing 150 mM NaCl, and pH = 6.

0.

6. The plant nanovesicles loaded with paeoniflorin according to claim 1, characterized in that, The average particle size of the nanovesicles is 80–160 nm, and the zeta potential is -10–+10 mV.

7. The application of a plant nanovesicle loaded with paeoniflorin in the preparation of a drug for regulating rheumatoid arthritis, characterized in that, Based on MALAT1 regulation of the ERK / MAPK signaling pathway, the expression of IL-2, IL-6 and TNF-α is reduced.

8. The application according to claim 7, characterized in that, The formulation of the drug may be selected from any of the following forms: using plant nanovesicles loaded with paeoniflorin as an additive; using plant nanovesicles loaded with paeoniflorin as an additive for the active ingredient; injectable; oral; patch; and preparing an enhanced patch by gelling and loading plant nanovesicles loaded with paeoniflorin.

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