Drug-loaded callicarpa nudiflora-derived extracellular vesicle as well as preparation method and application thereof

Abstract

The invention provides a drug-loaded callicarpa nudiflora-derived extracellular vesicle as well as a preparation method and application thereof, and relates to the technical field of pharmaceutical preparations. The drug-loaded callicarpa nudiflora-derived extracellular vesicle has relatively high encapsulation efficiency and drug loading rate and good gastrointestinal tract stability and biocompatibility, can target a colitis disease part to reduce adverse reaction of a drug and improve the curative effect, solves the problem that the drug is insoluble in water, and can be used for preparing the drug-loaded callicarpa nudiflora-derived extracellular vesicle. The invention provides a novel drug delivery strategy for treating the ulcerative colitis, and is expected to become an effective strategy for clinically treating the ulcerative colitis, thereby achieving the effects of synergistically treating the ulcerative colitis and improving the effectiveness and safety of drug delivery.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a drug-loaded extracellular vesicle derived from Callicarpa nudiflora, and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic, recurrent inflammatory disease of the gastrointestinal tract involving the large intestine or colon. It continuously damages the rectal mucosa, usually leading to erosions and ulcers. It is one of the two main forms of inflammatory bowel disease (IBD) and one of the most common causes of colorectal cancer. The pathogenesis of the disease is complex, but the exact mechanism is still unclear. It is generally believed to be related to the combined effects of intestinal flora disorders, genetic susceptibility, environmental factors, intestinal epithelial barrier defects, immune factors, and inflammatory mediators. At the same time, the disease has a long course, is prone to recurrence, is difficult to cure, and is prone to cancer. It is currently recognized as one of the more difficult diseases to cure.

[0003] The main treatment for UC is currently drug therapy. Aminosalicylates are the main treatment option for mild to moderate ulcerative colitis, topical and systemic steroids can be used to treat ulcerative colitis attacks, while immunosuppressants and new biologics, as well as an ever-increasing number of cell signaling pathway inhibitors, are used to treat moderate to severe ulcerative colitis. Although conventional drug therapy can relieve the initial symptoms of most UC patients, as the disease progresses, patients are prone to drug tolerance and side effects from high-dose drug use. Therefore, the development of a formulation that can specifically and effectively deliver low-dose drugs to sites of inflammation is crucial for the treatment of UC.

[0004] Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles that can be released by almost all types of cells, carrying important biological molecules such as nucleic acids, proteins and lipids, including exosomes, microvesicles and apoptotic bodies. Compared with mammalian EVs, plant-derived EVs have similar morphology (nanovesicles), composition (lipids, RNA and proteins) and functions, but their research is still in its infancy. Plant EVs have unique morphological and compositional characteristics as natural nanocarriers and have great potential to be developed into the next generation of biotherapy and drug delivery platforms: (1) Plant EVs are lipid bilayer structures that can encapsulate hydrophilic drugs in the aqueous core and accommodate hydrophobic drugs in the lipid bilayer, thereby improving the insolubility and stability of macromolecular drugs and thus enhancing the efficacy of drugs; (2) Plant EVs do not contain zoonotic or human pathogens and have almost no toxicity or immunogenicity; (3) Plant EVs have intrinsic therapeutic activities, including anti-cancer, anti-inflammatory, and antioxidant activities; (4) Plant sources are cost-effective, environmentally friendly, and economical, and are sustainable and renewable resources; (5) Plant EVs have excellent cellular uptake, gastrointestinal stability, and specific targeting capabilities.

[0005] Callicarpa nudiflora Hook. & Arn. is a plant of the Verbenaceae family. According to traditional Chinese medicine theory, it has anti-inflammatory, anti-depression, anti-ulcer, astringent hemostasis, and treatment of burns and scalds. Modern pharmacological studies have confirmed that it has anti-inflammatory, anti-thrombotic, neuroprotective, analgesic and antioxidant activities. However, the research on the chemical composition of Callicarpa nudiflora Hook. & Arn. started relatively late at home and abroad, and there is no report on extracellular vesicles derived from Callicarpa nudiflora. Therefore, a solution of loading extracellular vesicles derived from Callicarpa nudiflora with UC therapeutic drugs can be studied to solve the above problems. Summary of the invention

[0006] The present invention aims to provide a drug-loaded extracellular vesicle derived from Callicarpa nudiflora and a preparation method and application thereof. The drug-loaded extracellular vesicle derived from Callicarpa nudiflora has a high encapsulation rate and drug loading rate as well as good gastrointestinal stability and biocompatibility, can target the site of colon inflammation, solve the problem of drug insolubility in water, improve the therapeutic effect, and reduce adverse reactions caused by systemic medication.

[0007] In the first aspect, the present invention provides a drug-loaded extracellular vesicle derived from Callicarpa spp., wherein the drug-loaded extracellular vesicle derived from Callicarpa spp. comprises extracellular vesicles derived from Callicarpa spp. and a drug for treating ulcerative colitis loaded in the extracellular vesicles derived from Callicarpa spp.; the loading amount of the drug for treating ulcerative colitis in the drug-loaded extracellular vesicles derived from Callicarpa spp. is 4.39%~8.72%.

[0008] When used as a drug delivery vehicle for the treatment of ulcerative colitis, extracellular vesicles derived from Callicarpa nudiflora not only show advantages in stability, biocompatibility and cellular internalization, but also have intrinsic therapeutic activity. They can synergize with drugs to play anti-inflammatory and antioxidant effects. At the same time, targeting the inflammatory site of the colon can reduce the adverse reactions of the drug and improve the efficacy, thus ensuring the effectiveness and safety of drug delivery.

[0009] Optionally, the particle size of the drug-loaded Callicarpa nudiflora-derived extracellular vesicles is 100 nm to 200 nm.

[0010] Optionally, the ζ potential of the drug-loaded Callicarpa spp. derived extracellular vesicles is -30 mV~-10 mV, which can ensure that the drug-loaded Callicarpa spp. derived extracellular vesicle system is in a relatively stable state.

[0011] Optionally, the drug-loaded Callicarpa nudiflora-derived extracellular vesicles are double-layer phospholipid vesicle structures, and thus can encapsulate hydrophilic drugs and accommodate hydrophobic drugs, thereby improving the insolubility and stability of macromolecular drugs and enhancing drug efficacy.

[0012] Optionally, the ulcerative colitis treatment drug includes at least one of aminosalicylic acid drugs, glucocorticoids, immunosuppressive drugs and biological agents.

[0013] Optionally, the aminosalicylic acid drug includes at least one of mesalazine and sulfasalazine.

[0014] Optionally, the glucocorticoid includes at least one of dexamethasone, budesonide and prednisone.

[0015] Optionally, the immunosuppressive drug includes at least one of azathioprine and mercaptopurine.

[0016] Optionally, the biological agent includes at least one of infliximab and adalimumab.

[0017] In a second aspect, the present invention provides a method for preparing drug-loaded extracellular vesicles derived from Callicarpa nudiflora, comprising the following steps: The juice of Callicarpa nudiflora was squeezed and filtered to obtain a filtrate, and the filtrate was subjected to differential centrifugation and sucrose density gradient centrifugation in sequence to obtain extracellular vesicles derived from Callicarpa nudiflora; Drugs are encapsulated on extracellular vesicles derived from Callicarpa nudiflora to obtain drug-loaded extracellular vesicles derived from Callicarpa nudiflora.

[0018] The preparation method provided by the present invention can provide a reference for constructing other plant-derived extracellular vesicle carrier platforms, and has good innovation and development and application prospects.

[0019] Optionally, the differential centrifugation includes: centrifuging the filtrate at 1,000×g at 4°C for 10 min~20 min to obtain supernatant one; centrifuging supernatant one at 5,000×g at 4°C for 20 min~40 min to obtain supernatant two; centrifuging supernatant two at 10,000×g at 4°C for 40 min~60 min to obtain supernatant three; centrifuging supernatant three at 150,000×g at 4°C for 1.5 h~2 h, resuspending the precipitate with 2 mL~5 mL PBS solution to obtain a crude extract of extracellular vesicles derived from Callicarpa nudiflora.

[0020] The differential centrifugation method utilizes the differences in sedimentation coefficients of components in the sample to apply different centrifugal forces to different particles. The centrifugal speed is gradually increased during multiple centrifugations to allow different particles to settle in turn, thereby obtaining extracellular vesicles from Callicarpa nudiflora of similar size. Specifically, low-speed centrifugation at 1,000×g, 5,000×g, and 10,000×g can remove large particle impurities and cell fragments from Callicarpa nudiflora leaves in the filtrate, and ultracentrifugation at 150,000×g can remove large molecular proteins and soluble impurities in the filtrate.

[0021] Optionally, the sucrose density gradient centrifugation includes: adding a crude extract of extracellular vesicles derived from Callicarpa nudiflora to a mixed sucrose solution, centrifuging the solution at 150,000×g at 4°C for 1.5 h to 2 h to obtain a band containing extracellular vesicles derived from Callicarpa nudiflora, adding an equal amount of PBS solution, mixing the solution, centrifuging the solution at 150,000×g at 4°C for 1 h to 1.5 h, resuspending the precipitate with 2 mL to 5 mL of PBS solution and filtering the solution through a 450 nm filter membrane to obtain extracellular vesicles derived from Callicarpa nudiflora.

[0022] The sucrose density gradient centrifugation method utilizes the density difference of sucrose at different concentrations to form a density gradient of the sample in a mixed sucrose solution. Centrifugation accelerates the sedimentation of the sample and allows molecules of different densities to be stratified in the density gradient to achieve the purpose of separation and purification, thereby obtaining high-purity extracellular vesicles from Callicarpa nudiflora.

[0023] Optionally, the mixed sucrose solution includes sucrose solutions with concentrations of 8%, 30%, 45% and 60%, wherein the volume ratio of the 8%, 30%, 45% and 60% sucrose solutions is 3:3:2:2.

[0024] Optionally, the band containing extracellular vesicles derived from Callicarpa nudiflora is located between the concentration gradient layers of the 30% sucrose solution and the 45% sucrose solution.

[0025] Optionally, the encapsulating the drug on the extracellular vesicles derived from Callicarpa nudiflora includes: mixing the extracellular vesicles derived from Callicarpa nudiflora and the drug in a feed ratio of (20~1):1, incubating at 37°C for 1 h~8 h, and then stirring at 55°C~60°C for 30 min~60 min; wherein the feed ratio of the extracellular vesicles derived from Callicarpa nudiflora and the drug is preferably 10:1, and the co-incubation time is preferably 4 h.

[0026] Since hydrophobic drugs are insoluble in water, they need to be dissolved in ethanol before being loaded onto extracellular vesicles derived from Callicarpa nudiflora. After loading, the drugs are stirred at 55 ℃~60 ℃ to evaporate the ethanol in the solution, and finally filtered through a 450 nm filter membrane to remove unencapsulated free drugs.

[0027] The co-incubation method is the simplest and most commonly used method to load drugs into extracellular vesicles. Drugs are encapsulated into extracellular vesicles through hydrophobic effects, diffusion or electrostatic effects, and can also be loaded into extracellular vesicles along a concentration gradient. The drug loading efficiency depends on the hydrophobicity of the drug molecules. Hydrophobic drugs can interact with the lipid layer of the vesicle membrane and improve the drug loading efficiency.

[0028] In a third aspect, the present invention provides a use of the above-mentioned drug-loaded Callicarpa nudiflora-derived extracellular vesicles in the preparation of a drug for treating ulcerative colitis.

[0029] The drug-loaded Callicarpa nudiflora-derived extracellular vesicles provided by the present invention can target the site of colon inflammation, reduce adverse drug reactions and improve efficacy, provide a new drug delivery strategy for the treatment of ulcerative colitis, and are expected to become an effective strategy for the clinical treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly introduce the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0031] Figure 1 This is a band diagram formed after sucrose density gradient centrifugation of the crude CN-EVLP extract prepared in Example 1. The purified CN-EVLP is mainly distributed in the middle layer of the sucrose solution with a concentration of 30% and 45%; Figure 2 Dynamic light scattering test results of CN-EVLP in performance testing; Figure 3 Dynamic light scattering test results of CN-EVLP-Bud in performance testing; Figure 4 This is the morphology of CN-EVLP under H-600 transmission electron microscope during performance testing; Figure 5This is the morphology of CN-EVLP-Bud under H-600 transmission electron microscope in performance testing; Figure 6 The results of storage stability test of CN-EVLP at 4 ℃, -20 ℃, -80 ℃ and freeze-dried conditions in performance testing; Figure 7 The results of storage stability test of CN-EVLP-Bud at 4 ℃ and -80 ℃ in performance testing; Figure 8 The results of the gastrointestinal stability test of CN-EVLP in the performance test; Fig. 9 The results of the gastrointestinal stability test of CN-EVLP-Bud in the performance test; Fig.10 The scavenging ability of Bud, CN-EVLP and CN-EVLP-Bud on DPPH at 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL and 20 μg / mL in the in vitro antioxidant experiment for performance testing; Fig.11 The Trolox equivalent antioxidant capacity of Bud, CN-EVLP, and CN-EVLP-Bud at 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL in the in vitro antioxidant assay for performance testing; Fig.12 Effects of 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL of Bud, CN-EVLP, and CN-EVLP-Bud on the survival rate of Caco-2 cells in the cytological evaluation for performance testing; Fig.13 Effects of 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL of Bud, CN-EVLP, and CN-EVLP-Bud on the viability of RAW 264.7 cells in cytological evaluation for performance testing; Fig.14 Effects of 2.5 μg / mL, 5 μg / mL, and 10 μg / mL of Bud, CN-EVLP, and CN-EVLP-Bud on NO concentration in RAW 264.7 cells in cytological evaluation of performance testing (n=3; "*", P<0.05; "**", P<0.01; "***", P<0.001; "****", P<0.0001; significant compared with the LPS group); Fig.15Effects of 5 μg / mL Bud, CN-EVLP, and CN-EVLP-Bud on TNF-α concentration in RAW264.7 cells in cytological evaluation of performance testing (n=3; "*", P<0.05; "**", P<0.01; "***", P<0.001; "****", P<0.0001; significant compared with the LPS group); Fig.16 Effects of 5 μg / mL Bud, CN-EVLP, and CN-EVLP-Bud on IL-6 concentration in RAW264.7 cells in the cytological evaluation of performance testing (n=3; "*", P<0.05; "**", P<0.01; "***", P<0.001; "****", P<0.0001; significant compared with the LPS group). DETAILED DESCRIPTION

[0032] The following examples are further listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are only examples in a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0033] The present invention provides a drug-loaded extracellular vesicle derived from Callicarpa nudiflora, wherein the drug-loaded extracellular vesicle derived from Callicarpa nudiflora comprises extracellular vesicles derived from Callicarpa nudiflora and a drug for treating ulcerative colitis loaded in the extracellular vesicles derived from Callicarpa nudiflora; the loading amount of the drug for treating ulcerative colitis in the drug-loaded extracellular vesicles derived from Callicarpa nudiflora is 4.39%~8.72%.

[0034] In some embodiments, the particle size of the drug-loaded Callicarpa nudiflora-derived extracellular vesicles is 100 nm to 200 nm.

[0035] In some embodiments, the zeta potential of the drug-loaded Callicarpa nudiflora-derived extracellular vesicles is -30 mV to -10 mV.

[0036] In some embodiments, the drug-loaded Callicarpa nudiflora-derived extracellular vesicles are double-layer phospholipid vesicle structures.

[0037] In some embodiments, the ulcerative colitis treatment drug includes at least one of an aminosalicylic acid drug, a glucocorticoid, an immunosuppressant, and a biologic.

[0038] In some embodiments, the aminosalicylic acid drug includes at least one of mesalazine and sulfasalazine.

[0039] In some embodiments, the glucocorticoid comprises at least one of dexamethasone, budesonide, and prednisone.

[0040] In some embodiments, the immunosuppressive drug comprises at least one of azathioprine and mercaptopurine.

[0041] In some embodiments, the biologic comprises at least one of infliximab and adalimumab.

[0042] The present invention also provides a method for preparing drug-loaded extracellular vesicles derived from Callicarpa nudiflora, comprising the following steps: S1. Juicing Callicarpa nudiflora and filtering to obtain a filtrate, and subjecting the filtrate to differential centrifugation and sucrose density gradient centrifugation in sequence to obtain Callicarpa nudiflora-derived extracellular vesicles; S2. Encapsulating drugs on extracellular vesicles derived from Callicarpa nudiflora to obtain drug-loaded extracellular vesicles derived from Callicarpa nudiflora.

[0043] In fact, when executing step S1, the following sub-steps are included: S1.1. Mix equal amounts of Callicarpa nudiflora leaves and PBS solution, squeeze juice with a wall-breaking machine, and filter with gauze to obtain a filtrate; centrifuge the filtrate at 1,000×g for 10 min~20 min at 4°C to obtain supernatant 1; centrifuge supernatant 1 at 5,000×g for 20 min~40 min at 4°C to obtain supernatant 2; centrifuge supernatant 2 at 10,000×g for 40 min~60 min at 4°C to obtain supernatant 3; centrifuge supernatant 3 at 150,000×g for 1.5 h~2 h at 4°C, resuspend the precipitate with 2 mL~5 mL PBS solution to obtain a crude extract of extracellular vesicles from Callicarpa nudiflora; S1.2. Add the mixed sucrose solution to the crude extract of extracellular vesicles derived from Callicarpa nudiflora obtained in step S1.1, centrifuge at 150,000×g at 4°C for 1.5 h~2 h to obtain a band containing extracellular vesicles derived from Callicarpa nudiflora. Add an equal amount of PBS solution to the extract, mix well, centrifuge at 150,000×g at 4°C for 1 h~1.5 h, resuspend the precipitate with 2 mL~5 mL of PBS solution and filter through a 450 nm filter membrane to obtain extracellular vesicles derived from Callicarpa nudiflora.

[0044] In some embodiments, when performing step S1.2, the mixed sucrose solution includes sucrose solutions with concentrations of 8%, 30%, 45% and 60%, wherein the volume ratio of the 8%, 30%, 45% and 60% sucrose solutions is 3:3:2:2.

[0045] In some embodiments, when performing step S1.2, the band containing extracellular vesicles derived from Callicarpa nudiflora is located between the concentration gradient layers of the 30% sucrose solution and the 45% sucrose solution.

[0046] In some embodiments, when executing step S2, it includes: mixing the extracellular vesicles derived from Callicarpa nudiflora and the drug in a feed ratio of (20~1):1, incubating at 37°C for 1 h~8 h, and stirring at 55°C~60°C for 30 min~60 min; wherein, the feed ratio of the extracellular vesicles derived from Callicarpa nudiflora and the drug is preferably 10:1, and the co-incubation time is preferably 4 h.

[0047] In some embodiments, since hydrophobic drugs are insoluble in water, the drugs need to be pre-dissolved in ethanol before being encapsulated on extracellular vesicles derived from Callicarpa nudiflora. After encapsulation, the drugs are stirred at 55°C to 60°C to evaporate the ethanol in the solution, and finally filtered through a 450 nm filter membrane to remove unencapsulated free drugs.

[0048] The present invention also provides an application of the above-mentioned drug-loaded Callicarpa nudiflora-derived extracellular vesicles in the preparation of a drug for treating ulcerative colitis.

[0049] Unless otherwise specified, the various reagents and raw materials used in the examples of the present invention are commodities that can be purchased from the market or products that can be prepared by known methods.

[0050] Example 1

[0051] Example 1 of the present invention provides a method for preparing extracellular vesicles derived from Callicarpa nudiflora carrying budesonide, comprising the following steps: S1.1. Mix 500 g of Callicarpa nudiflora leaves with 500 mL of PBS solution, squeeze the juice with a wall-breaking machine for 3 min, and filter the filtrate with gauze; centrifuge the filtrate at 1,000×g for 15 min at 4°C to obtain supernatant 1; centrifuge supernatant 1 at 5,000×g for 30 min at 4°C to obtain supernatant 2; centrifuge supernatant 2 at 10,000×g for 60 min at 4°C to obtain supernatant 3; centrifuge supernatant 3 at 150,000×g for 2 h at 4°C, remove the precipitate and take the supernatant to obtain the crude extract of extracellular vesicles (CN-EVLP) derived from Callicarpa nudiflora; S1.2. Prepare sucrose solutions with concentrations of 8%, 30%, 45% and 60% respectively, and add them to the ultracentrifuge tube in the order of v(8%): ​​v(30%): v(45%): v(60%)=3:3:2:2. Then add an equal amount of PBS solution to resuspend the CN-EVLP crude extract, and centrifuge at 150,000×g for 2 h at 4°C to obtain CN-EVLP bands. Figure 1As shown; the CN-EVLP strips were mixed with PBS solution and centrifuged at 150,000×g for 1 h at 4°C to obtain a precipitate. 3 mL of PBS solution was taken to blow the precipitate and then filtered through a 450 nm filter to obtain a CN-EVLP solution; S2. Dissolve 1 mg of budesonide (Bud) in 1 mL of ethanol to prepare a 1 mg / mL Bud solution. Mix 0.1 mL of Bud solution with 1 mL of CN-EVLP solution, incubate on a shaker at 37 °C and 100 r / min for 4 h, stir at 55 °C for 35 min, and filter through a 450 nm filter to obtain extracellular vesicles derived from Callicarpa nudiflora (CN-EVLP-Bud) loaded with budesonide.

[0052] Example 2

[0053] Example 2 of the present invention provides a method for preparing extracellular vesicles derived from Callicarpa nudiflora carrying budesonide, which is different from Example 1 in that, in step S2: the volume ratio of the Bud solution and the CN-EVLP solution is 1:1; the incubation time of the Bud solution and the CN-EVLP solution is 2 h.

[0054] Example 3

[0055] Example 3 of the present invention provides a method for preparing extracellular vesicles derived from Callicarpa nudiflora carrying budesonide, which is different from Example 1 in that, in step S2: the volume ratio of the Bud solution and the CN-EVLP solution is 1:5; and the incubation time of the Bud solution and the CN-EVLP solution is 2 h.

[0056] Example 4

[0057] Example 4 of the present invention provides a method for preparing extracellular vesicles derived from Callicarpa nudiflora carrying budesonide, which is different from Example 1 in that, in step S2: the volume ratio of the Bud solution and the CN-EVLP solution is 1:10; the incubation time of the Bud solution and the CN-EVLP solution is 2 h.

[0058] Example 5

[0059] Example 5 of the present invention provides a method for preparing extracellular vesicles derived from Callicarpa nudiflora carrying budesonide, which is different from Example 1 in that, in step S2, the volume ratio of Bud and CN-EVLP solution is 1:20; and the incubation time of Bud solution and CN-EVLP solution is 2 h.

[0060] Example 6

[0061] Example 6 of the present invention provides a method for preparing extracellular vesicles derived from Callicarpa nudiflora carrying budesonide, which is different from Example 1 in that, in step S2: the volume ratio of Bud and CN-EVLP solution is 1:5; and the incubation time of Bud solution and CN-EVLP solution is 1 h.

[0062] Example 7

[0063] Example 7 of the present invention provides a method for preparing extracellular vesicles derived from Callicarpa nudiflora carrying budesonide, which is different from Example 1 in that, in step S2, the volume ratio of Bud and CN-EVLP solution is 1:5; and the incubation time of Bud solution and CN-EVLP solution is 4 h.

[0064] Example 8

[0065] Example 8 of the present invention provides a method for preparing extracellular vesicles derived from Callicarpa nudiflora carrying budesonide, which is different from Example 1 in that, in step S2, the volume ratio of Bud and CN-EVLP solution is 1:5; and the incubation time of Bud solution and CN-EVLP solution is 8 h.

[0066] Performance Testing

[0067] The CN-EVLP and CN-EVLP-Bud prepared in Example 1 were subjected to performance testing, including: (1) The average particle size, polydispersity coefficient and Zeta potential of CN-EVLP and CN-EVLP-Bud were measured using a dynamic laser scattering particle size analyzer. The measurements were repeated three times. The results are shown in Table 1.

[0068] Table 1 Average particle size, polydispersity index and potential value of CN-EVLP and CN-EVLP-Bud

[0069] Figure 2 This is the dynamic light scattering detection result of CN-EVLP. Figure 3 This is the dynamic light scattering test result of CN-EVLP-Bud. The results show that there is no significant difference in the particle size of CN-EVLP and CN-EVLP-Bud and both are consistent with the distribution range of plant-derived extracellular vesicles. They have good dispersion and normal distribution. The Zeta potential is similar and consistent with that of plant-derived extracellular vesicles.

[0070] (2) Transmission electron microscopy

[0071] After CN-EVLP and CN-EVLP-Bud were diluted with PBS solution, 10 μL of the diluted CN-EVLP and CN-EVLP-Bud were respectively pipetted onto the microporous copper mesh. After natural drying, they were stained with 2% phosphotungstic acid for 3 min. The residual phosphotungstic acid was washed with distilled water and dried with filter paper. The copper mesh was placed under an H-600 transmission electron microscope to observe the morphological structure and photographed for preservation.

[0072] The transmission electron microscopy results of CN-EVLP are as follows Figure 4 As shown in Figure 1, it was found that CN-EVLP has a phospholipid bilayer structure, with a nearly round shape and different particle sizes, but all distributed within the definition of plant-derived extracellular vesicles; the transmission electron microscopy results of CN-EVLP-Bud are shown in Figure 1. Figure 5 As shown, it was observed that CN-EVLP-Bud also had a phospholipid bilayer structure, and its shape was approximately circular, almost consistent with the shape and size of CN-EVLP.

[0073] (3) Storage stability test

[0074] Four portions of 10 mL of CN-EVLP were taken into four EP tubes, one was stored at 4 ℃, one was stored at -20 ℃, one was stored at -80 ℃, and one was freeze-dried. The particle sizes of CN-EVLP under the four storage conditions were measured at 1 d, 3 d, 5 d, 7 d, 9 d, 11 d, and 14 d, respectively. Two portions of 10 mL of CN-EVLP-Bud were taken into two EP tubes, one was stored at 4 ℃, and the other was stored at -80 ℃. The particle sizes of CN-EVLP-Bud under the two storage conditions were measured at 1 d, 3 d, 5 d, 7 d, 9 d, 11 d, 14 d, and 30 d, respectively. When measuring the particle size using a nanoparticle sizer, the solution must be filtered through a 450 nm filter membrane.

[0075] Freeze-drying conditions: Weigh 1.8917 g of trehalose and dissolve it in 100 mL of deionized water to prepare a 50 mM trehalose solution. Mix 300 mg / mL CN-EVLP solution and trehalose solution in equal volumes, equilibrate at 37 °C for 30 min, pre-freeze at -80 °C for 12 h, and place in a freeze dryer; freeze-dry in a freeze dryer for 24 h and store in a dry place at room temperature. When reconstituted, add deionized water equal to the volume of CN-EVLP and quickly place in a 37 °C water bath, and shake rapidly to dissolve.

[0076] The stability results of CN-EVLP are as follows Figure 6As shown in the figure, it was found that the particle size of CN-EVLP did not change much within 14 days under the four storage conditions. The particle size change was slightly larger under the freeze-dried condition, and the particle size change was the smallest at 4°C and -80°C. Therefore, CN-EVLP can be stored at 4°C for short-term storage and -80°C for long-term storage for subsequent drug loading experiments.

[0077] The stability results of CN-EVLP-Bud are shown in Figure 7 As shown in the figure, it was found that the particle size of CN-EVLP-Bud did not change much within 30 days under the two storage conditions of 4 ℃ and -80 ℃. Therefore, CN-EVLP-Bud can be stored for subsequent experiments by short-term storage at 4 ℃ and long-term storage at -80 ℃.

[0078] (4) Gastrointestinal stability test

[0079] 1.34 μL of 18.5% HCl (pH=2.0) and 24 μL of 80 mg / mL pepsin solution (pH=2.0 adjusted with 0.1 g / L HCl) were mixed to simulate gastric conditions, and 1 mL of CN-EVLP and CN-EVLP-Bud resuspended in PBS solution were added, respectively, and incubated at 37 °C for 1 h, 2 h, 3 h, and 4 h, respectively, and the particle size and Zeta potential were measured.

[0080] After adding 80 μL of a mixture containing 24 mg / mL bile extract and 4 mg / mL pancreatic enzyme to 0.1 g / L NaHCO3, the pH of the mixture was adjusted to 6.5 with 1 g / L NaHCO3 to simulate small intestine conditions. 1 mL of CN-EVLP and CN-EVLP-Bud resuspended in PBS solution were added and incubated at 37 °C for 1 h, 2 h, 3 h, 4 h, 6 h and 12 h, respectively, and the particle size and Zeta potential were measured.

[0081] The results of CN-EVLP are as follows Figure 8 As shown in the results, it was found that after 4 h of gastric digestion, the particle size of CN-EVLP increased from 126.03 nm to 160.44 nm, and the surface charge increased from -10.76 mV to -6.05 mV; after 12 h of intestinal digestion, the particle size of CN-EVLP increased to 205.50 nm, and the surface charge decreased to about -31.42 mV, which was consistent with the change law in gastrointestinal fluid.

[0082] The results of CN-EVLP-Bud are as follows Fig. 9As shown in the results, it was found that after 4 h of gastric juice digestion, the particle size of CN-EVLP-Bud changed from 132.02 nm to 126.68 nm, and the surface charge increased from -10.20 mV to -2.56 mV; after 12 h of intestinal juice digestion, the particle size of CN-EVLP-Bud increased to 351.47 nm, and the surface charge decreased to about -27.71 mV, which was consistent with the change law in gastrointestinal juice.

[0083] (5) Determination of encapsulation efficiency and drug loading rate

[0084] 1 mL of CN-EVLP-Bud was mixed with 3 mL of acetonitrile and then subjected to water bath ultrasound for 2 h to release the drug. The encapsulation efficiency and drug loading rate of the drug in CN-EVLP were determined by HPLC. The average values ​​of the three parallel measurements were obtained, and the encapsulation efficiency was 76.74% and the drug loading rate was 7.67%.

[0085] (6) In vitro antioxidant test

[0086] To evaluate the antioxidant capacity of CN-EVLP and CN-EVLP-Bud, free radical scavenging assays of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were performed. The antioxidant activities of Bud, CN-EVLP and CN-EVLP-Bud at 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL and 20 μg / mL were measured by simulating physiological free radical conditions (the concentration of Bud was used as the basis for calculation).

[0087] DPPH exists stably in organic solvents in the form of nitrogen-containing free radicals at room temperature. It turns purple in ethanol and is reduced to colorless in the presence of antioxidant molecules. Different concentrations of Bud, CN-EVLP and CN-EVLP-Bud were mixed with equal volumes of 0.05 mg / mL DPPH ethanol solution, incubated in the dark for 30 min, and the absorbance of the samples was measured at 517 nm. The DPPH scavenging rate was calculated using the following formula: , Among them, A sample is the absorbance value of the drug-administered preparation group, A control is the absorbance value of the control group, A blank is the absorbance value of the blank group.

[0088] ABTS can be oxidized to green ABTS + , in the presence of antioxidant molecules ABTS + The production of ABTS will be inhibited, the reaction system will fade and the absorbance will decrease. Therefore, the ability of the sample to scavenge ABTS free radicals can be reflected by the determination of absorbance. The ABTS solution and the oxidant solution in the kit were mixed in equal volumes and stored in the dark at room temperature for 12 h as the working solution; 10 μL of Bud, CN-EVLP and CN-EVLP-Bud of different concentrations were mixed with 200 μL of ABTS working solution respectively, and the absorbance was measured at 734 nm after standing for 6 min. The Trolox-Equivalent Antioxidant Capacity (TEAC) was used to represent the ABTS free radical scavenging ability.

[0089] DPPH scavenging rate and ABTS antioxidant capacity were Fig.10 and Fig.11 As shown, the results showed that the free radical scavenging activity of CN-EVLP and CN-EVLP-Bud varied in a dose-dependent manner in the range of 0.625 μg / mL~20 μg / mL, while Bud had almost no free radical scavenging activity.

[0090] (7) Cytological evaluation

[0091] ① The CCK-8 method was used to screen the safe concentrations of Bud, CN-EVLP and CN-EVLP-Bud for Caco-2 cells and RAW 264.7 cells. The specific steps included: Caco-2 cells were cultured at 5 × 10 3 RAW 264.7 cells were cultured at 2×10 4 After the cells were attached to the wall, the old culture medium was removed, and 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL and 20 μg / mL of Bud solution, CN-EVLP solution and CN-EVLP-Bud solution were added respectively (the concentration of Bud was used as the basis for calculation). Six replicate wells were set for different concentrations of different samples. After incubation for 24 h, the supernatant was discarded and the cells were washed twice with PBS. After adding 100 μL of CCK-8 culture medium solution (basic culture medium: CCK-8=9:1) to each well, the plates were shielded from light and placed in an incubator for 4 h. The 96-well plates were taken out to avoid light, and the absorbance at 450 nm was measured. The cell survival rate was calculated according to the following formula: , Among them, OD sample is the OD value of the drug administration group, OD controlis the OD value of the control group, OD blank is the OD value of the blank group.

[0092] The viability of Caco-2 cells and RAW 264.7 cells were Fig.12 and Fig.13 As shown in the figure, the results showed that Caco-2 cells and RAW 264.7 cells had no obvious cytotoxicity in the range of 1.25 μg / mL~20 μg / mL after being treated with CN-EVLP for 24 h, and had no obvious cytotoxicity in the range of 1.25 μg / mL~10 μg / mL after being treated with CN-EVLP-Bud for 24 h, while no obvious cytotoxicity was observed in the range of 1.25 μg / mL~5 μg / mL after Bud treatment. However, certain toxicity was observed in the CN-EVLP-Bud group and the Bud group when the concentration was greater than this range.

[0093] ② Take RAW 264.7 cells that are in good growth condition and in the logarithmic growth phase and use 2×10 4 The cells were seeded into a 24-well plate at a density of 100 cells / well, and 0.5 mL of cell suspension was added to each well and cultured in a cell culture incubator. After the cells adhered to the wall, they were washed three times with PBS and 1 μg / mL of LPS was added to induce M1 phenotype macrophages. After the macrophage polarization was completed, the concentrations of NO, TNF-α and IL-6 were measured.

[0094] After 1 μg / mL of LPS was incubated with different preparations (Bud, CN-EVLP and CN-EVLP-Bud) of different concentrations (2.5 μg / mL, 5 μg / mL and 10 μg / mL) for 24 h, 50 μL of the supernatant was taken and added to a 96-well plate with the gradient diluted standard solution. Griess Reagent I and II reagents restored to room temperature were added, and the absorbance of each group was measured at 540 nm. The corresponding NO content was calculated according to the standard curve.

[0095] After incubation with 1 μg / mL LPS and 5 μg / mL Bud, CN-EVLP, and CN-EVLP-Bud for 24 h, the samples were processed according to the steps of the ELISA kit, the absorbance of each group was measured at 540 nm, and the corresponding TNF-α and IL-6 contents were calculated according to the standard curve.

[0096] The results of NO, TNF-α and IL-6 contents were Fig.14 , Fig.15 and Fig.16As shown in the figure, the concentrations of NO, TNF-α and IL-6 in the supernatant of cells in the LPS stimulation group were significantly higher than those in the normal group, proving the successful establishment of the inflammation model. After treatment with Bud, CN-EVLP and CN-EVLP-Bud, the secretion of NO, TNF-α and IL-6 was significantly inhibited, and the anti-inflammatory effect of CN-EVLP-Bud was better than that of the other two groups, further verifying the importance of CN-EVLP as a drug carrier. At the same time, it was observed that Bud, CN-EVLP and CN-EVLP-Bud inhibited the production of NO in a concentration-dependent manner.

[0097] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. A drug-loaded extracellular vesicle derived from Callicarpa nudiflora, characterized in that: The drug-loaded extracellular vesicles derived from Callicarpa nudiflora include extracellular vesicles derived from Callicarpa nudiflora and drugs for treating ulcerative colitis loaded in the extracellular vesicles derived from Callicarpa nudiflora; the loading amount of the drugs for treating ulcerative colitis in the drug-loaded extracellular vesicles derived from Callicarpa nudiflora is 4.39%~8.72%.

2. The drug-loaded extracellular vesicles derived from Callicarpa nudiflora according to claim 1, characterized in that: The particle size of the drug-loaded extracellular vesicles derived from Callicarpa nudiflora is 100 nm~200 nm; the ζ potential of the drug-loaded extracellular vesicles derived from Callicarpa nudiflora is -30 mV~-10 mV; the drug-loaded extracellular vesicles derived from Callicarpa nudiflora is a double-layer phospholipid vesicle structure; the ulcerative colitis treatment drug includes at least one of aminosalicylic acid drugs, glucocorticoids, immunosuppressants and biological agents.

3. The drug-loaded extracellular vesicles derived from Callicarpa nudiflora according to claim 2, characterized in that: The aminosalicylic acid drugs include at least one of mesalazine and sulfasalazine; the glucocorticoids include at least one of dexamethasone, budesonide and prednisone; the immunosuppressive drugs include at least one of azathioprine and mercaptopurine; and the biological agents include at least one of infliximab and adalimumab.

4. A method for preparing drug-loaded extracellular vesicles derived from Callicarpa nudiflora, characterized in that: The following steps are involved: The juice of Callicarpa nudiflora was squeezed and filtered to obtain a filtrate, and the filtrate was subjected to differential centrifugation and sucrose density gradient centrifugation in sequence to obtain extracellular vesicles derived from Callicarpa nudiflora; Drugs are encapsulated on extracellular vesicles derived from Callicarpa nudiflora to obtain drug-loaded extracellular vesicles derived from Callicarpa nudiflora.

5. The preparation method according to claim 4, characterized in that: The differential centrifugation includes: centrifuging the filtrate at 1,000×g for 10 min~20 min at 4°C to obtain supernatant one; centrifuging supernatant one at 5,000×g for 20 min~40 min at 4°C to obtain supernatant two; centrifuging supernatant two at 10,000×g for 40 min~60 min at 4°C to obtain supernatant three; centrifuging supernatant three at 150,000×g for 1.5 h~2 h at 4°C, resuspending the precipitate with 2 mL~5 mL PBS solution to obtain a crude extract of extracellular vesicles derived from Callicarpa nudiflora.

6. The preparation method according to claim 4 or 5, characterized in that: The sucrose density gradient centrifugation includes: adding a crude extracellular vesicle extract from Callicarpa nudiflora to a mixed sucrose solution, centrifuging the solution at 150,000×g for 1.5 h to 2 h at 4°C to obtain a band containing extracellular vesicles from Callicarpa nudiflora, adding an equal amount of PBS solution, mixing the solution, centrifuging the solution at 150,000×g for 1 h to 1.5 h at 4°C, resuspending the precipitate with 2 mL to 5 mL of PBS solution, and filtering the solution through a 450 nm filter membrane to obtain extracellular vesicles from Callicarpa nudiflora.

7. The preparation method according to claim 6, characterized in that: The mixed sucrose solution includes sucrose solutions with concentrations of 8%, 30%, 45% and 60%; the strip containing extracellular vesicles derived from Callicarpa nudiflora is located between the concentration gradient layers of the 30% sucrose solution and the 45% sucrose solution.

8. The preparation method according to claim 4, characterized in that: The method of encapsulating drugs on extracellular vesicles derived from Callicarpa nudiflora includes: mixing the extracellular vesicles derived from Callicarpa nudiflora and the drug in a feed ratio of (20~1):1, incubating at 37°C for 1 h~8 h, stirring at 55°C~60°C for 30 min~60 min, and filtering through a 450 nm filter membrane.

9. Use of the drug-loaded extracellular vesicles derived from Callicarpa nudiflora as described in any one of claims 1 to 3, or the drug-loaded extracellular vesicles derived from Callicarpa nudiflora prepared by the preparation method described in any one of claims 4 to 8, in the preparation of drugs for treating ulcerative colitis.

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