Plant cell vesicle and preparation method thereof

Abstract

The invention discloses a plant cell vesicle and a preparation method thereof, and belongs to the technical field of biology. The preparation method comprises the steps of plant tissue pretreatment (adding cane sugar as an osmotic pressure protective agent), high-pressure homogenization (100-300MPa, 2-3 times), composite enzymolysis (mixing cellulase and pectinase, 35-45 DEG C, 10-30 minutes), differential centrifugation and ultracentrifugation (100,000 * g, 2 hours) combined purification. The average particle size of the obtained vesicle is 50-200nm, and the vesicle can be used for drug molecule or nucleic acid delivery. By optimizing parameters and additives in each step, the yield, structural integrity and functional characteristics of the vesicles are remarkably improved, and technical support is provided for application of the plant-derived vesicles in the field of nano-drug delivery.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a plant cell vesicle and a preparation method thereof. BACKGROUND

[0002] Cell vesicles are nanoscale vesicular structures secreted by cell membranes or endomembrane systems, with low immunogenicity, natural targeting and good biocompatibility, and have broad application prospects in the fields of drug delivery, gene therapy, etc. Compared with animal-derived vesicles (such as exosomes), plant cell vesicles are widely available, low-cost and free of ethical controversy, and have become a research hotspot in recent years. However, the existing preparation methods of plant cell vesicles have the following key problems:

[0003] Firstly, the traditional purification method relies on single ultracentrifugation, which is difficult to effectively remove cell debris, large particle impurities and protein complexes, resulting in low vesicle purity (usually < 70%); secondly, the pre-treatment step lacks osmotic pressure protection, and plant cells are prone to rupture or autolysis due to osmotic pressure change during homogenization, affecting the structural integrity of the vesicles; thirdly, the enzymolysis conditions (such as enzyme type, temperature, time) are not standardized, and a single enzyme (such as cellulase) is difficult to break the polysaccharide-pectin complex structure of plant cell wall, resulting in low vesicle yield (usually < 5%); fourthly, there is a lack of surface marker verification, which cannot confirm the membrane source and functional characteristics of the vesicles. These problems limit the practical application of plant cell vesicles in the field of drug delivery, etc.

[0004] Therefore, it is urgent to develop a standardized, high-yield and high-purity plant cell vesicle preparation method to promote its clinical translation. SUMMARY

[0005] The present application first provides a separation method of plant cell vesicles, comprising the following steps:

[0006] a) pre-treatment of plant tissue or cells;

[0007] b) high-pressure homogenization treatment;

[0008] c) complex enzymolysis treatment;

[0009] d) differential centrifugation combined with ultracentrifugation purification.

[0010] In some embodiments, the pressure of high-pressure homogenization in step b) is 100-300 MPa, and the cycle number is 2-3 times.

[0011] In some embodiments, the complex enzyme in step c) is a mixture of cellulase and pectinase, the enzymolysis temperature is 35-45℃, and the time is 10-30 minutes.

[0012] In some embodiments, the ultracentrifugation conditions in step d) are 100,000 × g for 2 hours.

[0013] In some embodiments, sucrose, an osmotic pressure protectant, is added during the pretreatment step.

[0014] In some embodiments, the chelating agent EDTA is added to the enzymatic hydrolysis system.

[0015] In some embodiments, the average particle size of the resulting vesicles is 50-200 nm.

[0016] The present invention also provides plant cell vesicles prepared by the above method, which can be used for encapsulating drug molecules or delivering nucleic acids.

[0017] The present invention also provides a drug delivery system comprising the above-described plant cell vesicles as a carrier.

[0018] Finally, this invention provides the application of the above-mentioned plant cell vesicles in the preparation of antitumor or anti-inflammatory nanomaterials.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] (1) Adding sucrose during the pretreatment stage: maintains the osmotic pressure balance inside and outside the cell, reduces cell rupture and leakage of contents during homogenization, and increases the integrity rate of vesicle structure by more than 30%.

[0021] (2) Optimization of high pressure homogenization parameters: The pressure range of 100-300MPa can effectively destroy the cellulose-pectin network of plant cell walls, while avoiding excessive pressure that leads to vesicle aggregation. The vesicle yield is 2-3 times higher than that of traditional homogenization.

[0022] (3) Composite enzymatic hydrolysis system: Cellulase and pectinase work together to specifically degrade the main components of the cell wall (cellulose and pectin), and the enzymatic hydrolysis efficiency is 40% higher than that of a single enzyme, with a vesicle yield of 8-12%.

[0023] (4) EDTA chelation aid: chelating Mg 2+ Ca 2+ The presence of metal ions relieves their inhibitory effect on enzymes, increasing enzyme activity by 25%.

[0024] (5) Ultracentrifugation purification: Centrifugation at 100,000×g for 2 hours can effectively separate vesicles and protein complexes, and the purity is 20% higher than that of ultracentrifugation alone.

[0025] The plant cell vesicles prepared by this invention have uniform particle size and good biocompatibility, and can be used to encapsulate drug molecules or nucleic acids, providing a novel plant-derived carrier for targeted delivery systems. Attached Figure Description

[0026] Figure 1 Transmission electron microscope images of peony vesicles.

[0027] Figure 2 1. Peony vesicle particle size distribution curve.

[0028] Figure 3 ROS inhibition rate of peony vesicles.

[0029] Figure 4 Promotes collagen synthesis; A represents the result for type I collagen; B represents the result for type III collagen. Detailed Implementation

[0030] The methods and applications of the present invention have been described through preferred embodiments. Those skilled in the art will be able to make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the technology of the present invention.

[0031] Example 1: Preparation of peony cell vesicles

[0032] Plant materials: The high-quality ornamental peony variety 'Yao Huang' (Paeonia suffruticosa 'Yao Huang') which is widely promoted in commercial cultivation was selected. This variety has golden yellow flowers and full flower shape. It is a traditional famous variety in my country with a high market share and has a stable genetic background and a mature commercial cultivation system.

[0033] Reagents and instruments:

[0034] Osmotic pressure protectant: sucrose (Sinopharm Chemical Reagent Co., Ltd., analytical grade);

[0035] Buffer solution: PBS buffer (pH 7.4, Hyclone, SH30256.01);

[0036] Complex enzymes: cellulase (10 U / mg, Sigma-Aldrich, C1184), pectinase (5 U / mg, Sigma-Aldrich, P2736);

[0037] Chelating agent: EDTA (Sigma-Aldrich, E6758);

[0038] Instruments: High-pressure homogenizer (ATS Engineering Inc., Model 100), ultracentrifuge (Beckman Coulter, Optima L-100XP), nanoparticle size analyzer (Malvern Panalytical, Nano ZS), flow cytometry (BDFACSCanto II).

[0039] step:

[0040] Pretreatment: Take 10g of peony leaves, cut them into small pieces, add 50mL of pre-cooled PBS buffer containing 0.5M sucrose (containing protease inhibitor PMSF, 1mM), homogenize 3 times (10,000rpm, 30 seconds / time) using a tissue homogenizer (IKA, T25), centrifuge at 4℃, 1,000×g for 10 minutes, discard the supernatant, and resuspend the precipitate in PBS to 20mL.

[0041] High-pressure homogenization: The cell suspension was homogenized three times at 200 MPa (with a 5-minute interval between each time) and stored at 4°C.

[0042] Compound enzymatic hydrolysis: Dilute the homogenate to 50 mL with PBS, add cellulase (final concentration 20 U / mL) and pectinase (final concentration 10 U / mL), mix well, add EDTA (final concentration 10 mM), and enzymatically hydrolyze for 30 minutes at 37°C with shaking (150 rpm).

[0043] Purification: The enzymatic hydrolysate was centrifuged at 1,000×g for 10 minutes at 4°C to remove unbroken cells, and then centrifuged at 10,000×g for 20 minutes to remove cell debris. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 100,000×g for 2 hours (4°C). The precipitate was resuspended in PBS to obtain peony cell vesicles. The electron micrographs of the black truffle vesicles are shown below. Figure 1 As shown; particle size and concentration results are as follows. Figure 2 As shown.

[0044] Example 2: Optimization of high-pressure homogenization parameters (control group: 100MPa vs 200MPa)

[0045] Grouping:

[0046] Control group 1: High-pressure homogenization pressure 100MPa, 3 times;

[0047] Experimental group 1: High-pressure homogenization pressure 200MPa, 3 times (same as Example 1).

[0048] Detection indicators: vesicle yield (total protein content measured by BCA method), average particle size (nanoparticle size analyzer).

[0049] Results: The vesicle yield in the control group was 4.2%, with an average particle size of 100-150 nm; the yield in the experimental group was 8.5%, with a particle size of 60-100 nm. This indicates that 200 MPa is more conducive to cell wall disruption, increasing yield and reducing particle size.

[0050] Example 3: Validation of ultracentrifugation conditions (control group: 100,000×g for 1 hour vs. 2 hours)

[0051] Grouping:

[0052] Control group 3: Ultracentrifugation at 100,000 × g for 1 hour;

[0053] Experimental group 3: Ultracentrifugation at 100,000×g for 2 hours (same as Example 1).

[0054] Detection indicators: protein impurity content (Bradford method), purity (Western blot detection of TSG101, internal control antibody purchased from Cell Signaling, catalog number: 5019S).

[0055] Results: The total protein content in the supernatant of control group 3 was 0.8 mg / mL, and the TSG101 band gray value was 0.3; the protein content in the supernatant of experimental group 3 was 0.3 mg / mL, and the TSG101 gray value was 0.8, indicating that extending the centrifugation time can effectively remove impurities and improve vesicle purity.

[0056] Example 4: Vesicle Encapsulation Capacity Test

[0057] The vesicles prepared in Example 1 were incubated with fluorescently labeled siRNA (FAM-siRNA, purchased from RiboBio, catalog number: siRNA-FAM-001) at a mass ratio of 1:10 for 30 minutes (37°C), and the fluorescence intensity of FAM within the vesicles was detected by flow cytometry. The results showed that the siRNA encapsulation efficiency within the vesicles reached 65±5%, indicating that it has good nucleic acid encapsulation capacity.

[0058] In Examples 2-4 above, control groups with single parameter changes (such as pressure, enzyme type, centrifugation time) were set up to verify the necessity and optimization effect of the parameters in each step of the present invention.

[0059] Example 5: Verification experiment on the inhibition of ROS generation by peony vesicles

[0060] Experimental objective: To verify the inhibitory effect of different concentrations of peony vesicles (MD) on the generation of intracellular reactive oxygen species (ROS) and the optimal concentration for this effect.

[0061] Experimental materials: human skin fibroblasts (HDFs), peony vesicle extract prepared in Example 1 (0.01%, 0.1%, 1%, denoted as MD-L, MD-M, MD-H, respectively), vitamin E (VE, 0.1%, positive control), DCFH-DA fluorescent probe, DMEM culture medium, fetal bovine serum (FBS), and trypsin digestion solution.

[0062] Experimental steps:

[0063] Cell culture: HDFs were seeded in DMEM medium containing 10% FBS and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%, the cells were passaged and the cells from the 3rd to 5th generations were used for experiments.

[0064] Grouping and treatment: Take cells in the logarithmic growth phase and divide them into groups of 5 × 10⁻⁶. 4 Cells / wel l were seeded in 6-well plates and cultured for 24 hours, then the medium was replaced with serum-free medium for synchronization for another 24 hours. They were randomly divided into 5 groups:

[0065] Control group (no treatment, containing only cells and culture medium);

[0066] Vitamin E group (final concentration 0.1%);

[0067] MD-L group (peony vesicles 0.01%);

[0068] MD-M group (peony vesicles 0.1%);

[0069] MD-H group (1% peony vesicles).

[0070] Each group has 3 replicates, and ROS is detected after 24 hours of treatment.

[0071] ROS assay: Discard the culture medium, add serum-free medium containing 10 μM DCFH-DA, incubate at 37°C for 30 min, and wash three times with PBS to remove excess probe. Detect the fluorescence intensity at excitation wavelength 488 nm and emission wavelength 525 nm using a fluorescence microplate reader, and calculate the ROS inhibition rate.

[0072] ROS inhibition rate = (1 - fluorescence intensity of control group / fluorescence intensity of experimental group) × 100%

[0073] Experimental parameters:

[0074] Cell density: 5×10 4 cells / well (6-well plate);

[0075] Processing time: 24 hours;

[0076] Probe concentration: 10 μM;

[0077] Incubation temperature: 37℃;

[0078] Detection wavelength: excitation 488nm, emission 525nm.

[0079] Experimental results are as follows Figure 3 As shown, compared with the Control group, the VE group, MD-M group and MD-H group all significantly reduced the ROS fluorescence intensity (P<0.05, P<0.01, P<0.001), among which the MD-H group had the highest inhibition rate (30.0%), indicating that the peony vesicles have a concentration-dependent inhibitory effect on ROS generation.

[0080] The inhibition rate of the MD-M group (0.1%) was 22.4%, which is close to that of the VE positive control group (17.9%), indicating that a moderate concentration of peony vesicles can achieve a relatively ideal effect; the MD-L group (0.01%) had a weaker inhibitory effect (4.0%), suggesting that the low concentration of vesicles had insufficient biological activity.

[0081] Example 6: Verification Experiment on the Promotion of Collagen Synthesis by Peony Vesicles

[0082] Experimental objective: To verify the promoting effect of peony vesicles (MD) on the synthesis of type I collagen (Col I) and type III collagen (Col III), and to compare the effect of growth factor TGF-β1.

[0083] Experimental materials: human skin fibroblasts (HDFs), peony vesicle extract prepared in Example 1 (0.01%, MD), TGF-β1 (0.01ppm, positive control), mouse anti-human ColⅠ ELISA kit, mouse anti-human ColⅢ ELISA kit, DMEM medium, fetal bovine serum (FBS).

[0084] Experimental steps:

[0085] Cell culture: Same as in Example 5.

[0086] Grouping and treatment: Take cells in the logarithmic growth phase and divide them into groups of 1×10⁻⁶. 5 Cells / wel l were seeded in 12-well plates and cultured for 24 hours, then the medium was replaced with serum-free medium for synchronization for another 24 hours. They were randomly divided into 3 groups:

[0087] Control group (no processing applied);

[0088] TGF-β1 group (final concentration 0.01 ppm);

[0089] MD group (peony vesicles 0.01%).

[0090] Each group was set up with 3 replicates. After 48 hours of treatment, the cell culture supernatant was collected for collagen content detection.

[0091] Collagen detection: ELISA was used, following the kit instructions, to determine the concentrations (ng / mL) of ColⅠ and ColⅢ in the supernatant. The control group was considered as 100%, and the relative expression levels for each group were calculated.

[0092] Relative expression level = (control group concentration / experimental group concentration) × 100%

[0093] Experimental parameters:

[0094] Cell density: 1×10 5 cells / well (12-well plate);

[0095] Processing time: 48 hours;

[0096] Growth factor concentration: 0.01 ppm;

[0097] Vesicle concentration: 0.01%;

[0098] Detection kit: ELISA method (sensitivity: ColⅠ≥1.0ng / mL, ColⅢ≥0.5ng / mL).

[0099] Experimental results are as follows Figure 4 As shown, the results of the type I collagen (Col I) test are as follows:

[0100] The relative expression level of ColⅠ in the Control group was 100%, in the TGF-β1 group it was 106.49% (↑6.49%), and in the MD group it was 116.91% (↑16.91%). The increase in ColⅠ expression in the MD group was more significant than that in the TGF-β1 group (+10.42%), indicating that the promoting effect of peony vesicles on ColⅠ synthesis was superior to that of growth factors.

[0101] Type III collagen (Col III) detection results: The relative expression level of Col III in the Control group was 100%, in the TGF-β1 group it was 113.35% (↑13.35%), and in the MD group it was 112.69% (↑12.69%). The effects of the MD group and the TGF-β1 group were similar (difference only 0.66%), and both were significantly higher than the control group (*P<0.05).

[0102] In summary, the peony vesicles prepared in this invention (MD group) significantly promoted the synthesis of both Col I and Col III. The increase in Col I (16.91%) was higher than that of TGF-β1 (6.49%), demonstrating its advantage in promoting type I collagen (primarily maintaining skin tension). The increase in Col III (12.69%) was comparable to that of TGF-β1 (13.35%), indicating that its effect on type III collagen (primarily involved in early wound repair) was on par with the positive control drug. This confirms that peony vesicles, by regulating fibroblast differentiation, bidirectionally promote the synthesis of type I and type III collagen, demonstrating potential for anti-wrinkle skincare applications.

[0103] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for isolating plant cell vesicles, characterized in that, The method comprises the following steps: a) pretreatment of plant tissue or cells; b) high pressure homogenization treatment; c) complex enzymatic hydrolysis treatment; d) differential centrifugation combined with ultracentrifugation purification.

2. The method of claim 1, wherein, The pressure of high pressure homogenization in step b) is 100-300 MPa, and the cycle number is 2-3 times.

3. The method of claim 1, wherein, In step c), the complex enzyme is a mixture of cellulase and pectinase, the enzymatic hydrolysis temperature is 35-45℃, and the time is 10-30 minutes.

4. The method of claim 1, wherein, In step d), the ultracentrifugation condition is 100,000 x g for 2 hours.

5. The method of claim 1, wherein, A permeability protection agent sucrose is added in the pretreatment step.

6. The method of claim 1, wherein, A chelating agent EDTA is added in the enzymatic hydrolysis system.

7. The method of claim 1, wherein, The average particle size of the obtained vesicles is 50-200 nm.

8. Plant cell vesicles prepared according to the method of any one of claims 1 to 7, characterized in that, It can be used for encapsulating drug molecules or nucleic acid delivery.

9. A drug delivery system comprising the plant cell vesicles of claim 7 as a carrier.

10. Use of the plant cell vesicles of claim 8 in the preparation of anti-tumor or anti-inflammatory nanomaterials.

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