Hybrid vesicles for targeting dendritic cells as well as preparation method and application of hybrid vesicles

Abstract

The invention relates to a hybrid vesicle for targeting dendritic cells as well as a preparation method and application of the hybrid vesicle, and relates to the technical field of biology. The hybrid vesicles for targeting the dendritic cells are obtained by hybridizing orange peel vesicles and mint leaf vesicles. The hybrid vesicle has relatively high content of mannose and menthol, can be effectively combined with CD206 on the surface of a dendritic cell in a targeting manner, can efficiently target the dendritic cell, and can effectively open tight connection of lymphatic endothelial cells, so that the hybrid vesicle can quickly and effectively enter the lymphatic vessel as a carrier, and can be used for treating the lymphatic endothelial cells. Therefore, lymphatic reflux and lymph node homing are effectively promoted.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to hybrid vesicles for targeting dendritic cells and a preparation method and application thereof. Background Art

[0002] Antigens and other components need lymphatic return and lymph node homing after entering the body, which is an important immune process of the body. Dendritic cells, as the most professional antigen-presenting cells in the body, are responsible for delivering vaccine antigens to lymph nodes. Clinical studies have shown that the efficacy of various vaccines is proportional to the amount of antigens presented by dendritic cells. Therefore, it is of great significance to promote the recognition of antigens by dendritic cells and accelerate the lymphatic return of dendritic cells. Extracellular vesicles are important communication media between biological cells and have an important regulatory effect on cellular biological processes. Plant cell vesicles present a typical disc-shaped vesicle structure. Studies have found that plant exosomes have important preventive and therapeutic effects in many major disease areas, providing important clues for the development of new drugs and new ideas for increasing the amount of antigens presented by dendritic cells. Summary of the invention

[0003] In view of the above problems, the present invention provides a hybrid vesicle for targeting dendritic cells. The hybrid vesicle has a high content of mannose and menthol, can effectively target and bind to CD206 on the surface of dendritic cells, can efficiently target dendritic cells, and can also effectively open the tight junctions of lymphatic endothelial cells, so that the hybrid vesicle can quickly and effectively enter the lymphatic vessels as a carrier, thereby effectively promoting lymphatic return and lymph node homing.

[0004] The invention provides a hybrid vesicle for targeting dendritic cells. The hybrid vesicle is obtained by hybridizing orange peel vesicles and mint leaf vesicles.

[0005] Mint is a perennial herbaceous plant of the genus Mentha in the family Lamiaceae. It has a pungent and cool sweating and antipyretic effect. It has a good effect on the treatment of influenza, headache, red eyes, body heat, sore throat, gum swelling and pain, etc. It has a good effect on neuralgia, skin itching, rash and eczema when used externally. In addition, mint can also be used as tea, which has the effect of clearing the mind and improving eyesight. Orange peel is the peel of the sweet orange of the Rutaceae family. It has many functions such as maintaining normal vascular osmotic pressure, enhancing capillary toughness, lowering cholesterol, regulating immunity, and anti-cancer and anti-tumor. Mint leaf vesicles with mint can reduce the expression of tight junctions by lymphatic endothelial cells. Orange peel vesicles contain mannose components, which can effectively promote antigens to be recognized and targeted by dendritic cells. Most of the existing studies use mint leaf vesicles or orange peel vesicles alone, and combining the two can fully stimulate the lymphatic return and lymph node homing of dendritic cells, which will have a breakthrough significance for the efficacy of drugs including vaccines.

[0006] In one embodiment, the particle size of the orange peel vesicles is less than 0.22 μm.

[0007] The orange peel vesicles of the above-mentioned particle size have a higher content of mannose, which can effectively target and bind to CD206 on the surface of dendritic cells.

[0008] In one embodiment, the particle size of the peppermint leaf vesicles is 0.22-0.45 μm.

[0009] The mint leaf vesicles of the above-mentioned particle size have a high content of menthol, which can effectively open the tight junctions of lymphatic endothelial cells. Vesicles of different particle sizes have different effects on reducing the expression of proteins such as ZO-1. Therefore, it is necessary to screen the mint leaf vesicle particle size that best promotes the homing of dendritic cells to lymph nodes.

[0010] In one embodiment, the hybrid vesicles include mannose and menthol.

[0011] In one embodiment, the particle size of the hybrid vesicle is 120-200 nm.

[0012] The above particle size is conducive to the uptake of hybrid vesicles by dendritic cells.

[0013] The present invention also provides a method for preparing the hybrid vesicle, comprising the following steps:

[0014] Preparation of orange peel vesicles: taking orange peel, squeezing juice, filtering, taking filtrate, centrifuging, taking filtrate, low temperature ultracentrifugation, taking precipitate, density gradient centrifugation, removing impurities, separating according to particle size, and obtaining orange peel vesicles;

[0015] Preparation of mint leaf vesicles: taking mint leaves, squeezing juice, filtering, taking filtrate, centrifuging, taking filtrate, performing low-temperature ultracentrifugation, taking precipitate, performing density gradient centrifugation, removing impurities, and separating according to particle size to obtain mint leaf vesicles;

[0016] Preparation of hybrid vesicles: Orange peel vesicles and mint leaf vesicles were hybridized by freeze-thaw and ultrasonic method to obtain hybrid vesicles.

[0017] In one embodiment, the hybridization includes the following steps: mixing orange peel vesicles and mint leaf vesicles to obtain mixed vesicles, heating the mixed vesicles in a water bath, placing them in liquid nitrogen, repeating the process several times, and performing ultrasonic treatment to obtain hybrid vesicles.

[0018] In one embodiment, the water bath heating temperature is 35-39°C, and the water bath heating time is 3-7 minutes; the liquid nitrogen standing temperature is -196°C, and the liquid nitrogen standing time is 3-7 minutes, and the number of repetitions is 2-4 times; the ultrasonic treatment power is 150-250W, and the ultrasonic treatment time is 2-4 minutes.

[0019] In one embodiment, in the step of preparing orange peel vesicles, the centrifugation includes: initial centrifugation and secondary centrifugation; the speed of the initial centrifugation is 3000g and the time is 2h, and the speed of the secondary centrifugation is 10000g and the time is 2h.

[0020] In one embodiment, in the step of preparing orange peel vesicles, the rotation speed of the low-temperature ultracentrifugation is 100,000 g, the time of the low-temperature ultracentrifugation is 2 h, and the temperature is 4°C.

[0021] In one embodiment, in the step of preparing mint leaf vesicles, the centrifugation includes: initial centrifugation and secondary centrifugation; the speed of the initial centrifugation is 3000g and the time is 2h, and the speed of the secondary centrifugation is 10000g and the time is 2h.

[0022] In one embodiment, in the step of preparing peppermint leaf vesicles, the rotation speed of the low-temperature ultracentrifugation is 100,000 g, the time of the low-temperature ultracentrifugation is 2 h, and the temperature is 4°C.

[0023] The present invention also provides application of the hybrid vesicle in drug preparation.

[0024] The present invention also provides a medicine, comprising a medicine active ingredient and a carrier, wherein the carrier comprises the hybrid vesicle.

[0025] In one embodiment, the drug includes a vaccine, the vaccine includes a carrier, and the carrier includes the hybrid vesicle.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The hybrid vesicle for targeting dendritic cells of the present invention, and its preparation method and application, the hybrid vesicle has a high content of mannose and menthol, can effectively target and bind to CD206 on the surface of dendritic cells, and can also effectively open the tight junction protein of lymphatic endothelial cells, can efficiently target dendritic cells, and the hybrid vesicle can quickly and effectively enter the lymphatic vessels as a carrier, thereby effectively promoting lymphatic return and lymph node homing. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of particle size analysis of different subpopulations of orange peel vesicles (OPNV) in the embodiment, wherein A is sOPNV, <0.22 μm, B is mOPNV, 0.22-0.45 μm, and C is lOPNV, 0.45-1 μm;

[0029] Figure 2The results of the determination of mannose content in orange peel vesicles (OPNV) in the embodiment are shown in Figure 1, wherein A is sOPNV, B is mOPNV, and C is lOPNV;

[0030] Figure 3 The results of particle size analysis of different subpopulations of peppermint leaf vesicles (MLNV) in the embodiment, wherein A is sMLNV, <0.22 μm, B is mMLNV, 0.22-0.45 μm, and C is lMLNV, 0.45-1 μm;

[0031] Figure 4 The results of the determination of menthol content in peppermint leaf vesicles (MLNV) in the embodiment are shown in FIG. A is sMLNV, B is mMLNV, and C is lMLNV;

[0032] Figure 5 TEM characterization image of hybrid vesicles;

[0033] Figure 6 The figure shows the result of hybrid vesicles reducing the expression of ZO-1 protein in lymphatic endothelial cells;

[0034] Figure 7 The result diagram shows that hybrid vesicles reduce the expression of Occludin protein in lymphatic endothelial cells;

[0035] Figure 8 The results show the ability of hybrid vesicles to target dendritic cells. DETAILED DESCRIPTION

[0036] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] source:

[0039] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0040] Example

[0041] 1. Prepare orange peel vesicles and mint leaf vesicles.

[0042] 1. Prepare orange peel vesicles.

[0043] (1) Use ultrapure water to clean the orange peel, use a juicer or grinder to collect the juice and remove the residue;

[0044] (2) using gauze to filter out large particles of impurities in the juice and collect the filtrate;

[0045] (3) Initial centrifugation: centrifuge at 3000 g for 2 hours to remove large particles in the filtrate. Centrifuge again at 10,000 g for 2 hours and collect the filtrate;

[0046] (4) Low-temperature ultracentrifugation: ultracentrifuge at 100,000 g for 2 h at 4°C, collect the precipitate, and resuspend in PBS;

[0047] (5) Density gradient centrifugation at a speed of 100,000 g for 1 h, using VesPura plant exosome purification reagent to purify the vesicles and remove non-vesicle impurities.

[0048] (6) OPNVs of different sizes were separated by membrane filters with pore sizes of 0.22 and 0.45 μm, thereby obtaining three subpopulations of apoptotic vesicles: sOPNV, <0.22 μm; mOPNV, 0.22-0.45 μm and lOPNV, 0.45-1 μm.

[0049] 2. Prepare peppermint leaf vesicles.

[0050] (1) Wash mint leaves with ultrapure water, use a juicer or grinder to collect the juice and remove the residue;

[0051] (2) using gauze to filter out large particles of impurities in the juice and collect the filtrate;

[0052] (3) Initial centrifugation: centrifuge at 3000 g for 2 hours to remove large particles in the filtrate. Centrifuge again at 10,000 g for 2 hours and collect the filtrate;

[0053] (4) Low-temperature ultracentrifugation: ultracentrifuge at 100,000 g for 2 h at 4°C, collect the precipitate, and resuspend in PBS;

[0054] (5) Density gradient centrifugation at a speed of 100,000 g for 1 h, using VesPura plant exosome purification reagent to purify the vesicles and remove non-vesicle impurities.

[0055] (6) MLNVs of different sizes were separated by membrane filters with pore sizes of 0.22 and 0.45 μm, thereby obtaining three subpopulations of apoptotic vesicles: sMLNV, <0.22 μm; mMLNV, 0.22-0.45 μm and lMLNV, 0.45-1 μm).

[0056] 2. Determination of vesicle particle size and PDI.

[0057] The prepared orange peel vesicles and mint leaf vesicles were diluted 20-50 times with distilled water, and the particle size of the vesicles was measured by Malvern laser potential particle size analyzer, and the particle size and particle size distribution (PDI) were recorded. The results are as follows: Figure 1 , 3 shown.

[0058] 3. Determination of mannose in orange peel vesicles.

[0059] The content of mannose in orange peel vesicles was detected by high performance liquid chromatography. The liquid phase method was as follows: mobile phase: methanol / water (v:v / 30:70), flow rate: 1.0mL / min, detection wavelength: 202nm. 0.05, 0.1, 0.3, 0.5, 1 and 2mg of mannose were weighed and dissolved in 1mL of mobile phase solution, and the standard curve was prepared by detection. The concentration of mannitol in orange peel vesicles was calculated by comparing the peak area of ​​orange peel vesicles with the peak area of ​​the corresponding concentration on the standard curve.

[0060] The results are as follows Figure 2 As shown, sOPNV had the highest mannose content.

[0061] 4. Determination of menthol in peppermint leaf vesicles.

[0062] The content of menthol in mint leaf vesicles was analyzed by gas chromatography-mass spectrometry. Accurately weigh 0.2 mL of mint leaf vesicles and add 1 mL of methanol solution. Ultrasonic treatment was performed for 30 min. The damaged mint leaf vesicles were oscillated by an oscillator and ultrasound for 60 min. After filtering and loading, the menthol content was analyzed by gas chromatography-mass spectrometry. Accurately weigh 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 mg of menthol and dissolve them in 1 mL of methanol solution to prepare a series of menthol standard solutions. The menthol content was analyzed by gas chromatography-mass spectrometry, and a standard curve was drawn. On this basis, the menthol content in mint leaf vesicles was calculated.

[0063] The results are as follows Figure 4 As shown, mMLNV had the highest menthol content.

[0064] 5. Preparation of hybrid vesicles.

[0065] sOPNV and mMLNV were selected and hybridized using abnormal freeze-thaw and ultrasound methods as follows:

[0066] 1 ml of sOPNV and 1 ml of mMLNV were mixed, and then kept in a 37°C water bath for 5 min and then in liquid nitrogen for 5 min, and repeated 3 times. The resulting mixed vesicles were sonicated at 200 W for 3 min.

[0067] 6. Characterization of hybrid vesicles.

[0068] The prepared hybrid vesicles were diluted 10 times with physiological saline and dropped on the copper grid, stained with 2% uranyl acetate saturated ethanol solution in the dark for 8 minutes, washed 3 times with 70% ethanol, washed 3 times with ultrapure water; stained with 2.6% lead citrate solution in the dark for 8 minutes; washed 3 times with ultrapure water, slightly dried with filter paper, and dried at room temperature overnight for use. The images of the hybrid vesicles were collected using a transmission electron microscope (TEM) at a voltage of 80 kV.

[0069] The results are as follows Figure 5 As shown,

[0070] VII. Detection of the effect of hybrid vesicles on the expression of tight junction proteins in lymphatic endothelial cells

[0071] The hybrid vesicles were diluted with DMEM medium (containing 5% fetal bovine serum) to a gradient concentration of 0, 20, 50, and 100 μg / mL. 5 Mouse lymphatic endothelial cells were co-incubated with 100 μg / mL of hybrid vesicles in DMEM medium (containing 5% fetal bovine serum) for 6 hours, and 100 μg / mL of DMSO solution was used as a control. Real-time fluorescence quantitative PCR was used to investigate the effect of hybrid vesicles on the expression of tight junction proteins ZO-1 and Occludin in mouse lymphatic endothelial cells at the gene and protein levels.

[0072] The results are as follows Figure 6 , 7 shown.

[0073] 8. Evaluation of the targeting ability of hybrid vesicles to dendritic cells.

[0074] The cell slides were soaked in 0.01% lysine for 5 min and then placed in a 24-well cell culture plate. 4 Dendritic cells of cells / well were added to the well plate, and the hybrid vesicles diluted with cell culture medium RPMI 1640 were incubated with cells for 6 hours. The hybrid vesicles were labeled with CMFDA, a chloromethyl derivative of diacetate fluorescein. Finally, the cell slides were carefully rinsed with PBS twice, fixed with paraformaldehyde, and then rinsed with PBS to label the nucleus with DAPI, and the cytoskeleton was labeled with rhodamine-conjugated phalloidin for 1.5 hours, and then the slides were sealed with mounting medium and observed under a confocal microscope.

[0075] The results are as follows Figure 8 shown.

[0076] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0077] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A hybrid vesicle for targeting dendritic cells, characterized in that: The hybrid vesicle is obtained by hybridizing orange peel vesicles and mint leaf vesicles.

2. The hybrid vesicle according to claim 1, characterized in that The particle size of the orange peel vesicle is less than 0.22 μm.

3. The hybrid vesicle according to claim 1, characterized in that The particle size of the peppermint leaf vesicle is 0.22-0.45 μm.

4. The hybrid vesicle according to any one of claims 1 to 3, characterized in that The hybrid vesicles include mannose and menthol.

5. The hybrid vesicle according to claim 4, characterized in that The particle size of the hybrid vesicle is 120-200 nm.

6. The method for preparing the hybrid vesicle according to any one of claims 1 to 5, characterized in that: The following steps are involved: Preparation of orange peel vesicles: taking orange peel, squeezing juice, filtering, taking filtrate, centrifuging, taking filtrate, low temperature ultracentrifugation, taking precipitate, density gradient centrifugation, removing impurities, separating according to particle size, and obtaining orange peel vesicles; Preparation of mint leaf vesicles: taking mint leaves, squeezing juice, filtering, taking filtrate, centrifuging, taking filtrate, performing low-temperature ultracentrifugation, taking precipitate, performing density gradient centrifugation, removing impurities, and separating according to particle size to obtain mint leaf vesicles; Preparation of hybrid vesicles: Orange peel vesicles and mint leaf vesicles were hybridized by freeze-thaw and ultrasonic method to obtain hybrid vesicles.

7. The preparation method according to claim 6, characterized in that: In the step of preparing orange peel vesicles, the centrifugation includes: initial centrifugation and secondary centrifugation; the speed of the initial centrifugation is 3000g and the time is 2h, and the speed of the secondary centrifugation is 10000g and the time is 2h.

8. The preparation method according to claim 6, characterized in that: In the step of preparing peppermint leaf vesicles, the centrifugation includes: initial centrifugation and secondary centrifugation; the speed of the initial centrifugation is 3000g and the time is 2h, and the speed of the secondary centrifugation is 10000g and the time is 2h.

9. Use of the hybrid vesicle according to any one of claims 1 to 5 in drug preparation.

10. A drug, characterized in that The invention comprises a pharmaceutically active ingredient and a carrier, wherein the carrier comprises the hybrid vesicle according to any one of claims 1 to 5.

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