CN111905107A - Aloe exosome-like vesicle capable of protecting aloe active ingredients and serving as drug carrier - Google Patents
Aloe exosome-like vesicle capable of protecting aloe active ingredients and serving as drug carrier Download PDFInfo
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Abstract
The invention discloses an aloe exosome-like vesicle which can protect an aloe active ingredient and can be used as a drug carrier; relates to an aloe exosome-like vesicle which can protect an aloe active ingredient and can be used as a drug carrier. The exosome-like vesicle can be obtained by separating from aloe cortex and its gel, and has a diameter of 30-300 nm. More specifically, the exosome-like vesicle contains active ingredients such as aloe-emodin and aloesin, and also contains beta-sitosterol which has protection and stabilization effects on lipid structures, so that the stability of the aloe-exosome-like vesicle is improved, and the aloe-emodin-like vesicle has good protection effects on internal biological and pharmacological active substances. In addition, the exosome-like vesicle can be loaded with anticancer drug adriamycin, acts on breast cancer cells, shows a remarkable cell killing effect, has good drug carrier potential, and has important significance for the transmission of clinical antitumor drugs and cancer treatment.
Description
Technical Field
The invention relates to the technical field of separation and extraction of plant exosome-like vesicles and research on drug delivery carriers, in particular to extraction of aloe cortex and aloe gel layer exosome-like vesicles containing active drug ingredients and feasibility research on the exosome-like vesicles as drug carriers.
Background
1. Exosome is a nano-scale vesicle actively secreted by cells, carries a lot of genetic information derived from parental cells, such as nucleic acid, protein and the like, is used as an important medium for long-distance communication among cells, and participates in various biological processes. Meanwhile, the phospholipid bilayer structure has a certain protection effect on internal active ingredients, is easy to modify, and is widely concerned in the field of drug delivery carrier research.
2. In plants, however, recently, researchers have obtained exosome-like vesicles isolated from ginger, grapefruit, dendrobium officinale and other plants, and research has shown that the exosome-like vesicles isolated from plants are rich in lipid, protein, nucleic acid and other components, as are exosomes from mammals. Meanwhile, the plant exosome-like vesicle contains pharmacological active ingredients of plants, for example, the ginger exosome-like vesicle contains active ingredients such as shogaol and gingerol, so that research on the action of the active ingredients in treatment of various diseases gradually becomes an attention focus of clinical researchers.
3. Aloe is a traditional Chinese medicine, has a long medicinal history, and its medicinal value is recorded in book of materia medica summary, book of materia medica Jing Shu, etc., Aloe is rich in pharmacological active components, such as aloe-emodin, aloesin, etc., and has pharmacological effects of resisting inflammation, resisting bacteria, etc. At present, many researches are carried out to separate and obtain active ingredients such as aloe-emodin from aloe, however, the anthraquinone ingredients are easy to be influenced by environmental factors such as temperature and the like due to containing phenolic hydroxyl groups, so that the pharmacological activity of the anthraquinone ingredients is influenced. The aloe exosome-like vesicle has a bilayer structure similar to exosome, is a natural protective cover and can reduce the influence of the external environment on the internal components of the aloe exosome-like vesicle. In addition, the aloe contains a common phytosterol, namely beta-sitosterol, which is widely present in cell membranes of various plants, has obvious inhibition effect on the flow of phospholipid fatty acyl side chains, can enhance the sequencing effect among phospholipid molecules, and has obvious lipid oxidation resistance and membrane structure stabilizing performance. The aloe exosome-like vesicles are secreted by each part of cells in aloe and can carry beta-sitosterol in aloe cell membranes, so that the phospholipid bilayer structure is more stable, and the biological and pharmacological activities of the internal active ingredients are more favorably maintained.
4. Although exosomes have great potential in drug delivery vector research, the separation of the lactation exosomes always has the problems of high cost, time consumption, low yield and the like, and the application of the exosomes in the drug delivery vector research field is greatly limited. The aloe exosome-like vesicle has the biological characteristics similar to exosomes, has the advantages of simplicity in preparation, low cost, considerable yield and the like, can be used as a good substitute of exosomes, and plays a role in the field of drug carrier research.
Disclosure of Invention
The invention aims to provide an aloe exosome-like vesicle which can protect an aloe active ingredient and can be used as a drug carrier.
1) The invention relates to an exosome-like vesicle separated from aloe cortex and gel, which contains aloe-emodin, aloesin and other effective aloe active ingredients, contains beta-sitosterol capable of enhancing lipid oxidation resistance and stability, has obvious protection effect on the internal effective ingredients, and can maintain the stability of pharmacological active ingredients for a long time;
2) the exosome-like vesicles obtained by separating the aloe cortex and the aloe gel have certain differences in properties such as active ingredients and protein distribution;
3) after the aloe exosome-like vesicle obtained by the invention is loaded with the anti-cancer drug, compared with a free drug, the aloe exosome-like vesicle has a more remarkable killing effect on breast cancer cells.
4) The aloe exosome-like vesicle obtained by the invention can penetrate the stratum corneum of the skin, and compared with free dye, the aloe exosome-like vesicle carrying the dye has better penetrating effect on the skin.
Solution technical scheme
In order to achieve the purpose, the invention provides the following technical scheme:
step a, aloe peel and aloe gel juice:
weighing fresh aloe peel and aloe gel 100g respectively, cleaning, cutting into small pieces, adding 50g of prepared PBS ice and 400mL of precooled PBS, placing into a juicer, and mincing, pausing every 30s at intervals of 10s for 240s in total. Obtaining fresh aloe peel and aloe gel juice, filtering with warp cloth, and standing at 4 deg.C;
preparing aloe peel and aloe gel exosome-like vesicles:
centrifuging fresh Aloe peel and Aloe gel juice at 300 × g for 10min, discarding precipitate, and collecting supernatant A1 and A2; centrifuging the supernatant A1 and A2 at 1000 Xg for 10min to obtain supernatant B1 and B2; centrifuging the supernatants B1 and B2 at 3000 Xg for 20min to obtain supernatants C1 and C2; and centrifuging the supernatant C1 and C2 at 11000 Xg for 40min to obtain supernatant D1 and D2, filtering the supernatant D1 and D2 by a 0.22 mu m filter membrane, centrifuging at 50000 Xg for 90min to obtain precipitates E1 and E2, re-suspending the precipitates E1 and E2 by a proper amount of PBS to obtain suspensions F1 and F2, centrifuging the suspensions F1 and F2 at 50000 Xg for 90min to obtain precipitates G1 and G2, and re-suspending by PBS to respectively obtain the aloe cortex and the aloe gel exosome-like vesicles.
Preferably, the finally obtained aloe exosome-like vesicle is precipitated and resuspended in 1xPBS buffer solution (containing 25mM trehalose and 1.5. mu.g/mL protease inhibitor) to obtain aloe exosome-like vesicles with good dispersibility, and meanwhile, membrane surface proteins of the aloe exosome-like vesicles are protected, and the influence of low-temperature freezing conditions on the aloe exosome-like vesicles is reduced.
Further, the prepared aloe cortex and aloe gel exosome-like vesicles are resuspended by using a proper amount of methanol, ultrasonically dissolved, centrifuged to leave supernatant, filtered and stored at 4 ℃ to be analyzed for internal aloe active ingredients.
Preferably, in order to accurately detect the content of each active component in the aloe exosome-like vesicle, the total amount of protein for each component identification is fixed with the total protein amount of the aloe exosome-like vesicle as a quantitative standard. Centrifuging at 4 deg.C and 50000 Xg to obtain precipitate, resuspending the precipitate with 1mL of chromatographic grade methanol solution, ultrasonic treating for 20min to dissolve effective components in methanol, centrifuging at 11000 Xg for 10min, collecting supernatant, filtering with 0.22 μm organic phase filter membrane, sealing the bottle mouth with sealing membrane, and storing at 4 deg.C.
Preferably, the conditions of the mobile phase of the aloe-emodin, the aloesin and the beta-sitosterol are acetonitrile-water (50: 50), acetonitrile-water (14: 76) and methanol-water (85: 15) respectively when the aloe-emodin, the aloesin and the beta-sitosterol are analyzed by high performance liquid chromatography, the column temperature is 30 ℃, and the detection wavelengths are 254nm, 254nm and 205nm respectively.
Further, the content of aloe-emodin in the aloe-emodin-like vesicles separated in different time periods is analyzed through high performance liquid chromatography, and the stability of the aloe-emodin in the aloe-emodin-like vesicles is verified.
Preferably, the aloe exosome-like vesicles are exosome-like vesicles which are extracted and stored at-80 ℃ for 0, 30, 60, 90, 120 and 180 days.
Further, the obtained aloe cortex and aloe gel secretion-like vesicles are analyzed and characterized by SDS-PAGE gel electrophoresis, and the protein distribution of the aloe cortex and the aloe gel secretion-like vesicles is observed.
Preferably, the concentration of the prepared SDS-PAGE gel is 10%, 6 μ L of 5x protein loading buffer is added into 30 μ L of the aloe cortex and the aloe gel exosome-like vesicles with different concentrations, the mixture is fully vortexed and then reacts in a metal bath at 95 ℃ for 10min, and the final loading of the exosome-like vesicles with different protein concentrations is kept consistent by adjusting the loading volumes of the two exosome-like vesicles.
Further, the cell nucleus and the cell membrane of the mouse mammary cancer 4T1 cell and the aloe exosome-like vesicle are stained by a fluorescent dye staining method through a blue cell nucleus fluorescent Dye (DAPI), a green cell membrane fluorescent Dye (DiO) and a red cell membrane fluorescent Dye (DiI), and the uptake of the cell to the aloe exosome-like vesicle is verified through laser confocal microscope imaging.
Preferably, the concentrations of DAPI, DiO and DiI dyes used are 10. mu.g/mL, 10. mu. mol/mL and 10. mu. mol/mL, respectively, and all the dyes are placed in 1 XPBS. Mixing the aloe exosome-like vesicle and DiI dye, shaking to react with shaking table at room temperature for 20min, centrifuging at 50000 Xg and 4 ℃ for 90min, re-suspending the obtained precipitate with 1XPBS, centrifuging at 50000 Xg and 4 ℃ for 90min, washing with PBS again, re-suspending with whole cell culture medium, and preserving at 4 ℃.
Preferably, the cell fluorescent staining step is: taking out cells pre-cultured in a laser confocal culture dish from an incubator, sucking away culture solution, washing for 2 times by using PBS, adding 400 mu L of DiI/ADNs solution, and incubating for 12 hours in the incubator at 37 ℃; taking out the cell culture dish after incubation in the first step from the incubator, sucking and removing the DiI/ADNSS solution, washing the cell culture dish for 2 times by PBS, adding 400 mu L of 4% paraformaldehyde solution, continuing to cultivate the cell culture dish for 20min, sucking and removing the paraformaldehyde, washing the cell culture dish for 2 times by PBS, adding 400 mu L of DAPI staining solution, and putting the cell culture dish into the cell culture box for incubation for 15 min; taking out the incubated cell culture dish, sucking away the DAPI dye solution, washing with PBS for 2 times, adding 400 mu L DiO dye solution, incubating in a cell culture box for 15min, taking out, sucking away the DiO dye solution, washing with PBS for 2 times, and observing under a confocal laser microscope.
Furthermore, the cytotoxicity of the aloe exosome-like vesicle on tumor cells is verified by an MTT method, meanwhile, Doxorubicin (DOX) is loaded into the aloe exosome-like vesicle by an incubation method, the doxorubicin and free doxorubicin solution with corresponding concentration respectively act on the tumor cells, and the killing effect of the tumor cells is observed.
Preferably, the step of loading doxorubicin into the aloe exosome-like vesicles is as follows: dissolving 0.5mg of aloe exosome-like vesicles in 1mL of adriamycin-PBS solution at 200 mu g/mL, fully and uniformly mixing, incubating at 400rpm and 37 ℃ for 30min, centrifuging at 50000 Xg and 4 ℃ for 90min, re-suspending the obtained precipitate with 1XPBS, centrifuging at 50000 Xg and 4 ℃ for 90min, washing with PBS again until the supernatant is colorless, and re-suspending with 1mL of PBS buffer solution, thus obtaining the adriamycin-loaded aloe exosome-like vesicles (DOX/ADNs) solution which are stored at 4 ℃.
Preferably, murine mammary carcinoma 4T1 cells are selected as the research object, the cells are digested by trypsin, centrifuged at 4 ℃, 1000rpm for 10min, the supernatant is discarded, the precipitate is suspended in RPMI-1640 complete cell culture medium, after counting, 5 ten thousand/mL cell suspension is prepared, 200 muL/well cell suspension is added into a 96-well plate, the 96-well plate is placed at 37 ℃, and 5% CO is added2After the cells are attached to the wall, the administration concentration of the DOX/AND solution is set to be 0, 0.5, 0.8, 1, 3, 5, 8 AND 10 mu g/mL (the concentration is the concentration of DOX in the AND), 5 auxiliary holes are set for each concentration, 200 mu L/hole, AND the cells are placed in a cell incubator for 24 hours. Selecting 1xPBS to prepare 5mg/mL MTT solution, filtering the MTT solution by a 0.22-micron microporous filter membrane, taking out a 96-pore plate from an incubator, absorbing and discarding the drug solution, washing the MTT solution for 2 times by PBS, adding the MTT solution with 20 muL/pore, placing the MTT solution in a cell incubator for reaction for 4 hours, absorbing and discarding the MTT solution, adding 150 muL dimethyl sulfoxide (DMSO), placing the MTT solution on a shaking table, shaking at 800rpm for 10 minutes, and measuring the absorbance of the MTT solution at 490nm by using a microplate reader.
The invention has the advantages of
Compared with the prior art, the invention has the advantages that: 1) the preparation method for separating the exosome-like vesicles from the aloe peel and the aloe gel is simple and convenient to operate, the materials are low in price and easy to obtain, and the exosome-like vesicles resuspended by 1xPBS buffer solution containing 1.5 mu g/mL protease inhibitor and 25mM trehalose have good dispersibility; 2) the aloe cortex and gel layer exosome-like vesicles prepared by the invention contain pharmacologically active ingredients such as aloe-emodin and aloesin, and beta-sitosterol with stable lipid structure and oxidation resistance, so that the active ingredients such as aloe-emodin can be maintained stable for a long time; 3) the aloe exosome-like vesicle can be used as a drug carrier and can be used for loading anti-cancer drugs to effectively kill tumor cells; 4) the aloe exosome-like vesicle obtained by the invention can penetrate the stratum corneum of the skin and has important significance in the field of treatment and research of skin care products, skin diseases and even skin cancer.
Drawings
Fig. 1 is a graph of the characterization results of two aloe exosome-like vesicles isolated in example 1. In the figure: A. dynamic light scattering characterizes the particle size distribution of the exosome-like vesicles; B. appearance of exosome-like vesicles under a transmission electron microscope; C. protein distribution of the aloe cortex and the gel layer exocrine body-like vesicles under SDS-PAG E gel electrophoresis;
fig. 2 is a high performance liquid chromatography analysis chart of the pharmacologically active ingredient in the aloe exosome-like vesicles of example 2. In the figure: A. high performance liquid chromatography analysis of aloe-emodin in aloe exosome-like vesicles; B. high performance liquid chromatography analysis of aloesin in the aloe exosome-like vesicles; C. high performance liquid chromatography analysis of aloe exosome-like beta-sitosterol in vesicles; D. graph of aloe-emodin content in aloe exosome-like vesicles at different time periods.
Fig. 3 is a fluorescence-stained confocal laser imaging of the entry of aloe exosome-like vesicles into cells provided in example 3; A. imaging of nuclei of 4T1 cells with DAPI dye; B. imaging the cell membrane of 4T1 cells by DiO dye; C. 4T1 cells were imaged for the uptake of DiI/ADNs; D. 4T1 cell morphology imaging under bright field and other laser channels.
Fig. 4 is a graph comparing the results of aloe exosome-like vesicles as drug carriers for tumor cell killing in example 4.
FIG. 5 is a graph of laser confocal microscopy characterization of permeation of aloe exosome-like vesicles into mouse skin in example 5. A. The permeation condition of the aloe exosome-like vesicle loaded with the green fluorescent dye DiO in the rat skin; penetration of free green fluorescent dye DiO in rat skin.
Detailed Description
In order to explain the technical content, characterization and performance analysis methods of the present invention in detail, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used are commercially available.
Example 1:
example 1 of the present invention provides a method for separating exosome-like vesicles from aloe cortex and aloe gel, comprising the following steps:
step a, preparing fresh aloe peel and aloe gel juice:
taking 100g of fresh aloe peel, washing, cutting into small pieces, adding 50g of prepared PBS ice blocks and 400mL of ice PBS, and placing into a juicer for juicing, pausing every 30s at intervals of 10s for 240s in total. Obtaining fresh aloe peel and aloe gel juice, filtering with warp cloth, and standing at 4 deg.C;
preparing aloe peel and aloe gel exosome-like vesicles:
centrifuging fresh Aloe peel and Aloe gel juice at 300 × g for 10min, discarding precipitate to obtain supernatant A1 and A2; centrifuging the supernatant A1 and A2 at 1000 Xg for 10min to obtain supernatant B1 and B2; centrifuging the supernatants B1 and B2 at 3000 Xg for 20min to obtain supernatants C1 and C2; and centrifuging the supernatant C1 and C2 at 11000 Xg for 40min to obtain supernatant D1 and D2, filtering the supernatant D1 and D2 by a 0.22 mu m filter membrane, centrifuging at 50000 Xg for 90min to obtain precipitates E1 and E2, re-suspending the precipitates E1 and E2 by a proper amount of PBS to obtain suspensions F1 and F2, centrifuging the suspensions F1 and F2 at 50000 Xg for 90min to obtain precipitates G1 and G2, and re-suspending by PBS to respectively obtain the aloe cortex and the aloe gel exosome-like vesicles. As shown in fig. 1, a. dynamic light scattering characterizes the particle size distribution of exosome-like vesicles; B. appearance of exosome-like vesicles under a transmission electron microscope; C. protein distribution of aloe cortex and gel layer secretion-like vesicles under SDS-PAGE gel electrophoresis.
Example 2:
the process of analyzing the active ingredients in the aloe cortex and aloe gel exosome-like vesicles prepared in example 1 by high performance liquid chromatography was as follows:
after the protein content of the aloe cortex and the aloe gel exosome-like vesicle is respectively measured by a BCA protein quantification method, exosome-like vesicle solution with corresponding volume is taken according to the actual protein concentration of two exosome-like vesicles, the total protein mass is 1mg, the exosome-like vesicle solution is centrifuged for 90min at 4 ℃ and 50000 Xg to obtain exosome-like vesicle sediment, 1mL of chromatographic grade methanol solution is used for resuspending the sediment, ultrasonic treatment is carried out for 20min to fully dissolve the effective components in the methanol, then the exosome-like vesicle solution is centrifuged for 10min at 11000 Xg, supernatant is retained, the supernatant is filtered by a 0.22 mu m filter membrane, a bottle mouth is sealed by a sealing membrane, and the solution is stored for later use at 4 ℃.
The mobile phases of acetonitrile-water (1: 1) and acetonitrile-water (14: 86) are respectively used, the flow rate is 1mL/min, the column temperature is 30 ℃, the aloe-emodin, the aloesin standard product and the aloe-exosome-like vesicle are analyzed, and the obtained analysis results are shown in figure 2, the content of the aloe-emodin in the aloe-skin exosome-like vesicle is 2.209 g/kg, the content of the aloesin is 0.658 g/kg, the content of beta-sitosterol is 100.7g/kg, the content of the aloe-emodin in the aloe-gel-exosome-like vesicle is 0.187 g/kg, the content of the aloesin is 0.631 g/kg, and the content of beta-sitosterol is 275.56 g/kg. (the unit g/kg here is mActive pharmaceutical ingredient/mExosome-like vesicle protein amount). As shown in fig. 2, a. aloe-emodin hplc analysis map within aloe exosome-like vesicles; B. high performance liquid chromatography analysis of aloesin in the aloe exosome-like vesicles; C. high performance liquid chromatography analysis of aloe exosome-like beta-sitosterol in vesicles; D. graph of aloe-emodin content in aloe exosome-like vesicles at different time periods.
Example 3:
the uptake of aloe exosome-like vesicles (ADNs) obtained in example 1 by tumor cells was verified using fluorescence staining and confocal laser imaging techniques. Mouse breast cancer cell line was purchased from Biochemical cell Bank of Chinese academy of sciences, cultured in RPMI-1640 (Haikeleng) culture medium containing 10% fetal bovine serum (Haikeleng), and placed at 37 ℃ under 5% CO2Culturing in a cell culture box. The cell nucleus, cell membrane and ADNs of the mouse mammary cancer 4T1 cell are stained by DAPI, DiO and DiI through a fluorescent dye staining method, and the condition of the uptake of the cell to the aloe exosome-like vesicle is verified through laser confocal microscope imaging. Detailed description of the inventionThe process is as follows:
a) 1xPBS is selected to prepare DAPI, DiO and DiI dye solutions with the concentrations of 10 mu g/mL, 10 mu mol/mL and 10 mu mol/mL respectively. 1mg of ADNs and DiI dye are taken and fully mixed, incubated at 37 ℃ and 400rpm for 30min, centrifuged for 90min at 50000 Xg and 4 ℃, the obtained precipitate is resuspended by 1XPBS, centrifuged for 90min at 50000 Xg and 4 ℃, then repeatedly washed by PBS until the supernatant is colorless, and then resuspended by using a whole cell culture medium to prepare DiI/ANDs solution, and stored at 4 ℃.
b) The cell fluorescent staining step comprises: taking out cells pre-cultured in a laser confocal culture dish from an incubator, sucking away culture solution, washing for 2 times by using PBS, adding 400 mu L of DiI/ADNs solution, and incubating for 12 hours in the incubator at 37 ℃; taking out the cell culture dish after incubation in the first step from the incubator, sucking and removing the DiI/ADNSS solution, washing the cell culture dish for 2 times by PBS, adding 400 mu L of 4% paraformaldehyde solution, continuing to cultivate the cell culture dish for 20min, sucking and removing the paraformaldehyde, washing the cell culture dish for 2 times by PBS, adding 400 mu L of DAPI staining solution, and putting the cell culture dish into the cell culture box for incubation for 15 min; taking out the incubated cell culture dish, sucking away the DAPI dye solution, washing with PBS for 2 times, adding 400 mu L DiO dye solution, incubating in a cell culture box for 15min, taking out, sucking away the DiO dye solution, washing with PBS for 2 times, and observing under a confocal laser microscope. Fig. 3 is a fluorescent-stained confocal laser imaging of the entry of aloe exosome-like vesicles into cells provided in example 3. A. Imaging of nuclei of 4T1 cells with DAPI dye; B. imaging the cell membrane of 4T1 cells by DiO dye; C. 4T1 cells were imaged for the uptake of DiI/ADNs; D. 4T1 cell morphology imaging under bright field and other laser channels.
Example 4:
the cytotoxicity of ADNs, DOX and DOX/ANDs to 4T1 is verified by MTT method respectively. The method comprises the following specific steps:
a) cell plating: 4T1 cells are digested by trypsin, centrifuged at 1000rpm for 10min at 4 ℃ to remove supernatant, the precipitate is suspended in RPMI-1640 complete cell culture medium, counted to prepare 5 ten thousand/mL cell suspension, 200 mu L/well cell suspension is added into a 96-well plate, and the cell suspension is cultured in a cell culture box at 37 ℃ and 5% CO 2.
b) And (3) administration, namely setting three experimental groups of ANDs, DOX and DOX/ANDs respectively, wherein each experimental group is provided with different concentration gradients. Wherein the ADNs concentration is respectively as follows: 0. 3, 4.8, 6, 18, 48, 60. mu.g/mL, free DOX AND DOX/AND solutions were administered at concentrations of 0, 0.5, 0.8, 1, 3, 5, 8, 10. mu.g/mL (the concentrations here are the concentrations of DOX entrapped in ANDs), 5 sub-wells were set at each concentration, 200. mu.L/well, AND incubated in a cell incubator for 24 h.
c) MTT, taking a 96-well plate out of a cell culture box, absorbing and discarding a drug solution in a super clean bench, washing the plate with PBS for two times, adding an MTT solution (5 mg/mL and 20 mu L) filtered by a 0.22 mu m microporous membrane into each well, placing the plate in the cell culture box for reaction for 4 hours, absorbing and discarding the MTT solution, adding 150 mu L of dimethyl sulfoxide (DMSO), placing the plate on a shaking bed at 800rpm, shaking the plate for 10 minutes, and measuring the absorbance at 490nm by using a microplate reader. Fig. 4 is a graph comparing the results of aloe exosome-like vesicles as drug carriers for tumor cell killing in example 4.
Example 5:
the permeation of the aloe exosome-like vesicles (ADNs) obtained in example 1 to the mouse skin was verified using fluorescence staining and confocal laser imaging techniques. The method comprises the following specific steps:
a) 1mg of ADNs and 1mL of DiO dye solution (30 mu mol/L) are taken to be fully mixed, incubated at 37 ℃ and 400rpm for 30min, centrifuged at 50000 Xg and 4 ℃ for 90min, the obtained precipitate is resuspended by 1XPBS, centrifuged at 50000 Xg and 4 ℃ for 90min, the supernatant is washed by PBS again until no color exists, then resuspended by 1 mLPBS buffer solution, the precipitate is fully dispersed to prepare DiO/ANDs solution, and the DiO/ANDs solution is stored at 4 ℃.
Mice were anesthetized with 4% chloral hydrate (10. mu.L/g) and then treated with 9% Na2Removing hair on mouse back with S solution as depilatory, intermittently cleaning with clear water to reduce skin damage, and repeatedly cleaning with clear water to remove residual Na on skin2S solution, fixing the mice after 1h, respectively dripping DiO/ANDs and free DiO solution on the mouse skin, killing the mice by a spondylolisthesis method after 1.5h of infiltration, taking down the skin of the dripping part, and scanning the infiltration condition of the DiO/ANDs and the free DiO solution under a laser confocal microscope. As shown in figure 5 of the drawings,A. the permeation condition of the aloe exosome-like vesicle loaded with the green fluorescent dye DiO in the rat skin; penetration of free green fluorescent dye DiO in rat skin.
Claims (10)
1. A method for preparing fresh aloe peel and aloe gel juice comprises the following steps:
a. preparing fresh aloe peel and aloe gel juice:
cleaning fresh Aloe, separating Aloe cortex and inner gel, cutting into small pieces, soaking in PBS solution, juicing in a juicer to obtain fresh Aloe cortex and gel juice, filtering with warp cloth, and standing at 4 deg.C;
b. preparation of aloe peel and gel exosome-like vesicles:
centrifuging the fresh aloe peel and gel juice obtained in step a at 300 Xg for 10min, discarding the precipitate to obtain supernatant A1 and A2; centrifuging the supernatant A1 and A2 at 1000 Xg for 10min to obtain supernatant B1 and B2; centrifuging the supernatants B1 and B2 at 3000 Xg for 20min to obtain supernatants C1 and C2; and centrifuging the supernatant C1 and C2 at 11000 Xg for 40min to obtain supernatant D1 and D2, filtering the supernatant D1 and D2 by a 0.22 mu m filter membrane, centrifuging at 50000 Xg for 90min to obtain precipitates E1 and E2, re-suspending the precipitates E1 and E2 by a proper amount of PBS to obtain suspensions F1 and F2, centrifuging the suspensions F1 and F2 at 50000 Xg for 90min to obtain precipitates G1 and G2, and re-suspending by PBS to respectively obtain the aloe cortex and the aloe gel exosome-like vesicles.
2. The method for preparing fresh aloe vera skin and aloe vera gel juice according to claim 1, wherein the small pieces cut from the aloe vera skin layer and the inner gel in step a are mixed with the PBS ice cubes and the ice PBS solution in a mass ratio of 2:1:4, respectively, and then put into a juicer to extract juice; simultaneously, its procedure of squeezing juice does: pause every 30s for an interval of 10s for a total of 240 s.
3. The method for preparing fresh aloe peel and aloe gel juice according to claim 1, wherein the exosome-like vesicles finally obtained in step b are resuspended in 1xPBS buffer solution, and the 1xPBS buffer solution contains 25mM trehalose and 1.5 μ g/mL protease inhibitor, so that the aloe exosome-like vesicles with good dispersibility are obtained, membrane surface proteins of the exosome-like vesicles are protected, and the influence of low-temperature freezing conditions on the exosome-like vesicles is reduced.
4. The method of preparing fresh aloe vera skin and aloe vera gel juice according to claim 1, wherein the fresh aloe vera is selected from aloe vera, aloe barbadensis or aloe arborescens.
5. An aloe exosome-like vesicle prepared by the method of any one of claims 1 to 4.
6. A pretreatment method for analyzing active ingredients in aloe cortex and aloe gel secretion-like vesicles prepared by any one of the methods of claims 1 to 4, comprising the steps of:
centrifuging the obtained aloe cortex and aloe gel exosome-like vesicle at ultra high speed, discarding supernatant, re-suspending with appropriate amount of methanol, ultrasonically dissolving, centrifuging to collect supernatant, filtering, and storing at 4 deg.C.
7. The pretreatment method for analyzing the aloe cortex and the aloe gel exosome-like vesicles according to claim 6, wherein after the protein content of the exosome-like vesicles is measured by a BCA protein quantification method, exosome-like vesicle solution with a corresponding volume is taken according to the actual protein concentration of two exosome-like vesicles, the total protein mass of the exosome-like vesicle solution is 1mg, the exosome-like vesicles are obtained by centrifuging at 4 ℃ and 50000 Xg for 90min, 1mL of chromatographic grade methanol solution is used for resuspension and precipitation, ultrasonic treatment is carried out for 20min, effective components are fully dissolved in methanol, the methanol solution is centrifuged for 10min at 11000 Xg, supernatant is retained, the supernatant is filtered by a 0.22 μm filter membrane, the bottle mouth is sealed by a sealing membrane, and the supernatant is stored at 4 ℃ for later use.
8. The pretreatment method for analysis of aloe cortex and aloe gel exosome-like vesicles according to claim 6, wherein the two exosome-like vesicles each contain aloe-emodin, aloesin, and β -sitosterol.
9. The pretreatment method for analyzing aloe cortex and aloe gel exosome-like vesicles according to claim 7, wherein the two exosome-like vesicles have a protective effect on effective components such as aloe-emodin, aloesin and beta-sitosterol in the aloe cortex and aloe gel exosome-like vesicles, and can maintain the stability of the two exosome-like vesicles for a long time.
10. The aloe-exosome-like vesicle for use in pharmacy according to claim 5, wherein two kinds of aloe-exosome-like vesicles can be prepared into any one of granules, capsules, soft capsules, oral liquid preparations, injections and transdermal delivery preparations.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113846046A (en) * | 2021-10-22 | 2021-12-28 | 科丝美诗(中国)化妆品有限公司 | Dendrobium-derived exosome-like vesicle, preparation method thereof and application thereof in alopecia improvement |
| CN114159373A (en) * | 2021-10-22 | 2022-03-11 | 科丝美诗(中国)化妆品有限公司 | Dendrobium-derived exosome-like vesicle, preparation method thereof and application thereof in skin care cosmetics |
| CN115364176A (en) * | 2022-10-13 | 2022-11-22 | 中国医学科学院阜外医院深圳医院(深圳市孙逸仙心血管医院) | Application of dendrobium-derived nano vesicles in preparation of medicines for preventing and treating atherosclerosis diseases |
| CN115508492A (en) * | 2022-10-19 | 2022-12-23 | 陕西科技大学 | Fingerprint construction method and fingerprint of novel compound aloe capsule |
| CN116492279A (en) * | 2023-03-09 | 2023-07-28 | 广州拾纳客生物科技有限公司 | Exosome freeze-dried powder preparation method and application thereof |
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| WO2024108605A1 (en) * | 2022-11-25 | 2024-05-30 | 深圳先进技术研究院 | Preparation method for aloe-derived exosome and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007096924A2 (en) * | 2006-02-20 | 2007-08-30 | Maiello, Adolfo | Extraction, preparation and use of active principles of the aloe plant for producing self-meditaion devices such as controlled-release gauzes, bandages, stockings, elastic bands and arch supports |
| CN103479682A (en) * | 2012-06-14 | 2014-01-01 | 苏州恒宇生物科技有限公司 | Preparation method for plant source active component nano-scale membrane type vesicle |
| US20140161874A1 (en) * | 2011-05-23 | 2014-06-12 | Micaela Carvajal Alcaraz | Process of preparation and uses of plasma membrane vesicles extracted from plants enriched in membrane transport proteins |
| KR102059626B1 (en) * | 2019-05-10 | 2019-12-26 | 주식회사 바이오뷰텍 | Multi-layered lamella vesicles capturing hot spring water and cosmetic composition with the effect of improving skin moisturizing and skin barrier thereof |
-
2020
- 2020-08-19 CN CN202010833788.XA patent/CN111905107B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007096924A2 (en) * | 2006-02-20 | 2007-08-30 | Maiello, Adolfo | Extraction, preparation and use of active principles of the aloe plant for producing self-meditaion devices such as controlled-release gauzes, bandages, stockings, elastic bands and arch supports |
| US20140161874A1 (en) * | 2011-05-23 | 2014-06-12 | Micaela Carvajal Alcaraz | Process of preparation and uses of plasma membrane vesicles extracted from plants enriched in membrane transport proteins |
| CN103479682A (en) * | 2012-06-14 | 2014-01-01 | 苏州恒宇生物科技有限公司 | Preparation method for plant source active component nano-scale membrane type vesicle |
| KR102059626B1 (en) * | 2019-05-10 | 2019-12-26 | 주식회사 바이오뷰텍 | Multi-layered lamella vesicles capturing hot spring water and cosmetic composition with the effect of improving skin moisturizing and skin barrier thereof |
Non-Patent Citations (4)
| Title |
|---|
| HARHAJI, L等: "Aloe emodin inhibits the cytotoxic action of tumor necrosis factor" * |
| SANTOSH PATIL等: "Comparative evaluation of natural antioxidants spirulina and aloe vera forthe treatment of oral submucous fibrosis" * |
| 吴菊萍等: "番茄外泌体的分离提取工艺优化及其作为药物载体的可行性分析" * |
| 王艳萍: "芦荟大黄素类脂囊泡的制备及其质量评价研究" * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113846046A (en) * | 2021-10-22 | 2021-12-28 | 科丝美诗(中国)化妆品有限公司 | Dendrobium-derived exosome-like vesicle, preparation method thereof and application thereof in alopecia improvement |
| CN114159373A (en) * | 2021-10-22 | 2022-03-11 | 科丝美诗(中国)化妆品有限公司 | Dendrobium-derived exosome-like vesicle, preparation method thereof and application thereof in skin care cosmetics |
| CN114159373B (en) * | 2021-10-22 | 2024-05-14 | 科丝美诗(中国)化妆品有限公司 | Dendrobium exosome-like vesicle and preparation method and application thereof in skin care cosmetics |
| CN115364176A (en) * | 2022-10-13 | 2022-11-22 | 中国医学科学院阜外医院深圳医院(深圳市孙逸仙心血管医院) | Application of dendrobium-derived nano vesicles in preparation of medicines for preventing and treating atherosclerosis diseases |
| CN115508492A (en) * | 2022-10-19 | 2022-12-23 | 陕西科技大学 | Fingerprint construction method and fingerprint of novel compound aloe capsule |
| WO2024108605A1 (en) * | 2022-11-25 | 2024-05-30 | 深圳先进技术研究院 | Preparation method for aloe-derived exosome and use thereof |
| CN116492279A (en) * | 2023-03-09 | 2023-07-28 | 广州拾纳客生物科技有限公司 | Exosome freeze-dried powder preparation method and application thereof |
| CN117205136A (en) * | 2023-11-09 | 2023-12-12 | 北京尧景基因技术有限公司 | Plant vesicle loaded cosmetic active small molecule compound, composition, method and application |
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