CN114982958B - Targeted modified astaxanthin-loaded cow milk exosome nano-preparation and preparation method thereof - Google Patents
Targeted modified astaxanthin-loaded cow milk exosome nano-preparation and preparation method thereof Download PDFInfo
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- KBPHJBAIARWVSC-XQIHNALSSA-N trans-lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C KBPHJBAIARWVSC-XQIHNALSSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C7/00—Other dairy technology
- A23C7/04—Removing unwanted substances other than lactose or milk proteins from milk
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C7/00—Other dairy technology
- A23C7/04—Removing unwanted substances other than lactose or milk proteins from milk
- A23C7/046—Removing unwanted substances other than lactose or milk proteins from milk by centrifugation without using chemicals, e.g. bactofugation; re-use of bactofugate
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
- A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Cosmetics (AREA)
Abstract
The invention relates to a targeted modification astaxanthin-loaded cow milk exosome nano-preparation and a preparation method thereof, belonging to the field of nano-preparation. The invention uses cow milk exosomes as carriers and uses amphiphilic distearoyl phosphatidylethanolamine-polyethylene glycol 2000 As a connecting structure, hyaluronic acid with a targeting function is modified on the surface of a phospholipid bilayer membrane of the milk exosome, and astaxanthin with biological activity is loaded in an ultrasonic manner to form a stable astaxanthin targeting carrier system. The prepared targeted modified astaxanthin-loaded cow milk exosome nano preparation has good comprehensive performance: the water solubility, the thermal stability, the oxidation resistance and the inhibition effect on cell oxidative damage caused by lipopolysaccharide induction are obviously improved compared with free astaxanthin, and meanwhile, the biological compatibility is good.
Description
Technical Field
The invention relates to a targeted modification astaxanthin-loaded cow milk exosome nano-preparation and a preparation method thereof, belonging to the field of nano-preparation.
Background
Astaxanthin is a biologically active substance of lutein, carotenoids, deep red color, mainly present in algae, yeasts and shrimps. Astaxanthin has various health care functions such as anti-diabetes, anti-inflammatory, immunoregulation and the like, and has wide application prospect in functional foods. In addition, astaxanthin has extremely strong antioxidant activity and extremely strong monooxygen quenching capacity, and is an incredible active oxygen scavenger. Due to the large number of unsaturated bonds present in its structure, is unstable in ultraviolet light and hot acidic and alkaline solutions and is easy to degrade. In addition, astaxanthin exists mainly in the form of esters, and has poor water solubility, thus limiting the bioavailability.
Disclosure of Invention
[ technical problem ]
The preparation method of the nano preparation for targeted delivery of astaxanthin based on the exosomes derived from natural cow milk is provided, and the prepared targeted modified astaxanthin-loaded cow milk exosomes nano preparation has good comprehensive performance: the water solubility, the thermal stability, the oxidation resistance and the inhibition effect on cell oxidative damage caused by lipopolysaccharide induction are obviously improved compared with free astaxanthin, and meanwhile, the biological compatibility is good.
Technical scheme
The first aim of the invention is to provide a preparation method of a targeted modification astaxanthin-loaded cow milk exosome nano-preparation, which comprises the following steps:
(1) Centrifuging the cow milk for the first time to remove fat impurities with larger particles in the cow milk;
(2) Taking the supernatant obtained in the step (1) to adjust the pH value to 4.6 so as to precipitate protein impurities;
(3) Centrifuging the system obtained in the step (2) for the second time, and filtering the supernatant to remove small-particle precipitated impurities;
(4) Centrifuging the filtrate obtained in the step (3) for the third time, and taking the precipitate as the milk exosome;
(5) Fully dissolving and uniformly mixing the cow milk exosomes with a neutral phosphate buffer solution to obtain cow milk exosome dispersion liquid;
(6) Preparation of hyaluronic acid-distearoyl phosphatidylethanolamine-polyethylene glycol 2000 Wherein hyaluronic acid and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 The mass ratio of (2) is 10:1;
(7) Mixing the cow milk exosome dispersion liquid obtained in the step (5) with the hyaluronic acid-distearoyl phosphatidylethanolamine-polyethylene glycol obtained in the step (6) 2000 Mixing according to a mass ratio of 20:1, and stirring for reaction to obtain a hyaluronic acid modified cow milk exosome dispersion;
(8) Dissolving astaxanthin in alcohol to obtain astaxanthin alcohol solution; mixing the hyaluronic acid modified bovine milk exosome dispersion liquid obtained in the step (7) with astaxanthin alcohol solution according to the mass ratio of the bovine milk exosome to the astaxanthin of (8-50): 1; intermittent ultrasonic treatment under ice bath conditions, incubation and circulation on ice for multiple times, and incubation to obtain the hyaluronic acid modified astaxanthin-loaded cow milk exosome dispersion;
(9) Centrifuging the hyaluronic acid modified astaxanthin-loaded cow milk exosome dispersion liquid obtained in the step (8) to remove the unencapsulated astaxanthin, and removing the solvent by a rotary steaming method to obtain the hyaluronic acid modified astaxanthin-loaded cow milk exosome nano preparation.
In certain embodiments, the first centrifugation in step (1) is performed at a rotational speed of 10000-13000 g at 10 ℃ for 30 min.
In certain embodiments, the second centrifugation in step (3) is performed at a rotational speed of 10000-13000 g at 10℃for 30 min.
In certain embodiments, the third centrifugation in step (4) is performed at a rotational speed of 135000-150000 g and an ultracentrifugation of 10 ℃ for 90 min.
In a preferred embodiment of the present invention, in step (8), the hyaluronic acid-modified milk exosome dispersion obtained in step (7) is mixed with an astaxanthin alcohol solution in a mass ratio of milk exosome to astaxanthin of 10:1.
As a preferred embodiment of the present invention, the step (6) specifically includes: firstly, activating carboxyl in hyaluronic acid; then distearoyl phosphatidylethanolamine-polyethylene glycol 2000 Dissolving in dimethylformamide, and dissolving the dimethylformamide in distearoyl phosphatidylethanolamine-polyethylene glycol 2000 And the activated hyaluronic acid solution is prepared according to hyaluronic acid and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 Mixing the components in a mass ratio of 10:1, stirring the mixture for 12 hours at room temperature, and obtaining the hyaluronic acid-distearoyl phosphatidylethanolamine-polyethylene glycol after dialysis and purification 2000 。
As a preferred embodiment of the present invention, a method for activating carboxyl groups in hyaluronic acid: the hyaluronic acid is dissolved in water, and then N-hydroxysulfosuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide are added to activate the carboxyl groups in the hyaluronic acid.
As a preferred embodiment of the invention, in the step (7), stirring reaction is carried out for 12-24 hours at the temperature of 4 ℃.
As a preferred embodiment of the present invention, in step (8): intermittent ultrasound and on-ice incubation cycles under ice bath conditions are specifically:
intermittent ultrasound: intermittently performing ultrasonic treatment for 2 minutes under the condition that the ultrasonic power is 200-250W and the ice bath, wherein an intermittent ultrasonic treatment program is performed according to the opening 15 s/closing 15 s;
incubation on ice: after intermittent ultrasound, incubation was continued on ice for 2 minutes;
the intermittent ultrasound and on-ice incubation steps described above were cycled 3 times.
As a preferred embodiment of the present invention, the ultrasonic power in step (8) is 200W.
As a preferred embodiment of the present invention, in step (8), the incubation is carried out at 37℃for 1 hour.
The second object of the invention is to provide a targeted modified astaxanthin-loaded cow milk exosome nano-preparation prepared by the method.
[ advantageous effects ]
(1) The invention uses bovine milk exosome which has a phospholipid bilayer structure similar to a cell membrane and is secreted by cells as a carrier and uses amphiphilic distearoyl phosphatidylethanolamine-polyethylene glycol 2000 As a connecting structure, hyaluronic acid with a targeting function is modified on the surface of a phospholipid bilayer membrane of the milk exosome, and astaxanthin with biological activity is loaded in an ultrasonic manner to form a stable astaxanthin targeting carrier system.
(2) According to the invention, the hyaluronic acid is modified on the surface of the cow milk exosome, so that the targeting capability of the cow milk exosome as a nano carrier can be improved; by distearoyl phosphatidylethanolamine-polyethylene glycol 2000 The modification of the hyaluronic acid on the cow milk exosomes is realized, and the technical problems that the compatibility of the hyaluronic acid and the cow milk exosomes is poor and the hyaluronic acid is difficult to compound are overcome.
(3) According to the preparation method of the nano preparation for targeted delivery of astaxanthin based on the exosomes derived from natural cow milk, the prepared targeted modified astaxanthin-loaded cow milk exosomes nano preparation has good comprehensive performance: the water solubility, the thermal stability, the oxidation resistance and the inhibition effect on cell oxidative damage caused by lipopolysaccharide induction are obviously improved compared with free astaxanthin, and meanwhile, the biological compatibility is good.
(4) Compared with free astaxanthin, the astaxanthin is entrapped by the cow milk exosomes, the invention has the advantages that the oxidation injury resistance protection of RAW264.7 cells caused by LPS induction and the reduction of the production of intracellular pro-inflammatory factors are improved, and the targeted carrier system formed by further modifying the astaxanthin has better oxidation resistance and anti-inflammatory activity compared with the independent cow milk exosomes.
(5) According to the research of the invention, the bovine milk exosome modified by hyaluronic acid can be used as an ideal delivery system of astaxanthin, so that the biological activity of astaxanthin can be effectively improved, and the stress resistance of astaxanthin can be increased.
Drawings
FIG. 1 is a transmission electron micrograph of the bovine milk exosomes obtained in step S4 of example 1 of the present invention.
FIG. 2 is a transmission electron microscope image of astaxanthin-loaded bovine milk exosomes obtained in step S5 of example 1 of the present invention.
FIG. 3 is a transmission electron micrograph of astaxanthin-loaded milk exosomes modified with hyaluronic acid obtained in example 2 of the present invention.
FIG. 4 is a transmission electron microscope image of the milk exosomes isolated by the differential centrifugation method alone of comparative example 1 of the present invention.
FIG. 5 is a graph showing comparison of astaxanthin entrapment rates measured in example 3 of the present invention.
FIG. 6 is an ultraviolet spectrum of astaxanthin after treatment of free astaxanthin with hyaluronic acid modified bovine milk exosomes obtained in step S6 of example 2 of the present invention.
FIG. 7 is a thermogravimetric analysis of free astaxanthin, astaxanthin-loaded bovine milk exosomes of example 1 of the present invention, and hyaluronic acid modified astaxanthin-loaded bovine milk exosomes of example 2.
FIG. 8 is a graph showing the effect of bovine milk-derived extracellular vesicles (bovine milk exosomes) of example 1 at different concentrations on cell viability according to the invention.
FIG. 9 is a graph showing the effect of astaxanthin-loaded bovine milk exosomes obtained in example 1 of the present invention at different concentrations on cell viability.
FIG. 10 is a graph showing the effect of different concentrations of hyaluronic acid modified astaxanthin-loaded milk exosomes of example 2 of the present invention on cell viability.
FIG. 11 is a graph showing the comparison of the fluorescence intensity of protection of Lipopolysaccharide (LPS) -induced inflammatory cell models by free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) under the same astaxanthin concentration conditions in the present invention.
FIG. 12 is a graph comparing inhibition of tumor necrosis factor of Lipopolysaccharide (LPS) -induced inflammatory cell models by free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) under the same astaxanthin concentration conditions in the present invention.
Detailed Description
The present application is not limited to the specific conditions and details of the following embodiments. The various specific features may be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the invention is not described in any way in which it is possible. Any person skilled in the art can make simple variants and substitutions within the technical scope of the description of the invention, depending on the circumstances, which are all within the scope of protection of the invention. Various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Examples of the present invention will be described in detail below with reference to the accompanying drawings, for the purpose of explaining the present invention, and the technical scheme is clearly and completely described without limitation to the reagents or instruments etc. used in the invention.
The invention is further illustrated by the following specific examples.
Purchase channel and product information for distearoyl phosphatidylethanolamine-polyethylene glycol 2000 in example 2: the purity of the product is more than or equal to 98 percent and is purchased from Shanghai Miclin Biochemical technology Co.
Example 1: preparation method of astaxanthin-loaded cow milk exosomes
S1, placing cow milk into a high-speed centrifugal bottle, and centrifuging for 30 min at the rotating speed of 13000 and g and the temperature of 10 ℃ to remove fat impurities with larger particles in the cow milk;
s2, taking the supernatant after centrifugation in the step S1, and regulating the pH value of the supernatant to 4.6 by using a 2 mol/L hydrochloric acid solution so as to precipitate protein impurities in the supernatant;
s3, centrifuging the system obtained in the step S2 at 10000 g and 10 ℃ for 30 min, taking supernatant, and then passing through a filter membrane with the pore size of 0.22 mu m to remove small particle precipitates such as cell fragments and the like;
s4, ultracentrifugating the filtered clear liquid obtained in the step S3 at 135000 and g ℃ for 90 min, and taking the precipitate as an exosome (cow milk exosome) of cow milk source, so that the exosome is completely dissolved in a neutral phosphate buffer solution to obtain a dispersion liquid of the cow milk exosome.
S5, mixing the dispersion liquid of the cow milk exosomes obtained in the step S4 with an ethanol solution of astaxanthin according to the mass ratio of the cow milk exosomes to the astaxanthin of 10:1, carrying out intermittent ultrasonic treatment for 2 minutes under the ice bath condition with the ultrasonic power of 200-250W by adopting an ultrasonic loading method, carrying out intermittent ultrasonic treatment program according to the opening 15S/closing 15S, and then continuously incubating on ice for 2 minutes; the intermittent ultrasonic incubation step is circulated for 3 times, and then the astaxanthin-loaded cow milk exosome dispersion liquid is obtained. The astaxanthin-loaded bovine milk exosome dispersion was incubated at 37℃for 1 hour to restore the stability of the bovine milk exosome membrane.
The astaxanthin-loaded cow milk exosome dispersion is centrifuged (3000 Xg, 5min,25 ℃) to remove the unencapsulated astaxanthin, and the ethanol is removed by rotary evaporation at 37 ℃ to obtain the astaxanthin-loaded cow milk exosome nano-preparation.
Example 2: preparation method of hyaluronic acid modified astaxanthin-loaded cow milk exosomes
Steps S1-S4 of example 2 are identical to steps S1-S4 of example 1;
s5, preparing hyaluronic acid-distearoyl phosphatidylethanolamine-polyethylene glycol 2000 : first, hyaluronic acid (900 mg,30. Mu mol) was completely dissolved in distilled water, and then N-hydroxysuccinimide (40.0 mu mol) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (50.0 mu mol) were added to obtain a mixed solution, which was continuously stirred at low temperature for 4 hours to activate carboxyl groups in hyaluronic acid; then distearoyl phosphatidylethanolamine-polyethylene glycol 2000 Dissolving in dimethylformamide, dissolving N, N-Dimethylformamide (DMF) in distearoyl phosphatidylethanolamine-polyethylene glycol 2000 And the activated hyaluronic acid solution is prepared according to hyaluronic acid and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 Mixing at a mass ratio of 10:1, stirring at room temperature for 12 hours, transferring the obtained viscous solution into 3500 Dalton dialysis bags for dialysis for 48 hours, and freeze-drying to obtain hyaluronic acid-distearoyl phosphatidylethanolamine-polyethylene glycol 2000 ；
S6, dispersing the cow milk exosomes obtained in the step S4 and the hyaluronic acid-distearoyl phosphatidylethanolamine-polyethylene glycol obtained in the step S5 2000 Mixing according to a mass ratio of 20:1, stirring for 12-24 hours at a temperature of 4 ℃, and obtaining a hyaluronic acid modified cow milk exosome dispersion liquid through self-assembly reaction;
s7, mixing the hyaluronic acid modified bovine milk exosome dispersion liquid obtained in the step S6 with an astaxanthin ethanol solution according to the mass ratio of the bovine milk exosome to the astaxanthin of 10:1; intermittently performing ultrasonic treatment for 2 minutes under the condition of ultrasonic power of 200-250W and ice bath by adopting an ultrasonic loading method, performing intermittent ultrasonic treatment program according to opening 15 s/closing 15 s, and then continuously incubating on ice for 2 minutes; the intermittent ultrasonic incubation step is circulated for 3 times, and then the hyaluronic acid modified astaxanthin-loaded cow milk exosome dispersion liquid is obtained. The hyaluronic acid modified astaxanthin-loaded bovine milk exosome dispersion was incubated at 37 ℃ for 1 hour to restore the stability of the bovine milk exosome membrane.
And (3) centrifuging the hyaluronic acid modified astaxanthin-loaded cow milk exosome dispersion liquid (3000 Xg, 5min,25 ℃) to remove unencapsulated astaxanthin, and removing ethanol at 37 ℃ by a rotary steaming method to obtain the hyaluronic acid modified astaxanthin-loaded cow milk exosome nano preparation.
Example 3: optimization of hyaluronic acid modified astaxanthin-loaded bovine milk exosomes-influence of mass ratio of bovine milk exosomes to astaxanthin on astaxanthin entrapment rate
Based on the example 2, the mass ratio of the bovine milk exosomes to the astaxanthin in the step S7 is adjusted, namely, the mass ratio of the bovine milk exosomes modified by the hyaluronic acid to the astaxanthin is respectively 10:1 and 50:1, and two hyaluronic acid modified astaxanthin-loaded bovine milk exosomes nano-preparations are respectively obtained.
As shown in fig. 5, the astaxanthin entrapment rate (74%) was higher at a mass ratio of 10:1 of cow's milk exosomes to astaxanthin than at a mass ratio of 50:1 (54%).
Comparative example 1: cow milk exosomes isolated by differential centrifugation only
A method for extracting milk exosomes, referring to example 1, differs only in that step S2 is omitted, i.e., milk exosomes isolated only by differential centrifugation.
The testing method comprises the following steps:
transmission electron microscope characterization: 20. Mu.L of the milk exosomes obtained in step S4 of example 1, the astaxanthin-loaded milk exosomes obtained in step S5 of example 1, the hyaluronic acid-modified astaxanthin-loaded milk exosomes obtained in example 2, and the milk exosomes obtained in comparative example 1, which were isolated only by differential centrifugation, were respectively dropped into copper mesh, and negative staining was performed with 2% uranyl acetate, and observation was performed under a transmission electron microscope.
Solubility test：
Mixed solution 1: 1 mg astaxanthin was mixed with 10. Mu.L of the hyaluronic acid-modified bovine milk exosomes obtained in step S6 of example 2, vortexed well for 10 minutes and incubated for 30 minutes, after which 1 ml ddH was added to the above solution 2 O. Mixed solution 2:1 mg astaxanthin was dissolved in 1 ml ddH 2 O. After the above two mixed solutions were allowed to stand on ice for 30 min, they were centrifuged at 5000 rpm for 5min to remove undissolved astaxanthin. Measurement of ultraviolet-to-visible spectrometer in the range of 250-700 nmIn the visible absorption spectrum, astaxanthin concentration was calculated to evaluate the solubility of astaxanthin in water.
Astaxanthin entrapment rate:
and (3) continuously carrying out ultrasonic treatment on the hyaluronic acid modified bovine milk exosome nano-preparation loaded with astaxanthin to be detected for 30 min so as to release the astaxanthin loaded in the bovine milk exosome, extracting by using dichloromethane/methanol (2:1, v/v), and measuring an ultraviolet-visible absorption spectrum in the range of 250-700 nm by using an ultraviolet-visible spectrometer so as to obtain the astaxanthin concentration, thereby determining the astaxanthin embedding rate.
Thermal stability test:
the Discovery TGA 550 instrument (TA, new Castle, USA) was used to perform thermogravimetric analysis (TGA) to characterize the thermal stability of free astaxanthin, astaxanthin-loaded bovine milk exosomes, hyaluronic acid modified astaxanthin-loaded bovine milk exosomes under the following test conditions: the temperature range in nitrogen atmosphere is 30 to 700 ℃ and the temperature rise rate is 30 ℃ per minute.
Characterization of cell viability: RAW264.7 cells were plated at 1X 10 cells per well 5 The density of individual cells was inoculated in 96-well plates, incubated for 24 hours to adhere the cells, after removal of the medium, the concentration of bovine milk exosomes (example 1) was quantified in terms of the concentration of bovine milk exosomes (0, 1, 2, 2.5, 5, 7.5, 10 μg/mL), the culture medium of astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) was incubated for 24 hours, 20 μl of thiazole blue at a concentration of 5 mg/mL was added after the incubation was completed, the absorbance value was measured at 570 nm after shaking for 10 minutes, and the absorbance value at 0 mg/mL was set as the cell viability value of 100%.
Intracellular active oxygen level assessment: RAW264.7 cells were plated at 1X 10 cells per well 5 The density of individual cells was seeded in twelve well plates. Quantification of astaxanthin concentration, free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) were dissolved in medium, incubated for 24 hours, and the medium was removed and used with the mediumThe medium with Lipopolysaccharide (LPS) at 1. Mu.g/mL was used instead, and the medium alone was used as a control. After 24 hours of treatment, the cells were stained with 0.5 mL dichlorofluorescein diacetate (DCFH-DA, ex=500 nm, em=525 nm) for 30 minutes. Immediately after three washes with PBS, the cells were observed under a fluorescence microscope and the fluorescence intensity results exhibited by the cells were analyzed by the imagine J software.
Evaluation of intracellular proinflammatory factor secretion level: RAW264.7 macrophages were 1X 10 per well 5 Density of individual cells were inoculated in twelve well plates and incubated for 24 hours to allow adhesion of cells to the plates, quantified at the same astaxanthin concentration, replaced with fresh medium containing astaxanthin alone, astaxanthin-loaded bovine milk exosomes (example 1), hyaluronic acid modified astaxanthin-loaded bovine milk exosomes (example 2), incubated for 24 hours after removal of the medium, replaced with medium containing lipopolysaccharide 1 μg/mL and incubated for 24 hours, and intracellular tumor necrosis factor (TNF-. Alpha.) expression levels were determined using a biochemical kit.
Performance comparison:
microscopic morphology: as shown in fig. 1, the bovine milk exosomes obtained in step S4 of example 1 showed a cup shape or a sphere shape, with a size of about 100 nm; as shown in fig. 2, the astaxanthin-loaded bovine milk exosomes obtained in step S5 of example 1 were in the form of spheres, increasing in size to about 145 a nm a; as shown in fig. 3, the hyaluronic acid modified astaxanthin-loaded bovine milk exosomes obtained in example 2 still showed a spherical shape, with a size of about 165 and nm; as shown in fig. 4, the purity of the cow's milk exosomes isolated only by the differential centrifugation method of comparative example 1 was reduced, and the density under electron microscopy was reduced.
Solubility: as shown in FIG. 6, the ultraviolet spectrum shows a characteristic absorption peak of astaxanthin in the wavelength range of 250-700 nm around 480 nm. Under the condition that the astaxanthin content is the same, the hyaluronic acid modified cow milk exosomes obtained in the example 2 show higher absorption peaks than the free astaxanthin after acting on the astaxanthin, and the hyaluronic acid modified cow milk exosomes are proved to be capable of greatly improving the solubility of the astaxanthin in water.
Thermal stability: as shown in the thermogravimetric analysis curve of fig. 7, both the astaxanthin-loaded milk exosomes (example 1) and the hyaluronic acid-modified astaxanthin-loaded milk exosomes (example 2) exhibited better thermal stability than the free astaxanthin: higher weight loss of free astaxanthin at about 300 ℃ indicates poor thermal stability of free astaxanthin above 300 ℃; and the astaxanthin-loaded cow's milk exosomes (example 1) and the hyaluronic acid-modified astaxanthin-loaded cow's milk exosomes (example 2) have small weight loss at 20-700 ℃, indicating excellent thermal stability at 20-700 ℃. Based on this, astaxanthin-loaded cow's milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded cow's milk exosomes (example 2) can be applied to thermal processing processes below 700 ℃, with wider application scenarios than free astaxanthin.
Cell viability assay showed: the cow milk exosomes (example 1) with the concentration of 1-10 mug/mL have no inhibition effect on cell viability, which indicates that the cow milk exosomes have good biocompatibility and can be used as a safe delivery carrier. The astaxanthin-loaded bovine milk exosomes (example 1) and the hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) showed a trend of increasing and then decreasing cell viability with increasing concentration of the bovine milk exosomes, and analysis revealed that both the astaxanthin-loaded bovine milk exosomes (example 1) and the hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) were effective in promoting cell proliferation in the range of 1 μg/mL to 5 μg/mL of the bovine milk exosomes. Preferably, for the astaxanthin-loaded bovine milk exosomes (example 1), when the concentration of the bovine milk exosomes is 1-2.5 μg/mL, the cell viability is significantly improved compared to the blank group without the addition of the bovine milk exosomes, especially when the concentration of the bovine milk exosomes is 2.5 μg/mL, the cell viability is improved the highest compared to the blank group without the addition of the bovine milk exosomes, and the improvement ratio reaches 134%. Preferably, for the hyaluronic acid modified astaxanthin-loaded bovine milk exosomes (example 2), when the concentration of the bovine milk exosomes is 2-5 μg/mL, the cell viability is improved by more than 10% compared to the blank group without the addition of the bovine milk exosomes, especially when the concentration of the bovine milk exosomes is 5 μg/mL, the cell viability is improved maximally, and the improvement ratio is up to 111% compared to the blank group without the addition of the bovine milk exosomes.
Intracellular active oxygen levels: the comparison of the protective fluorescence intensity of free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) against Lipopolysaccharide (LPS) -induced inflammatory cell models under the same astaxanthin concentration conditions is shown in fig. 11. Compared with the blank group, the lipopolysaccharide-induced group only shows the highest fluorescence intensity, which indicates that the oxidative damage in cells is increased and the active oxygen is increased; the treatment of free astaxanthin, astaxanthin-loaded milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded milk exosomes (example 2) showed a decrease in fluorescence intensity compared to the lipopolysaccharide-induced group, indicating a decrease in active oxygen content. Wherein the fluorescence intensity of example 2 was as low as 42%, closest to the blank (30%), and showed the strongest oxidation resistance, indicating that the hyaluronic acid modified astaxanthin-loaded bovine milk exosomes had a more effective protection against lipopolysaccharide-induced cell damage.
Intracellular proinflammatory factor secretion level: inhibition of tumor necrosis factor in Lipopolysaccharide (LPS) -induced inflammatory cell models by free astaxanthin, astaxanthin-loaded bovine milk exosomes (example 1) and hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes (example 2) under the same astaxanthin concentration conditions is shown in fig. 12. Compared to the blank group, the lipopolysaccharide-induced group alone showed the highest intracellular expression level of tumor necrosis factor (TNF- α). The intracellular expression levels of intracellular tumor necrosis factor (TNF- α) were significantly reduced compared to the lipopolysaccharide-induced group after treatment of astaxanthin-loaded (example 1) and hyaluronic acid-modified astaxanthin-loaded (example 2) bovine milk exosomes, wherein the intracellular expression levels of intracellular tumor necrosis factor (TNF- α) were as low as 104 pg/mL in the hyaluronic acid-modified astaxanthin-loaded bovine milk exosomes of example 2, approaching the blank group (90 pg/mL), indicating a more pronounced inhibition of intracellular pro-inflammatory factors and a more pronounced hyaluronic acid targeting effect.
Claims (8)
1. The preparation method of the targeted modification astaxanthin-loaded cow milk exosome nano preparation is characterized by comprising the following steps of:
(1) Centrifuging the cow milk for the first time to remove fat impurities with larger particles in the cow milk;
(2) Taking the supernatant obtained in the step (1), and adjusting the pH value to 4.6 so as to precipitate protein impurities;
(3) Centrifuging the system obtained in the step (2) for the second time, and filtering the supernatant to remove small-particle precipitated impurities;
(4) Centrifuging the filtrate obtained in the step (3) for the third time, and taking the precipitate as the milk exosome;
(5) Fully dissolving and uniformly mixing the cow milk exosomes with a neutral phosphate buffer solution to obtain cow milk exosome dispersion liquid;
(6) Preparation of hyaluronic acid-distearoyl phosphatidylethanolamine-polyethylene glycol 2000 Wherein hyaluronic acid and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 The mass ratio of (2) is 10:1;
(7) Mixing the cow milk exosome dispersion liquid obtained in the step (5) with the hyaluronic acid-distearoyl phosphatidylethanolamine-polyethylene glycol obtained in the step (6) 2000 Mixing according to a mass ratio of 20:1, and stirring for reaction to obtain a hyaluronic acid modified cow milk exosome dispersion;
(8) Dissolving astaxanthin in alcohol to obtain astaxanthin alcohol solution; mixing the hyaluronic acid modified bovine milk exosome dispersion liquid obtained in the step (7) with an astaxanthin alcohol solution according to the mass ratio of the bovine milk exosome to the astaxanthin of 10:1; intermittent ultrasonic treatment under ice bath conditions, incubation and circulation on ice for multiple times, and incubation to obtain the hyaluronic acid modified astaxanthin-loaded cow milk exosome dispersion;
(9) Centrifuging the hyaluronic acid modified astaxanthin-loaded cow milk exosome dispersion liquid obtained in the step (8) to remove the unencapsulated astaxanthin, and removing the solvent by rotary evaporation to obtain the hyaluronic acid modified astaxanthin-loaded cow milk exosome nano-preparation.
2. The method according to claim 1, wherein step (6) specifically comprises: firstly, activating carboxyl in hyaluronic acid; then distearoyl phosphatidylethanolamine-polyethylene glycol 2000 Dissolving in dimethylformamide, and dissolving the dimethylformamide in distearoyl phosphatidylethanolamine-polyethylene glycol 2000 And the activated hyaluronic acid solution is prepared according to hyaluronic acid and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 Mixing the components in a mass ratio of 10:1, stirring the mixture for 12 hours at room temperature, and obtaining the hyaluronic acid-distearoyl phosphatidylethanolamine-polyethylene glycol after dialysis and purification 2000 。
3. The method of claim 2, wherein the method of activating the carboxyl groups in the hyaluronic acid: the hyaluronic acid is dissolved in water, and then N-hydroxysulfosuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide are added to activate the carboxyl groups in the hyaluronic acid.
4. The method according to claim 1, wherein in the step (7), the reaction is carried out under stirring at 4 ℃ for 12-24 hours.
5. The method according to claim 1, wherein in step (8): intermittent ultrasound and on-ice incubation cycles under ice bath conditions are specifically:
intermittent ultrasound: intermittently performing ultrasonic treatment for 2 minutes under the condition that the ultrasonic power is 200-250W and the ice bath, wherein an intermittent ultrasonic treatment program is performed according to the opening 15 s/closing 15 s;
incubation on ice: after intermittent ultrasound, incubation was continued on ice for 2 minutes;
the intermittent ultrasound and on-ice incubation steps described above were cycled 3 times.
6. The method of claim 5, wherein the ultrasonic power in step (8) is 200W.
7. The method according to claim 1, wherein in step (8), the incubation is performed at 37 ℃ for 1 hour.
8. A targeted modified astaxanthin-loaded cow's milk exosome nanoformulation made by the method of any one of claims 1-7.
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