CN116650450A - Modified exosome and preparation method and application thereof - Google Patents
Modified exosome and preparation method and application thereof Download PDFInfo
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- CN116650450A CN116650450A CN202310568721.1A CN202310568721A CN116650450A CN 116650450 A CN116650450 A CN 116650450A CN 202310568721 A CN202310568721 A CN 202310568721A CN 116650450 A CN116650450 A CN 116650450A
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- milk
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Classifications
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/0602—Vertebrate cells
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses application of a modified exosome in preparation of a medicine for relieving xerophthalmia, wherein the modified exosome is milk exosome loaded with lutein. The invention also discloses a preparation method of the modified exosome and the milk exosome and an eye drop for relieving xerophthalmia. By loading lutein into the milk exosome, under the synergistic effect of the milk exosome and lutein, the prepared modified exosome can obviously improve the symptoms of xerophthalmia, solve the problems of poor water solubility and poor stability of lutein, and greatly improve the bioavailability of lutein. The exosomes used in the invention are milk exosomes, and the safe and high-yield milk exosomes with stable sources can be obtained by the method, so that the limitations of difficult culture, poor stability and the like of cell-derived exosomes in practical application are solved.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a modified exosome, a preparation method and application thereof.
Background
Dry eye (Dry eye disease), also known as keratoconjunctival dryness syndrome, refers to a general term for any abnormality in tear quality or quantity, or dynamic abnormality, that leads to a decrease in tear film stability, and is accompanied by ocular discomfort and/or damage to ocular surface tissue. The dry eye causes a lot of diseases, has wide people involved, has limited improving effect at present, greatly influences the life and work of patients and brings heavy burden to society.
Lutein (Lutein) is a carotenoid, and has chemical formula C 40 H 56 O 2 The dye is widely used in vegetables, fruits, flowers and other plants, and the chemical formula of the dye contains two ketone rings, so that the dye is a main pigment existing in the macular area of human eyes. For eyes, lutein can diminish inflammation, resist oxidation and inhibitThe activity of oxygen-producing free radicals prevents the damage of oxygen free radicals to normal cells. However, lutein is a lipophilic carotenoid, which results in poor water solubility and is susceptible to factors such as oxygen, light, heat, pH and the like, resulting in unstable structure.
Exosomes (Exosomes) are natural nanoscale vesicles secreted by cells, the surface of the Exosomes is composed of phospholipid bilayer, the inside of the Exosomes can contain various bioactive substances such as proteins, mRNA, miRNA and the like, and effective components such as proteins, drugs, miRNA and the like can be transferred into cells in a membrane fusion mode, so that the intercellular communication effect is achieved.
The exosome has the characteristics of small volume, stable structure, capability of protecting the content from degradation and the like, and is an ideal drug delivery carrier because the exosome is derived from cells and has the advantages of no cytotoxicity, low immunogenicity, high biocompatibility and the like. Due to the complicated operation and high cost of cell culture, mass production of exosomes is limited.
Disclosure of Invention
Aiming at the problems and the defects, the invention aims to provide a modified exosome and a preparation method and application thereof, wherein the modified exosome is a medicine carrying system based on a milk exosome, lutein is loaded by the milk exosome, under the synergistic effect of the exosome and the lutein, the modified exosome has excellent effect of improving xerophthalmia, and the xerophthalmia improving effect is realized in animal experiments, thereby effectively breaking through a plurality of limitations such as low yield of the cell exosome, and solving the problems such as poor effect when the lutein is directly applied to organisms.
In order to achieve the above purpose, the invention provides an application of a modified exosome in preparing a medicine for relieving xerophthalmia, wherein the modified exosome is milk exosome loaded with lutein.
In one embodiment of the invention, the milk exosomes comprise milk exosomes secreted by bovine mammary cells.
In one embodiment of the present invention, the method for preparing the milk exosomes comprises the following steps:
(1) Centrifuging milk for the first time to remove fat impurities, casein residues and cell debris with larger particles in the milk;
(2) Taking the supernatant obtained in the step (1) to adjust the pH value to 4.5-4.7 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) And (3) carrying out third centrifugation on the filtrate obtained in the step (3), and obtaining a precipitate which is the milk exosome.
In one embodiment of the present invention, in the step (1), the centrifugal rotational speed is 10,000 to 13,000g, the temperature at the time of centrifugation is 4 to 10 ℃, and the centrifugation time is 25 to 35 minutes.
In one embodiment of the present invention, in step (2), the pH of the supernatant is adjusted using an acid solution including hydrochloric acid, acetic acid, and the like.
In one embodiment of the present invention, in the step (3), the centrifugal speed is 10,000 to 13,000g, the centrifugal time is 25 to 40min, and the centrifugal force is 0.7X10 4 g。
In one embodiment of the present invention, in the step (3), the pore size of the filter membrane used in the filtration is 0.22 to 0.45. Mu.m.
In one embodiment of the present invention, in step (3), the filtration is divided into two steps, first using a 0.45 μm filter membrane and then using a 0.22 μm filter membrane.
In one embodiment of the present invention, in the step (4), the centrifugal speed is 130,000 to 150,000g, the centrifugal time is 1 to 1.5 hours, and the centrifugal force is 13X 10 4 g。
In one embodiment of the invention, the modified exosomes are prepared by the following steps:
(1) Fully dissolving and uniformly mixing milk exosomes with a phosphate buffer solution to obtain a cow milk exosome dispersion liquid;
(2) Mixing lutein with the milk exosome dispersion liquid obtained in the step (1) under ice bath condition, performing ultrasonic treatment, and incubating on ice to obtain a mixture, and circulating the step for 2-3 times to obtain the mixture;
(3) Centrifuging the mixture obtained in the step (2) at a high speed to remove unloaded lutein;
(4) And (3) placing the mixture obtained in the step (3) into a super-high-speed refrigerated centrifuge, and separating to obtain a modified exosome.
In one embodiment of the invention, the mass ratio of lutein to milk exosomes is 1:9-1:10.
In one embodiment of the invention, the power during ultrasonic treatment is 140-160W and the ultrasonic time is 30s.
In one embodiment of the invention, after incubation on ice for 1-2 min, the method further comprises the step of incubating the mixture in the dark at a temperature of 35-37 ℃ for 110-130 min.
In one embodiment of the present invention, in the step (3), the rotational speed at the time of high-speed centrifugation is 4000 to 6000g for 5 to 10 minutes.
In one embodiment of the present invention, in the step (4), the centrifugal force is 13.0X10 when the ultra-high-speed cryocentrifuge is used for centrifugation, the rotational speed is 120,000 to 130,000g, the centrifugation time is 1 to 1.5 hours, and the centrifugal force is 13.0X10 4 g, the centrifugal temperature is 4-10 ℃.
In one embodiment of the invention, the dry eye relief drug comprises an eye drop or eye ointment.
In one embodiment of the invention, the eye drop is prepared by dissolving the modified exosomes in phosphate buffer solution or physiological saline.
In one embodiment of the invention, the relief of dry eye is a relief, inhibition, improvement or alleviation of any one or more of the following symptoms, signs:
(1) Eye discomfort such as dry feeling, foreign body sensation, burning sensation and the like caused by too fast tear evaporation;
(2) Punctate shedding of cornea epithelium in the eyelid cleavage area, and fluorescein staining in the cornea epithelium defect area;
(3) Limbal epithelial cell dysfunction, corneal thinning, ulceration and even perforation caused by inflammation.
The invention discloses a modified exosome prepared by the method.
The invention also discloses an eye drop prepared by the method.
The invention has the beneficial effects that:
(1) Compared with single lutein or single milk exosome, the prepared modified exosome can obviously improve the symptoms of xerophthalmia, and the symptoms of xerophthalmia are improved under the synergistic effect of the milk exosome and lutein.
(2) The exosomes used in the invention are milk exosomes, and the safe and high-yield milk exosomes with stable sources can be obtained by the method, so that the limitations of difficult culture, poor stability and the like of cell-derived exosomes in practical application are solved.
(3) In the modified exosomes prepared by the method, the embedding rate of the exosomes to lutein is high and can reach 69.22%, the particle size of the modified exosomes is uniform, and the PDI dispersion coefficient is smaller than 0.2.
(4) The modified exosome prepared by the method can obviously increase tear content and tear film rupture time of benzalkonium chloride mice, obviously improve dry eye symptoms, simultaneously promote the increase of corneal epithelium thickness and the decrease of keratin expression content, accelerate lacrimal gland tissue recovery, and has an effect obviously superior to that of free lutein and milk exosomes.
Drawings
Fig. 1 is a characteristic diagram of the milk exosome prepared in example 1, wherein (a) is a conceptual diagram, (b) is a characteristic western blotting diagram, (c) is a particle size, and (d) is a transmission electron microscopy diagram.
FIG. 2 is a characteristic diagram of the modified exosome prepared in example 2, wherein (a) is a conceptual diagram, (b) is a particle size, and (c) is a transmission electron microscope.
FIG. 3 shows the encapsulation efficiency of lutein in modified exosomes prepared in example 2.
FIG. 4 is a schematic of the solubility of free lutein, milk exosomes and modified exosomes in PBS buffer.
Fig. 5 is a photograph of fluorescein staining of corneal tissue of benzalkonium chloride mice after instillation of various eye drops.
FIG. 6 is a chart showing the fluorescein staining scores of corneal tissue of benzalkonium chloride mice after instillation of various eye drops.
Fig. 7 is tear content of benzalkonium chloride mice before and after intervention with different eye drops.
Figure 8 is a graph showing tear film break up time in benzalkonium chloride mice before and after intervention with different eye drops.
Fig. 9 is a graph of corneal tissue thickness recovery levels of benzalkonium chloride mice before and after different eye drop interventions.
FIG. 10 shows the expression levels of keratoprotein K10 in benzalkonium chloride mice before and after intervention with different eye drops.
Fig. 11 is a graph showing the lacrimal gland size recovery levels of benzalkonium chloride mice following various eye drop dry prognosis.
Fig. 12 is a graph showing the lacrimal gland to body weight ratio of benzalkonium chloride mice with different eye drop dry prognosis.
Detailed Description
In order to make the objects, technical solutions and effects of the present invention more clear and clear, the present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The reagent and the raw materials used in the invention are commercially available or can be prepared according to a literature method, wherein the PBS buffer solution used in the examples is 1X PBS buffer solution produced by Beijing Soy Bao technology Co., ltd, and the commercial product used in the examples is lutein medical antipyretic gel produced by Shanxi Hancheng anecdotal medical instruments Co., ltd
EXAMPLE 1 preparation of milk exosomes
The raw milk is commercially available milk pasteurized from local trioatlas pasture.
A preparation method of milk exosomes comprises the following steps:
(1) Centrifuging milk for the first time to remove fat impurities, casein residues and cell debris with larger particles in the milk, collecting emulsion in the middle part to obtain skimmed milk, centrifuging at 4deg.C at 13,000g for 30min;
(2) Taking the supernatant obtained in the step (1), and adjusting the pH value to 4.6 by using hydrochloric acid with the concentration of 2M so as to precipitate protein impurities;
(3) Subjecting the system obtained in step (2) to secondary centrifugation at 10,000g for 40min (centrifugal force of 0.7X10 g) 4 g) Filtering the supernatant to remove small particle precipitation impurities, and filtering the supernatant with 0.45 μm and 0.22 μm filter membrane;
(4) And (3) performing third centrifugation on the filtrate obtained in the step (3), washing the obtained precipitate with phosphate buffer solution for 3 times to obtain the milk exosome, wherein the centrifugation speed is 130,000g, and the centrifugation time is 1.5h.
The particle size and potential of the milk exosome prepared by transmission electron microscope observation and dynamic light scattering measurement are shown in figure 1, the milk exosome shows a cup stand shape, the hydration particle size is 121nm, and the biological property is good.
EXAMPLE 2 preparation of modified exosomes
A method for preparing a modified exosome, comprising the steps of:
(1) Fully dissolving and uniformly mixing the milk exosomes with PBS buffer solution to obtain cow milk exosome dispersion liquid;
(2) Mixing lutein with the milk exosome dispersion liquid obtained in the step (1) under ice bath condition, treating for 30s under ultrasonic power of 150W, and incubating on ice for 1min, repeating the step for 3 times, and incubating at 37 ℃ for 120min in dark again to obtain a mixture;
(3) Centrifuging the mixture obtained in the step (2) at a high speed at 25 ℃ to remove unloaded lutein, wherein the rotation speed is 5000g during centrifugation, and the centrifugation time is 5min;
(4) Placing the mixture obtained in step (3) into a super-high speed refrigerated centrifuge, centrifuging at 130,000g for 1.5h (centrifugal force is 13.0X10) 4 g) And obtaining the modified exosome through separation.
Wherein, the mass ratio of lutein to milk exosomes is 1:9 for loading, and the preparation method of the milk exosomes is the same as in example 1.
The particle size and potential of the modified exosome are observed by a transmission electron microscope and measured by dynamic light scattering, the result is shown in a figure 2, wherein lutein-exosome is the modified exosome, milk exosome loaded with lutein shows a cup stand shape, the hydration particle size is 145nm, and the biological property is good.
Detecting light absorption value at 474nm by ultraviolet spectrophotometer, calculating supernatant concentration and encapsulation efficiency according to lutein standard curve, and calculating encapsulation efficiency as follows:
encapsulation efficiency = [ (absorbance of milk exosomes immediately after lutein addition-absorbance of supernatant after centrifugation)/absorbance of milk exosomes immediately after lutein addition ] ×100%.
The encapsulation efficiency of lutein in the modified exosomes prepared in example 2 was tested and calculated to be 69.22%, as shown in fig. 3.
The free lutein, the milk exosomes prepared according to the method of example 1 and the modified exosomes prepared according to the method of example 2 are respectively mixed with the PBS buffer solution, and as can be seen from fig. 4, the pure lutein has poor solubility in the PBS buffer solution, the lutein is basically sunk at the bottom of the test tube, after the lutein is loaded in the milk exosomes, the milk exosomes loaded with the lutein can be uniformly dispersed in the PBS buffer solution, and therefore, the water solubility of the lutein can be remarkably improved when the lutein is loaded in the milk exosomes.
Example 3 preparation of mouse Dry eye model and Dry eye symptomatic relief
(1) C57 mice of 6-8 weeks old were selected, and the slit lamp was examined for no abnormality, and the tear secretion amount and corneal fluorescein staining were not abnormal. Dividing the method into 5 groups by adopting a random grouping method, wherein each group is 20 as a treatment group;
(2) The model of dry eye was modeled using a benzalkonium chloride solution at a mass concentration of 0.2% for 5 weeks on both eyes of 5 mice of the treatment group, 1 time a day, 5 μl/eye each time.
(3) Five groups of mice were dosed with 2 μl each per eye, once daily, using PBS buffer, eye drops containing free lutein, milk exosomes, modified exosomes, and commercial products, starting on the first day after 5 weeks, and relevant dry eye index assays were performed at time points 1, 4, 7, 14, 21 days.
Wherein the preparation method of milk exosomes is the same as in example 1, the preparation method of modified exosomes is the same as in example 2, and the eye drops containing free lutein, milk exosomes and modified exosomes are prepared by dissolving the substances in PBS buffer solution, wherein the concentration of lutein in the eye drops containing free lutein is 126 mug/mL, and the concentration of milk exosomes in the eye drops containing milk exosomes is 3.5×10 11 The concentration of modified exosomes in the eye drops containing modified exosomes is 1.5 x 10 per mL 12 /mL。
The control group was normal mice, and no treatment was required.
The model group is a mouse after dry eye model modeling is carried out on normal mice, and the modeling process is to use benzalkonium chloride solution with the mass concentration of 0.2% to drop the two eyes of 5 mice in the treatment group for 5 weeks, wherein the two eyes of the 5 mice in the treatment group are used for 1 time per day, and each time is 5 mu L/eye.
Dry eye index detection
After eye drops are dropped, on days 1, 4, 7, 14 and 21, chloral hydrate with the mass concentration of 4% is injected into the abdominal cavity of two groups of mice, the injection dose is 8 mu L/g, the mice are anesthetized, and the corneal epithelial fluorescein staining score, the measurement of tear secretion, the recovery of the thickness of cornea tissue and the recovery of the weight of lacrimal gland after dry eye induction are detected, so that the results are shown in fig. 5-12, wherein lutein-exosomes in the figures are modified exosomes.
(1) Corneal fluorescent staining score: 3 mu L of liquid sodium fluorescein with the mass concentration of 2% is dripped into conjunctival sac of a mouse, after eyes are closed in a darkroom for 1min, the corneal epithelial lesions of each eye are photographed and observed under a slit lamp by using cobalt blue light. Cornea staining scoring was performed according to the corresponding content in the reference:
S.Li,Z.Lu,Y.Huang,Y.Wang,Q.Jin,X.Shentu,J.Ye,J.Ji,K.Yao,H.Han,Anti-Oxidative and Anti-Inflamma tory Micelles:Break the Dry Eye Vicious Cycle,Adv Sci(Weinh)9(17)(2022)e2200435.
(2) Measuring the amount of tear secretion: tear test strips were placed 1/3 of the way out of the conjunctival sac of the lower eyelid of the mouse with ophthalmic microshelpers, and after 1min the readings were taken out and the yellow portion of the strip was recorded in mm units of measurement. Each eye was tested 3 times per eye, 15min apart, with the final result being the average of the 3 measurements.
(3) Tear film break time: 3 μl of liquid sodium fluorescein with a mass concentration of 2% was dropped into the conjunctival sac of the mouse, and when the first black spot on the corneal surface was observed using a slit lamp imaging system under cobalt blue light, tear film rupture time was recorded in seconds by pausing a stopwatch. Each group was measured 3 times for 10 right eyes and averaged for analysis.
(4) Cornea tissue thickness recovery: mice were sacrificed by cervical scission, eyeballs were taken and fixed in 4% paraformaldehyde, paraffin sections of eyeballs were made, and H & E staining was performed. Corneal tissue was photographed with an inverted microscope and tissue thickness was calculated using ImageJ software. Each slice was tested 3 times and the final result was the average of 3 measurements.
(5) Corneal keratin K10 expression level: tissue fluorescent staining was performed on the mouse cornea sections with K10 protein fluorescent antibodies, and photographic observation was performed under a fluorescent inverted microscope.
(6) Lacrimal gland size recovery level: mice among different groups were sacrificed by cervical scission, lacrimal gland measurements were taken, photographed and observed, and the ratio of lacrimal gland to body weight was calculated.
The experimental results show that the milk exosomes are stable in shape and size before and after loading lutein, and the characteristic proteins are obvious (figures 1 and 2). The encapsulation effect of milk exosomes on lutein is good (figure 3). FIGS. 5 and 6 show that the modified exosome group had a clear healing trend in the area of corneal damage from day seven of improvement compared to the PBS group and lutein group (FIG. 5), and that the corneal epithelium of the PBS and lutein-improved groups still exhibited large area flaky fluorescein staining with prolonged improvement time; the improvement of milk exosomes, modified exosomes and commercial products is improved, the area of fluorescein staining is obviously reduced, and the improvement effect of the modified exosomes is most obvious. From fig. 7 and 8, it can be seen that the indexes of dry eye symptoms such as tear content of mice in the modified exosome group are obviously improved, wherein the intervention effect of milk exosome on dry eye symptoms is similar to that of the modified exosome, and the modified exosome has a certain improvement effect on dry eye symptoms. Cornea recovery is shown in fig. 9 and 10, wherein the keratin K10 group in fig. 10 is an image observed under a fluorescence inversion microscope after treatment of keratin with K10, the cell nucleus DAPI is an image observed under a fluorescence inversion microscope after treatment of cell nucleus with DAPI dye, and the mixed group is an image observed under a fluorescence inversion microscope after treatment of keratin and cell nucleus with K10 and DAPI dye, respectively. Compared with the control group and the model group, the PBS group and the lutein group have no obvious improvement effect, the cornea epithelium has deep color and is still in an inflammatory infiltration state, the thickness of the cornea epithelium is increased after the exosome group, the modified exosome group and the commercial product group are improved, the keratin expression content is obviously reduced, and the modified exosome group has the most obvious effect. As can be seen from fig. 11 and 12, in the early stages of improvement, inflammation of dry eye causes significant atrophy of lacrimal gland tissue, and as the improvement time is prolonged, lacrimal gland tissue of the dry prognosis of exosomes, modified exosomes and commercial product groups is significantly increased, the color is recovered to be normal, no obvious inflammatory infiltration symptoms exist, the lacrimal gland sizes of PBS group and lutein group are recovered slowly, and inflammatory infiltration symptoms still exist in part of tissues.
Therefore, the milk exosome loaded with lutein can obviously improve symptoms and signs of xerophthalmia. After extracting exosomes in fresh milk by using an ultracentrifugation method, lutein is loaded into the milk exosomes by utilizing hydrophobic interaction, the milk exosomes loaded with lutein are obtained, the problems that cell-derived exosomes are complex to cultivate in practical application and difficult to produce in quantity are solved, meanwhile, the problems that lutein is insoluble in water, low in-vivo bioavailability, poor in stability and the like are solved, the characteristic symptoms of xerophthalmia can be obviously improved, and the application range is wider.
The above detailed description describes the analysis method according to the present invention. It should be noted that the above description is only intended to help those skilled in the art to better understand the method and idea of the present invention, and is not intended to limit the related content. Those skilled in the art may make appropriate adjustments or modifications to the present invention without departing from the principle of the present invention, and such adjustments and modifications should also fall within the scope of the present invention.
Claims (10)
1. The application of the modified exosome in preparing the medicine for relieving xerophthalmia is characterized in that the modified exosome is milk exosome loaded with lutein.
2. Use according to claim 1, wherein the milk exosomes comprise milk exosomes secreted by bovine mammary cells, the method of preparing the milk exosomes comprising the steps of:
(1) Centrifuging milk for the first time to remove fat impurities, casein residues and cell debris with larger particles in the milk;
(2) Taking the supernatant obtained in the step (1) to adjust the pH value to 4.5-4.7 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) And (3) carrying out third centrifugation on the filtrate obtained in the step (3), and obtaining a precipitate which is the milk exosome.
3. The use according to claim 2, wherein the rotational speed is 10000-13000 g, the centrifugation time is 25-35 min, and the centrifugation temperature is 4-10 ℃ during the first centrifugation in step (1) and the second centrifugation in step (3); in the third centrifugation in the step (4), the rotation speed is 130000 ～ 150000g, and the centrifugation time is 1-1.5 h.
4. The use according to claim 1, wherein the modified exosomes are prepared by the following steps:
(1) Fully dissolving and uniformly mixing milk exosomes with a phosphate buffer solution to obtain a cow milk exosome dispersion liquid;
(2) Mixing lutein with the milk exosome dispersion liquid obtained in the step (1) under ice bath condition, performing ultrasonic treatment for 30s under the ultrasonic condition with the power of 140-160W, incubating on ice for 1-2 min, and circulating for 2-3 times to obtain a mixture;
(3) Centrifuging the mixture obtained in the step (2) at a high speed to remove unloaded lutein;
(4) And (3) placing the mixture obtained in the step (3) into a super-high-speed refrigerated centrifuge, and separating to obtain a modified exosome.
5. The use according to claim 4, wherein the mass ratio of lutein to milk exosomes is 1:9 to 1:10.
6. The method according to claim 4, wherein in the step (2), after the ultrasonic treatment and the incubation on ice, the method further comprises the step of incubating the mixture in the dark at a temperature of 35-37 ℃ for 110-130 min.
7. The method according to claim 4, wherein in the step (3), the rotational speed at the time of high-speed centrifugation is 4000 to 6000g for 5 to 10 minutes; in the step (4), when the ultra-high-speed refrigerated centrifuge is used for centrifugation, the rotating speed is 120000-130,000 g, the centrifugation time is 1-1.5 h, and the centrifugation temperature is 4-10 ℃.
8. The use according to claim 1, wherein the dry eye relief drug comprises an eye drop or an eye ointment.
9. A modified exosome, characterized in that it is prepared by the preparation method described in claims 4 to 7.
10. An eye drop for alleviating dry eye, which is prepared by dissolving the modified exosome according to claim 9 in phosphate buffer solution or physiological saline.
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| CN112870339A (en) * | 2019-12-05 | 2021-06-01 | 珠海天使干细胞生物科技有限公司 | Application of cell exosome in eye mask |
| WO2022108956A2 (en) * | 2020-11-18 | 2022-05-27 | Abbott Laboratories | Methods of increasing height and promoting linear bone growth |
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| CN114982958A (en) * | 2022-06-13 | 2022-09-02 | 大连工业大学 | Targeted modified astaxanthin-loaded cow milk exosome nano preparation and preparation method thereof |
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| CN112870339A (en) * | 2019-12-05 | 2021-06-01 | 珠海天使干细胞生物科技有限公司 | Application of cell exosome in eye mask |
| CN112094808A (en) * | 2020-09-16 | 2020-12-18 | 中山大学中山眼科中心 | MiR-204-containing exosome and preparation method and application thereof |
| WO2022108956A2 (en) * | 2020-11-18 | 2022-05-27 | Abbott Laboratories | Methods of increasing height and promoting linear bone growth |
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