CN117398389B - Mesenchymal stem cell exosome loaded with boswellic acid and preparation method and application thereof - Google Patents
Mesenchymal stem cell exosome loaded with boswellic acid and preparation method and application thereof Download PDFInfo
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- CN117398389B CN117398389B CN202311252743.3A CN202311252743A CN117398389B CN 117398389 B CN117398389 B CN 117398389B CN 202311252743 A CN202311252743 A CN 202311252743A CN 117398389 B CN117398389 B CN 117398389B
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Abstract
The invention relates to a mesenchymal stem cell exosome loaded with boswellic acid, which comprises a carrier and an active ingredient, wherein the carrier is the mesenchymal stem cell exosome, and the active ingredient is boswellic acid; the medicine carrying rate of the active ingredient is 5-15%; the active ingredient is loaded on the surface of the exosome and loaded inside the exosome. According to the invention, the hydrophobic and lipophilic boswellic acid is loaded on the exosomes in a chemical crosslinking mode, and the boswellic acid and the mesenchymal stem cell exosomes serving as the carrier have the functions of immunoregulation and tissue repair, so that the effective delivery and directional concentration of the medicine can be realized by utilizing the delivery characteristics of the exosomes, and meanwhile, the immunoregulation and tissue repair functions of the stem cell exosomes can be fully exerted, and a good treatment effect of treating and repairing acne can be realized.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a mesenchymal stem cell exosome loaded with boswellic acid, a preparation method and application thereof.
Background
Acne is a skin disease that is predominantly manifested by chronic inflammation of the pilo-sebaceous unit, and by 2019 worldwide it was estimated that 23.1 million people had acne. The prevalence of acne in China is 8.1%, and the skin damage of the acne can cause local pain and itching, but the biggest influence of the acne on patients is psychological stress. The onset of acne is mainly related to factors such as increased androgen level, excessive sebum secretion, excessive follicular keratinization, propionibacterium acnes proliferation, and genetic, dietary, and immune. Although the treatment methods of acne are various as medical research is continued, the existing treatment methods such as external and oral antibiotics, external tretinoin medicines, hormone treatment and the like still have various problems of tolerance, antibiotic resistance and the like.
Mesenchymal stem cells are multipotent stem cells derived from mesoderm, have self-replication and strong differentiation capacity, and can be obtained from various materials such as bone marrow, umbilical cord, placenta, fat and the like. Mesenchymal stem cells are low in immunogenicity and exert various functions mainly by paracrine means, and the paracrine substances include soluble cytokines, chemokines, exosomes and the like. The exosomes are nano-scale vesicles with the diameter of about 30-150nm and the density of about 1.10-1.14g/mL, are in a 'structure' under an electron microscope, and internally carry various bioactive substances such as protein, lipid, RNA and the like. Exosomes can transfer abundant substances between cells, and exosomes delivery to recipient cells is a key step in mediating changes in cellular behavior. In addition, they play an important role in intercellular communication.
The boswellic acid is a substance extracted from boswellia carterii, and the main component of the boswellic acid is 3 alpha-acetyl-11-keto-beta-boswellic acid, and clinical experiments prove that the boswellic acid can inhibit the activity of 5-lipoxygenase, thereby playing an anti-inflammatory role. Boswellic acid is also disclosed for skin microbiome normalization of sensitive skin. For example, chinese patent application CN116234554a discloses a composition for skin microbiome normalization of sensitive skin, a topical composition comprising boswellic acid, one or more boswellic acid derivatives or mixtures thereof, wherein the boswellic acid, boswellic acid derivatives or mixtures thereof are encapsulated in a nanoemulsion having a liquid lipid core and a negative surface charge; the size of the nano emulsion is in the range of 50-200 nm; the surface charge is in the range of-50 to-90 mV. The encapsulation structure is obtained by dissolving emulsifier (glycerolcitrate/lactate/linoleate/oleate), auxiliary emulsifier (diglycerol monooleate) in pentanediol, adding stabilizer, emollient and active ingredient (boswellia serrata extract with purity of boswellia serrata derivative exceeding 40%) and high pressure homogenizer (Microfluidizer) such as deionized water, and performing 2 cycles under 700bar pressure. Although this protocol discloses the use of boswellic acid or derivatives thereof to make a composition for the normalization of the microbiome of the skin of sensitive skin, it is not constructed with exosomes as carriers and the composition is mainly validated for the effects on the change of condition of sensitive skin and the microbiome profile and confirms that it is possible to normalize significantly the abundance of bacteria belonging to the phylum firmicutes, more particularly staphylococcus epidermidis, in the sensitive skin group, bringing the microbiome profile closer to that of healthy skin. By searching, no report on the drug taking the exosome as a carrier and taking the boswellic acid as an active ingredient is found at present, and how the drug taking the exosome as the carrier and taking the boswellic acid as the active ingredient has therapeutic activity cannot be predicted.
Disclosure of Invention
First, the technical problem to be solved
In view of the above-mentioned shortcomings and disadvantages of the prior art, the present invention provides a mesenchymal stem cell exosome loaded with boswellic acid, wherein hydrophobic lipophilic boswellic acid is loaded onto the exosome by chemical crosslinking, so as to improve the solubility and bioavailability of boswellic acid in a buffer solution; the boswellic acid and the mesenchymal stem cell exosomes serving as the carriers have the functions of immunoregulation and tissue repair, can realize the effective delivery and directional concentration of the medicaments by utilizing the exosome delivery characteristics, can fully play the functions of immunoregulation and tissue repair of the stem cell exosomes, and realize the good treatment effect of treating and repairing acne.
(II) technical scheme
In a first aspect, the present invention provides a mesenchymal stem cell exosome loaded with boswellic acid, comprising a carrier and an active ingredient, wherein the carrier is the mesenchymal stem cell exosome, and the active ingredient is boswellic acid; the medicine carrying rate of the active ingredient is 5-15%; the active ingredient is loaded on the surface of the exosome and loaded inside the exosome. Preferably, the active ingredient loading is from 10% to 15%, more preferably 14.1%.
In the present application, the definition of boswellic acid is: a substance extracted from boswellia, the main component of which is 3α -acetyl-11-keto- β -boswellic acid, and which includes but is not limited to β -boswellic acid, acetyl- β -boswellic acid, 11-keto- β -boswellic acid, and acetyl-11-keto- β -boswellic acid.
According to a preferred embodiment of the invention, the boswellic acid is cross-linked to the exosomes by a combination of NHS, EDC.
According to a preferred embodiment of the present invention, the mesenchymal stem cell exosome is a adipose mesenchymal stem cell exosome.
In a second aspect, the present invention relates to a solution of the above-mentioned boswellic acid-loaded mesenchymal stem cell exosomes, wherein the solvent of the solution is PBS buffer, and the solution contains the above-mentioned boswellic acid-loaded mesenchymal stem cell exosomes.
Preferably, the concentration of the mesenchymal stem cell exosomes loaded with boswellic acid in the solution is 1.0E-1.12e+10 parts per mL, more preferably 1.10e+10 parts per mL.
In a third aspect, the present invention also provides a method for preparing a boswellic acid-loaded mesenchymal stem cell exosome solution, comprising:
s1, extracting primary mesenchymal stem cells, culturing, separating and purifying exosomes in cell supernatant, and re-suspending to prepare exosome suspension;
S2, preparing a boswellic acid solution, adding a solution containing NHS and EDC, and performing incubation reaction at room temperature to obtain a pre-modified boswellic acid solution; the carboxyl groups of the boswellic acid in the pre-modified boswellic acid solution are activated by EDC and NHS to be loaded on the surface and inside of exosomes;
S3, mixing the exosome suspension with the pre-modified boswellia acid solution, filling the mixed solution into a syringe, and connecting the syringe to a filter unit; pushing a plunger of the injector to sequentially pass the mixed solution through a multi-stage filter membrane with a filter hole reduced from 400nm to 100nm to prepare a solution containing mesenchymal stem cell exosomes loaded with boswellic acid; the boswellic acid is loaded on the surface of the exosomes and on the inside of the exosomes.
According to a preferred embodiment of the present invention, S1 comprises: collecting the supernatant of the mesenchymal stem cells, centrifuging for a plurality of times, and sequentially increasing the centrifugation speed to remove cells, dead cells and cell fragments from the cell supernatant respectively; finally, taking supernatant and centrifuging at a centrifugation speed of 80000-120000Xg/min for 5070min to obtain exosome sediment, re-suspending the exosome sediment by using sterile PBS, and centrifuging at a centrifugation speed of 80000-120000Xg/min again to obtain exosome; the exosomes were resuspended in PBS to obtain exosome suspension.
Preferably, after collecting the supernatant of the mesenchymal stem cells, the cells are first isolated by centrifugation at 300 Xg/min for 10min at low speed; centrifuging the supernatant at 2000 Xg/min for 10min, and removing dead cells; centrifuging the rest supernatant at 10000 Xg/min for 30min to remove cell debris; then, taking the supernatant again, centrifuging at a super-high speed of 100000 Xg/min for 60min to obtain an exosome precipitate, re-suspending the exosome precipitate with sterile PBS, and centrifuging at a super-high speed of 100000 Xg/min for 70min again to obtain a pure exosome; in order to facilitate subsequent loading of boswellic acid, it may be resuspended in PBS to give an exosome suspension.
According to a preferred embodiment of the present invention, S2 comprises: dissolving boswellic acid in DMSO to prepare a boswellic acid solution with a concentration of 8-15mg/mL, weighing NHS and EDC according to a mass ratio of 1:1, and dissolving in PBS to prepare a NHS-EDC solution with a total concentration of 0.95-1.2 mg/mL; mixing the boswellic acid solution with the NHS-EDC solution in equal volume, and incubating for 15-30min at room temperature to obtain the pre-modified boswellic acid solution.
Preferably, the mass of boswellic acid is 1/2-1/5 of the mass of the exosome during mixing of the pre-modified boswellic acid solution with the exosome suspension for loading.
According to a preferred embodiment of the present invention, in S3, when loaded, the mixture of boswellic acid and exosomes is loaded into a syringe, connected to a filter unit; pushing the plunger of the injector to filter the mixed solution for 10 times by using a 400nm membrane, then passing through a 200nm membrane, 10 times by using a 200nm membrane, and finally passing through a 100nm membrane, and 10 times by using a 100nm membrane, thereby preparing the solution containing the mesenchymal stem cell exosomes loaded with the boswellic acid.
In a fourth aspect, the invention also provides an application of the mesenchymal stem cell exosome loaded with the boswellic acid in preparing a medicament for treating acne or improving skin. The efficacy of the treatment and improvement includes, but is not limited to: improving inflammatory environment of acne part, inhibiting propionibacterium acnes (P.acne) proliferation and inflammatory infiltration caused by propionibacterium acnes, treating acne vulgaris, improving skin effect, etc.
(III) beneficial effects
The technical effects of the invention include:
(1) The invention loads the mesenchymal stem cell exosome of the boswellic acid, its boswellic acid is loaded on the surface of exosome and loaded in the exosome, MSCexo-Bos acts on the skin, along with MSCexo is cracked under the skin, the boswellic acid that surface and internal carry is released thereupon, exert and inhibit propionibacterium acnes and reproduce and improve the effects of the inflammatory environment of focus position; meanwhile, the mesenchymal stem cell exosome MSCexo serving as a carrier can inhibit the proinflammatory factor IL-6 and promote the expression of the anti-inflammatory factor IL-10, has the effects of inhibiting inflammatory reaction, promoting tissue repair and the like, and can exert the synergistic effects of repairing and consolidating while treating by combining MSCexo and frankincense acid, so that a good treatment effect can be exerted on acne.
(2) The mesenchymal stem cell exosomes loaded with the boswellic acid are used for injecting rats with acne models, when the injection amount of the compound prepared by the invention (100 mug/mouse and 17.5 mug boswellic acid) is only half of the injection dosage of the single mesenchymal stem cell exosomes (200 mug/mouse) or the boswellic acid (35 mug/mouse), experimental results show that the injection of the mesenchymal stem cell exosomes loaded with the boswellic acid reduces the levels of pro-inflammatory factors IL-6 and CCL2 of the rats models, the level of anti-inflammatory factors IL-10 is increased, and the anti-inflammatory effect of the mesenchymal stem cell exosomes loaded with the boswellic acid is obviously higher than that of boswellic acid BOS and exosomes MSCexo; thus, it is demonstrated that the synergy is generated between the vector-carrying exosomes of the mesenchymal stem cell exosomes loaded with boswellic acid and the boswellic acid active ingredient.
In addition, the invention also uses common acne drug isotretinoin (MSCexo-SOT) loaded by exosomes of the same source for injecting acne model rats, and under the condition of the same injection dosage (100 mug/mouse), although the levels of the pro-inflammatory factors IL-6 and CCL2 of mice in MSCexo-SOT treatment group are reduced and the levels of the anti-inflammatory factors IL-10 are increased, the anti-inflammatory effect of the mesenchymal stem cell exosomes MSCexo-BOS loaded by the boswellic acid of the invention is still obviously higher than that of the mice in MSCexo-SOT group; therefore, the synergy between the exosome and the boswellic acid in the boswellic acid-loaded mesenchymal stem cell exosome provided by the invention is obviously superior to the synergy between MSCexo and SOT.
(3) The preparation method of the invention comprises the steps of using NHS and EDC as cross-linking agents, simultaneously loading the boswellic acid in the exosome and connecting the boswellic acid to the exosome surface by a repeated push-resistance method matched with a syringe and a filter membrane (confirmed by chromatographic experimental detection), and finding that the particle size distribution and the shape of the exosome before and after loading the boswellic acid are not obviously different by the particle size analysis of the particles before and after loading the boswellic acid of mesenchymal stem cell exosome, which indicates that MSCexo-BOS keeps the structure of the exosome in the preparation process.
However, in the existing common drug-loaded exosome technology, the co-incubation method is rapid and simple, has no influence on the morphology of exosomes, is more favorable for loading hydrophobic molecules, and has lower loading efficiency. Ultrasound is relatively efficient and can load hydrophobic and hydrophilic molecules, but can result in compromised exosome membrane integrity. Electroporation can be loaded with both hydrophilic and hydrophobic compounds, but can result in exosomes aggregating and affecting exosome membrane integrity. Extrusion can relatively efficiently load hydrophilic and hydrophobic compounds, but can also cause exosomes to aggregate, affecting exosome size. The method of the present invention overcomes the above-described problems.
(4) Isotretinoin solution (10 mg/mL) was combined with exosome suspension at 1:1, the boswellic acid solution (10 mg/mL) was mixed with the exosome suspension according to 1:4, and the final drug loading rate is only slightly lower than the drug loading rate (14.1%) of BOS in MSCexo-SOT in MSCexo-BOS in the final drug loading rate value, which indicates that when BOS is only 0.4 times of SOT in the loading process, a basically equivalent drug loading rate value can be obtained, and the binding capacity of BOS and an exosome MSCexo is proved to be better than that of SOT and MSCexo. In further experiments, the loading of isotretinoin SOT on exosomes MSCexo was slightly higher than boswellic acid BOS, but the therapeutic efficacy of MSCexo-BOS was still higher than MSCexo-SOT at the same injection dose to the acne mouse model, demonstrating that the synergy of MSCexo with BOS was superior to MSCexo with SOT; the exosomes can effectively improve the bioavailability of BOS.
(5) The boswellic acid-loaded mesenchymal stem cell exosome prepared by the invention has the advantages of firm bonding of boswellic acid to exosome, good efficacy stability, difficult exosome cracking problem in the storage process, no reduction of exosome compound efficacy after the prepared sample is placed in a continuous refrigerating chamber for 3 weeks, and the characteristics of effectively enhancing the effect of concentrating lactic acid to inflammatory parts and improving the bioavailability, and ensuring the efficacy of the boswellic acid-loaded mesenchymal stem cell exosome for external use or subcutaneous injection for treating acne.
Drawings
FIG. 1 shows the results of measuring the particle size of exosomes in a nanoflow;
Fig. 2 is a typical morphology of mesenchymal stem cell exosomes observed by transmission electron microscopy.
FIG. 3 shows the particle size distribution and particle concentration of mesenchymal stem cell exosomes before and after loading with boswellic acid by nano-flow detection;
FIG. 4 shows the particle size distribution and particle concentration before and after nano-flow detection of the loading of mesenchymal stem cell exosomes with isotretinoin;
FIG. 5 shows the drug loading of boswellic acid by HPLC detection MSCexo-BOS;
FIG. 6 is an HPLC detection MSCexo-SOT of isotretinoin drug loading;
FIG. 7 shows the therapeutic effect of ear injection MSCexo-BOS on acne;
FIG. 8 shows HE staining results from transection of rat ear tissue;
FIG. 9 is a graph showing the effect of MSCexo-BOS on IL-6 secretion by cells;
FIG. 10 is a graph showing the effect of MSCexo-BOS on secretion of cellular CCL 2;
FIG. 11 is a graph showing the effect of MSCexo-BOS on IL-10 secretion by cells.
Detailed Description
The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.
Example 1
This example includes the commercial purchase of primary mesenchymal stem cells for culture and isolation and purification of exosomes from the cell supernatant in two parts:
(1) Primary mesenchymal stem cells (provider: qiao Xin boat biosciences in Shanghai) were purchased commercially and resuscitated.
(2) Collecting the supernatant of the mesenchymal stem cells, centrifuging at a low speed of 300 Xg/min for 10min, and separating out the cells; the supernatant was centrifuged at 2000 Xg/min for 10min to remove dead cells. Centrifuging the rest supernatant at 10000 Xg/min for 30min, and removing cell debris; and centrifuging the supernatant at 100,000Xg/min for 60min to obtain exosome precipitate, re-suspending the exosome precipitate with sterile PBS, and centrifuging at 100,000Xg/min for 70min to obtain exosome MSCexo.
The exosome particle size was detected using nanoflow, the results are shown in figure 1; fig. 1 shows: the average particle size of the collected mesenchymal stem cell exosomes is about 80.0nm, and the particle concentration is 1.10E+10particles/mL. Morphology observation of exosomes was performed using a Transmission Electron Microscope (TEM), the results are shown in fig. 2; fig. 2 shows: the mesenchymal stem cell exosome mainly has a round or oval tea-tray-like structure.
Example 2
In the embodiment, the boswellic acid and the isotretinoin are respectively loaded by adopting the exosome MSCexo as a carrier, so as to obtain the boswellic acid-loaded exosome MSCexo-BOS and the isotretinoin-loaded exosome MSCexo-SOT. And the particle concentration, the particle diameter, the drug loading rate and the like are tested and analyzed.
(1) Preparation of boswellic acid-loaded exosomes MSCexo-BOS
In order to facilitate drug loading onto the exosomes, exosomes MSCexo may be resuspended in PBS to make exosome suspension.
Weighing a plurality of boswellic acids, dissolving in DMSO to prepare a solution with a concentration of 10mg/mL, weighing equal mass of NHS and EDC, and dissolving in PBS to prepare a 2mg/mL NHS/EDC solution.
The boswellic acid solution and the NHS/EDC solution were mixed in equal volumes and incubated at room temperature for 20min. Followed by mixing with exosome suspension according to 1:4 for loading. Filling the mixture into a syringe, connecting to a filter unit (micro-fluidic nano biotechnology in su zhou), slowly pushing the plunger of the syringe to squeeze 1ml of aqueous suspension of EV boswellic acid, passing through 400nm membrane, pushing resistance 10 times; passing through a 200nm film, and pushing resistance for 10 times; the mixture is passed through a 100nm membrane and pushed for 10 times, and the filtrate is a boswellic acid-loaded exosome MSCexo-BOS solution.
(2) Preparation of isotretinoin-loaded exosomes MSCexo-SOT
Weigh several weights of isotretinoin and dissolve in methanol to make a solution (ready-to-use as prepared) with a concentration of 10 mg/mL. Equal mass of NHS and EDC were weighed and dissolved in PBS to prepare a 2mg/mL NHS/EDC solution. The boswellic acid solution and the NHS/EDC solution were mixed in equal volumes and incubated at room temperature for 20min. Followed by mixing with exosome suspension according to 1:1 for loading. Filling the mixture into a syringe, connecting to a filter unit (micro-fluidic nano biotechnology in su zhou), slowly pushing the plunger of the syringe to squeeze 1ml of aqueous suspension of EV boswellic acid, passing through 400nm membrane, pushing resistance 10 times; passing through a 200nm film, and pushing resistance for 10 times; passing through 100nm film, pushing resistance for 10 times; the filtrate is the apocrine MSCexo-SOT solution loaded with boswellic acid.
(3) Particle concentration and particle size analysis of mesenchymal stem cell exosomes before and after loading with boswellic acid: the particle size distribution of mesenchymal stem cell exosomes (MSCexo) was examined using nanoflow, as well as the particle size distribution and concentration (used to calculate recovery) of the boswellic acid-loaded exosomes (MSCexo-BOS) and isotretinoin-loaded exosomes (MSCexo-SOT) and the change in average particle size of the exosomes before and after loading was compared. The test results are shown in FIGS. 3-4.
The recovery rate calculation formula is as follows:
recovery = post-load exosome particle concentration/pre-load exosome particle concentration x 100%.
As can be seen from FIG. 3, the average particle diameter of MSCexo prepared was 77.7nm, the average particle diameter after the boswellic acid was loaded was 83.0nm, and the recovery rate was 53.18%; as is clear from FIG. 4, MSCexo was prepared to have an average particle diameter of 77.9nm, an average particle diameter after loading isotretinoin of 89.8nm, and a recovery rate of 51.96%. The grain size distribution of the exosomes before and after the boswellic acid and the isotretinoin are loaded has no obvious difference, which indicates that MSCexo-BOS and MSCexo-SOT maintain the structure of the exosomes in the preparation process.
(4) Testing of drug loading of BOS in MSCexo-BOS and SOT in MSCexo-SOT
The concentration of the exosome protein before loading was detected by using the BCA protein detection kit, and the concentration of the exosome protein after loading was calculated by multiplying the recovery rate. Extracting and collecting exosomes in the loaded suspension by ultrafiltration, and re-suspending the exosomes in PBS, and mixing RIPA lysate with exosome suspension 1:1, mixing uniformly, standing for 15-20min at 4 ℃, and shaking uniformly for standby after cracking. The standard curve and the content of boswellic acid and isotretinoin in the samples were then measured by HPLC evaporative scattering detector.
① The boswellic acid detection conditions are as follows: chromatographic column: SYMMETRYC 18.5 μm, 4.6X1250 mm Column; column temperature: 30 ℃, mobile phase: a:0.5% glacial acetic acid-water; b, methanol; the elution gradient was as follows:
detection wavelength: 210nm; ELSD Detector settings: n 2 pressure: 50psi, atomizing mode: heating, power 60%, drift tube temperature: 60 ℃, gain: 1, a step of; sample preparation: accurately weighing appropriate amount of boswellic acid reference substance, dissolving with methanol, and making into 80 μg/mL sample solution.
Sample injection volume: and (3) respectively injecting 2 mu L, 4 mu L, 8 mu L, 16 mu L and 32 mu L, examining a linear relation, establishing a standard curve, and detecting the content of the boswellic acid in the sample.
The test results are shown in fig. 5. According to the calculation formula:
The drug loading rate of the olibanum acid is calculated:
Drug loading ratio = amount of boswellic acid loaded on exosomes/(amount of total protein of exosomes x recovery rate + amount of boswellic acid loaded on exosomes) x 100%,
The drug loading rate of the olibanum acid is calculated to be 14.1 percent.
② Isotretinoin detection conditions: chromatographic column: waters XBridge C18 (4.6X250 nm,5 μm)
Mobile phase: methanol-water-glacial acetic acid (v/v/v: 770/225/5); flow rate: 1mL/min; column temperature: 30 ℃; detection wavelength: 355nm; time: 60min;
the test results are shown in fig. 6. According to the calculation formula:
And (3) calculating the acid drug loading rate of isotretinoin:
Drug loading = exosome loaded isotretinoin amount/(exosome total protein amount x recovery + exosome loaded isotretinoin amount) x 100%,
The calculated isotretinoin drug loading rate is 15.7 percent.
Example 3
This example constructs a rat acne model for the subsequent efficacy testing of boswellic acid loaded exosomes MSCexo-BOS and isotretinoin loaded exosomes MSCexo-SOT.
Selecting a Wistar male adult rat with 6-8 weeks old and good growth condition, placing the adult rat in a Ruiwod anesthesia machine for anesthesia, taking out the rat after the rat breathes steadily, extracting 0.3mL of oleic acid, uniformly smearing the oleic acid on the inner side of the right auricle of the rat (note that the oleic acid does not flow into the auditory canal of the rat and too much oleic acid is smeared on the head and the back to cause dehairing), and then marking the tail; propionibacterium acnes was injected every other day, and 3X 10 6 CFU total propionibacterium acnes was withdrawn with a 1mL sterile syringe and injected subcutaneously in the right ear inner contour at multiple points; the molding was completed by continuously conducting for 21 days.
Example 4
An acne mouse model was constructed using example 3, which tests the therapeutic effects of boswellic acid BOS, exosomes MSCexo, MSCexo-BOS, MSCexo-SOT injected into acne mice:
Rats successfully modeled were randomly divided into five groups: after the protein concentrations in the model group, BOS group, MSCexo group, MSCexo-BOS group and MSCexo-SOT group, BSA were measured for MSCexo-Bos and MSCexo-Sot, split-packaging was performed for MSCexo-Bos and MSCexo-Sot. The rats were anesthetized in a revascularization machine, taken out after the rats breathe smoothly, photographed, recorded and the ear thickness measured. A1 mL sterile syringe was used to withdraw a 200. Mu.g total protein solution of MSCexo, 200. Mu. gBOS in PBS (containing 35. Mu.g boswellic acid), 100. Mu.g of MSCexo-SOT (containing 19.4. Mu.g SOT), MSCexo-BOS (containing 17.5. Mu.g boswellic acid) and 1mL volume of PBS (Model group) for subcutaneous multipoint injection in the right ear of five groups of rats, respectively. The materials were obtained after 3 consecutive administrations and used for HE staining. The test results are shown in fig. 7 and 8.
As shown in FIG. 7, MSCexo-BOS has more obvious effect on treating mouse acne than BOS, MSCexo and MSCexo-SOT, and is characterized by obvious reduction of auricle skin symptoms of rats, lighter color, soft touch texture, reduced skin temperature, obvious thinning of epidermis thickness and partial crust falling off; wherein the Sham group is a blank control group. As can be seen from FIG. 8, the anti-inflammatory effect of MSCexo-BOS was more pronounced than that of BOS, MSCexo and MSCexo-SOT, which was manifested by a significant decrease in the extent of follicular expansion in the ears of the rats after MSCexo-BOS treatment, a decrease in inflammatory cell infiltration, and a gradual restoration of their integrity by the keratinized layers.
Example 5
The present example is based on HacaT cell inflammation model, and uses enzyme-linked immunosorbent assay (ELISA) to test the anti-inflammatory properties of MSCexo-BOS, BOS, MSCexo, MSCexo-SOT and the like.
(1) Preparation of HacaT cell inflammation model
HacaT cells with good growth conditions are selected for the seed plates. Cells were seeded in 12-well plates, 10 ten thousand cells per well. And (5) performing modeling when the cell fusion degree reaches 70-80%.
Cells were divided into 6 groups: blank, model, BOS, MSCexo, MSCexo-SOT and MSCexo-BOS groups, each with 4 wells for 24 wells. The treatment modes of each group were as follows (LPS is bacterial lipopolysaccharide):
① Blank control group: culturing in DMEM basal medium for 24h;
② Model group: LPS (1. Mu.g/mL) +DMEM basal medium for 24h;
③ BOS group: BOS (9.4. Mu.g/mL) +DMEM basal medium is pre-incubated for 6h, followed by LPS (1. Mu.g/mL) +DMEM basal medium for 24h;
④ MSCexo groups: MSCexo (100. Mu.g/mL) +DMEM basal medium was pre-incubated for 6h, followed by LPS (1. Mu.g/mL) +DMEM basal medium for 24h;
⑤ MSCexo-BOS group: MSCexo-BOS (100. Mu.g/mL) +DMEM basal medium was pre-incubated for 6h, followed by LPS (1. Mu.g/mL) +DMEM basal medium for 24h;
⑥ MSCexo-SOT group: MSCexo-SOT (100. Mu.g/mL) +DMEM basal medium was pre-incubated for 6h, followed by LPS (1. Mu.g/mL) +DMEM basal medium for 24h.
The ③-⑥ was cultured in advance with BOS, MSCexo, MSCexo-BOS and MSCexo-SOT for 6 hours, and then cultured with LPS and DMEM. After modeling/administration, clear solution was collected for detection of inflammatory and anti-inflammatory factors. MSCexo-BOS and MSCexo-SOT used in this example were prepared as in example 2.
(2) The levels of IL-6 and CCL2 in HacaT cell supernatants were determined using a double antibody sandwich method.
① Pro-inflammatory factor IL-6: mu.L of assay diluent was added to the 96-well plate, after which standards or samples were added and incubated for 2h at room temperature. The 96-well plate was washed 3 times with wash buffer (PBS buffer containing 0.05% Tween-20), 200. Mu.L of Human IL-6 detection antibody solution was added, and incubated at room temperature for 2 hours. The washing step was repeated, followed by adding 200. Mu.L of the color-developing solution, incubating at room temperature for 20min, and protected from light. Accordingly, 50. Mu.L of TMB substrate was added to each well under light-shielding conditions. Absorbance was measured at 450nm using a microplate reader.
② Pro-inflammatory factor CCL2: 200. Mu.L of standard or sample was added to a 96-well plate and incubated for 2h at room temperature. The 96-well plate was washed 3 times with wash buffer (PBS buffer containing 0.05% tween-20), 200 μl of Human CCL2 detection antibody solution was added and incubated for 1h at room temperature. The washing step was repeated, followed by adding 200. Mu.L of a color developing solution, incubating at room temperature for 30min, and protecting from light. Accordingly, 50 μ LTMB substrate was added to each well under light-shielding conditions. Absorbance was measured at 450nm using a microplate reader.
③ Anti-inflammatory factor IL-10: the levels of IL-10 in HacaT cell supernatants were determined using chemiluminescence. The bar code is scanned into an instrument for identification and input, 500 mu L of diluent is added into each calibration product, and the mixture is gently shaken and mixed uniformly, and balanced for 30min. 100 mu L of calibration product is added into the reagent strip, and the sample is checked by an on-machine.
Sample detection: the cell supernatant was centrifuged, 100. Mu.L was added to the strip, and the sample was checked on the machine. When stimulated by LPS, the inflammatory signal pathway of the cells is activated, and then the inflammatory related factors IL-6 and CCL2 are secreted, so that the cells are induced to generate inflammatory response, and the test results are shown in FIG. 9, FIG. 10 and FIG. 11 (M represents model group, S represents blank group or sham operation group). HacaT produced large amounts of IL-6 and CCL2 at 1 μg/mL LPS stimulation, which was very significantly different from the blank, indicating that the cells exhibited a stronger inflammatory response. After BOS, MSCexo, MSCexo-BOS and MSCexo-SOT are given, the levels of pro-inflammatory factors IL-6 and CCL2 are reduced, the level of anti-inflammatory factors IL-10 is increased, and the action effect of MSCexo-BOS is obviously higher than that of BOS, MSCexo, MSCexo-SOT and the like; it is shown that MSCexo in MSCexo-BOS produces a synergistic effect with BOS that is superior to the synergistic effect between MSCexo and SOT.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (5)
1. The mesenchymal stem cell exosome for treating acne and loaded with the boswellic acid is characterized by comprising a carrier and an active ingredient, wherein the carrier is the mesenchymal stem cell exosome, and the active ingredient is the boswellic acid; the medicine carrying rate of the active ingredient is 5-15%; the active ingredient is loaded on the surface of the exosome and is loaded in the exosome; the mesenchymal stem cell exosome is a adipose mesenchymal stem cell exosome; the preparation method of the mesenchymal stem cell exosome loaded with the boswellic acid comprises the following steps:
s1, extracting primary mesenchymal stem cells, culturing, separating and purifying exosomes in cell supernatant, and re-suspending to prepare exosome suspension;
S2, preparing a boswellic acid solution, adding a solution containing NHS and EDC, and performing incubation reaction at room temperature to obtain a pre-modified boswellic acid solution; the carboxyl groups of the boswellic acid in the pre-modified boswellic acid solution are activated by EDC and NHS to be loaded on the surface and inside of exosomes;
S3, mixing the exosome suspension with the pre-modified boswellia acid solution, filling the mixed solution into a syringe, and connecting the syringe to a filter unit; pushing a plunger of the injector to sequentially pass the mixed solution through a multi-stage filter membrane with a filter hole reduced from 400nm to 100nm to prepare a solution containing mesenchymal stem cell exosomes loaded with boswellic acid; the boswellic acid is loaded on the surface of the exosome and on the inside of the exosome.
2. The boswellic acid-loaded mesenchymal stem cell exosome of claim 1, wherein S1 further comprises: collecting the supernatant of the mesenchymal stem cells, centrifuging for a plurality of times, and sequentially increasing the centrifugation speed to remove cells, dead cells and cell fragments from the cell supernatant respectively; finally, taking supernatant and centrifuging at a centrifugation speed of 80000-120000Xg/min for 50-70min to obtain exosome sediment, re-suspending the exosome sediment by using sterile PBS, and centrifuging at a centrifugation speed of 80000-120000Xg/min again to obtain exosome; the exosomes were resuspended in PBS to obtain exosome suspension.
3. The boswellic acid-loaded mesenchymal stem cell exosome of claim 1, wherein S2 comprises: dissolving boswellic acid in DMSO to prepare a boswellic acid solution with a concentration of 8-15mg/mL, weighing NHS and EDC according to a mass ratio of 1:1, and dissolving in PBS to prepare a NHS-EDC solution with a total concentration of 0.95-1.2 mg/mL; mixing the boswellic acid solution with the NHS-EDC solution in equal volume, and incubating for 15-30min at room temperature to obtain the pre-modified boswellic acid solution.
4. The boswellic acid-loaded mesenchymal stem cell exosome of claim 1, wherein in S3, the pre-modified boswellic acid solution is mixed with the exosome suspension for use in the loading process, wherein the mass of boswellic acid is 1/3-1/5 of the mass of the exosome when mixed; loading the mixed solution of boswellic acid and exosome into a syringe, and connecting to a filter unit; pushing the plunger of the injector to filter the mixed solution for 10 times by using a 400nm membrane, then passing through a 200nm membrane, 10 times by using a 200nm membrane, and finally passing through a 100nm membrane, and 10 times by using a 100nm membrane, thereby preparing the solution containing the mesenchymal stem cell exosomes loaded with the boswellic acid.
5. Use of the boswellic acid-loaded mesenchymal stem cell exosome of any one of claims 1-4 in the manufacture of a medicament for treating acne.
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