Targeted modification method of rose exosome, hyaluronic acid modified rose exosome and application of hyaluronic acid modified rose exosome

Abstract

The invention belongs to the technical field of targeting exosome preparation, and particularly relates to a targeting modification method of a rose exosome, a hyaluronic acid modified rose exosome and application of the hyaluronic acid modified rose exosome. The method comprises the following steps: dissolving hyaluronic acid in a rose exosome, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, carrying out a reaction for 8-16 h, carrying out centrifugation, and carrying out resuspension precipitation so as to obtain a hyaluronic acid modified rose exosome; the volume mass ratio of the hyaluronic acid to the rose exosome solution is 1 mg: (0.5-1) ml, and the hyaluronic acid, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and the N-hydroxysuccinimide are in an equal molar ratio. The characteristic that hyaluronic acid is highly compatible with an inflammatory cell CD44 receptor is utilized, the targeting property of the rose exosome to inflammatory cells is enhanced, and the utilization rate and the anti-inflammatory effect of the rose exosome are further improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of targeted exosome preparation, and specifically relates to a targeted modification method of rose exosomes, hyaluronic acid-modified rose exosomes and applications thereof. Background Art

[0002] On January 3, 2022, scientists from the Institute of Oncology at the University of Oviedo in Spain and the University of Paris in France published a review paper titled: Hallmarks of aging: An expanding universe in the journal Cell. This review creatively proposed twelve hallmarks of aging for the first time on the basis of previous studies, which is of great significance for understanding the occurrence and development of aging and how to intervene in the aging process. The original nine major aging markers have been updated and improved, and three new aging markers, chronic inflammation, intestinal microbial dysbiosis, and macroautophagy inactivation, have been added. As one of the twelve major markers of aging, chronic inflammation is inseparable from aging. The significant feature of inflammatory aging is that the aging body has an imbalance of inflammatory homeostasis with age, cells and the body gradually tilt towards the pro-inflammatory process, and the anti-inflammatory effect is increasingly weakened, which eventually manifests as a chronic inflammatory state. From a molecular perspective, after cell aging, pro-inflammatory factors will be secreted, namely the senescence-associated secretory phenotype (SASP). These inflammatory factors will not only affect the surrounding normal cells, but also recruit a large number of inflammatory cells, affecting tissue repair and destroying tissue homeostasis. In recent years, many studies have shown that eliminating senescence-related cells or reversing the senescence state of cells can significantly delay the pathological progression of aging-related diseases and prolong the lifespan of the host.

[0003] In recent years, plant-derived extracellular vesicles have attracted great attention from the biological and medical communities. They have a phospholipid bilayer structure similar to that of animal-derived exosomes and carry abundant proteins, lipids, nucleic acids, and plant-specific secondary metabolites. The extraction and efficacy verification of various types of plant exosomes have been carried out at home and abroad. Plant exosomes have been proven to have good biosafety and oral safety and can cross biological barriers.

[0004] In the previous study, the inventor successfully extracted rose exosomes and confirmed their cross-border regulatory ability, which has good repair, soothing and anti-inflammatory effects, especially in inhibiting the expression of inflammatory factors IL-1α, IL-8, TNF-α, etc. However, in practical applications, rose exosomes do not have targeting properties. After acting on the skin, they will also be taken up by normal cells, and the utilization rate is low. Therefore, how to transform natural rose exosomes to improve their targeting properties is the technical problem to be solved by the present invention. Summary of the invention

[0005] In order to solve the above technical problems, the present invention first provides a targeted modification method of rose exosomes.

[0006] A targeted modification method of rose exosomes of the present invention comprises the following steps:

[0007] Dissolve hyaluronic acid in rose exosomes, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, react for 8h-16h, centrifuge, and resuspend the precipitate to obtain hyaluronic acid-modified rose exosomes;

[0008] The volume mass ratio of the hyaluronic acid and rose exosome solution is 1 mg: 0.5 ml-1 ml, and the hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) are in an equal molar ratio.

[0009] The present invention extracts natural rose exosomes and couples the carboxyl groups on hyaluronic acid (HA) with the amino groups on the surface of exosomes under the catalysis of EDC / NHS, so that HA is distributed on the outer surface of rose exosomes. The high affinity of HA to the CD44 receptor of inflammatory cells is utilized to enhance the targeting of rose exosomes to inflammatory cells, thereby further improving the utilization rate of rose exosomes.

[0010] Furthermore, the concentration of the rose exosomes is 0.8 mg / mL-1 mg / mL.

[0011] Furthermore, the preparation method of rose exosomes comprises the following steps:

[0012] 1) Wash and dry fresh rose petals, mix with an equal amount of PBS solution and homogenize to obtain a homogenate;

[0013] 2) Centrifuge the homogenate obtained in 1) at 3000g-6000g for 15-30min, and retain the upper wine-red transparent solution;

[0014] 3) Centrifuge the wine red transparent solution in 2) at 8000g-12000g for 40min-60min, and retain the supernatant;

[0015] 4) Centrifuge the supernatant from 3) at 140000g-150000g for 60min-80min, and retain the precipitate;

[0016] 5) resuspending the precipitate obtained in 4) with PBS solution and centrifuging at 140000g-150000g for 60min-80min, and retaining the precipitate;

[0017] 6) Resuspend the precipitate from 5) with PBS solution and centrifuge at 3800g-4000g for 8min-10min, discard the insoluble precipitate, retain the supernatant, obtain rose exosomes, and adjust the concentration to 0.8mg / mL-1mg / mL.

[0018] Based on the same inventive concept, the present invention also provides hyaluronic acid-modified rose exosomes prepared by the above method.

[0019] Based on the same inventive concept, the present invention also provides the use of the rose exosomes in the preparation of anti-inflammatory drugs.

[0020] Furthermore, the drug targets inflammatory cells and inhibits the release of inflammatory factors.

[0021] Furthermore, the inflammatory factors include TNF-α.

[0022] Based on the same inventive concept, the present invention also provides an anti-inflammatory drug, which uses the hyaluronic acid-modified rose exosomes as an effective ingredient.

[0023] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0024] The auxiliary material is an aqueous or oil / fat buffer isotonic with the physiological environment, including one or more of physiological saline, phosphate buffer, Hank's buffer, DMSO, and vegetable oil.

[0025] Based on the same inventive concept, the present invention also provides the use of the rose exosomes in the preparation of cosmetics for improving skin inflammatory aging, wherein the cosmetics are essence or cream.

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

[0027] (1) In practical applications, the probability of natural rose exosomes being taken up by all skin cells is the same, so the utilization rate is not high. The present invention utilizes the high affinity of hyaluronic acid (HA) with CD44 to modify rose exosomes with HA to improve their targeting to inflammatory cells, thereby further improving the utilization rate of rose exosomes. The present invention solves the problem that natural rose exosomes do not have inflammatory cell targeting in cosmetic applications.

[0028] (2) The modification method provided by the present invention has a mild reaction, significant effect, and great application potential. It can be used to prepare cosmetics for improving inflammatory aging of the skin, such as essence or cream. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is an electron micrograph of rose exosomes.

[0030] Figure 2 This is an electron micrograph of HA-modified rose exosomes.

[0031] Figure 3 This is the particle size distribution diagram of rose exosomes.

[0032] Figure 4 This is the particle size distribution diagram of HA-modified rose exosomes.

[0033] Figure 5 is the initial potential of rose exosomes.

[0034] Figure 6 Potential for HA-modified rose exosomes.

[0035] Figure 7 These are the staining electron micrographs of macrophages Raw264.7 after treatment with rose exosomes and HA-modified rose exosomes.

[0036] Figure 8 Quantification of rose exosomes and HA-modified rose exosomes targeting macrophage Raw264.7, A is the green fluorescence intensity, and B is the proportion of targeted cells.

[0037] Fig. 9 The content of pro-inflammatory factor TNF-α in macrophage Raw264.7 after treatment with different concentrations of HA-modified rose exosomes. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0039] Example 1: Targeted modification of rose exosomes

[0040] 1. Reagents

[0041] Hyaluronic acid (HA-TLM 20-40), Bloomage Biotechnology; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), McLean; N-hydroxysuccinimide (NHS), McLean. PBS phosphate buffer, Solebol.

[0042] 2 Methods

[0043] (1) Obtaining rose exosomes: Take fresh rose petals, rinse them with clean water, and then rinse them with sterile PBS. After drying, weigh 200 g of rose petals and pour them into a juicer. Add sterile PBS solution at a mass ratio of 1:1. After fully juicing and homogenizing, transfer them to a centrifuge tube; centrifuge at 4000g, 4℃ for 20 min, carefully transfer the upper wine red transparent solution to a new centrifuge tube, centrifuge at 10000g, 4℃ for 60 min, transfer the supernatant to an ultracentrifuge tube after centrifugation, balance the centrifuge tube, and ultracentrifuge at 150000g, 4℃ for 70 min. Discard the supernatant, resuspend the precipitate with 1 mL of sterile PBS solution, fill the ultracentrifuge tube with sterile PBS solution again after resuspension, balance, and ultracentrifuge at 150000g, 4℃ for 70 min; resuspend the precipitate with 1 mL of sterile PBS solution after centrifugation. After centrifugation again (4000g, 4°C for 10 min), the insoluble precipitate was discarded and the supernatant was retained to obtain rose exosomes. The content of rose exosomes was determined by the BCA protein quantification method. The rose exosome content was adjusted to 1 mg / mL and stored in a -80°C refrigerator.

[0044] (2) HA-modified rose exosomes: 500 μL of rose exosomes was added with 1 mg of hyaluronic acid (HA). After the HA was fully dissolved, 0.33 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 0.495 mg of N-hydroxysuccinimide (NHS) were added in sequence. The mixture was reacted at room temperature (20°C-25°C) in the dark for 12 h, and then centrifuged twice at 150,000 g for 70 min. The precipitate was resuspended in PBS buffer to obtain HA-modified rose exosomes.

[0045] To demonstrate the effect of the HA-modified rose exosomes of the present invention, the following verification experiments were performed:

[0046] Characterization of HA-modified rose exosomes:

[0047] (1) Structural identification of rose exosomes: adjust the sample concentration, take 10uL sample and drop it on the copper grid, let it stand at room temperature for 10min, wash it with deionized water three times, negatively stain the copper grid with phosphotungstic acid for 30s, wash it with deionized water three times, dry it naturally at room temperature, observe the morphology and structure of the vesicles under a transmission electron microscope, and take pictures and record them.

[0048] (2) Rose exosome particle size detection: exosomes were collected for NTA testing.

[0049] (3) Detection of zeta potential changes of rose exosomes is shown in Table 1:

[0050] Table 1 Detection results of zeta potential changes of rose exosomes

[0051]

[0052] Result description: Figure 1 This is an electron micrograph of rose exosomes. Figure 2 This is an electron micrograph of HA-modified rose exosomes. Figure 3 This is the particle size distribution diagram of rose exosomes. Figure 4 This is the particle size distribution diagram of HA-modified rose exosomes. Figure 5 is the initial potential of rose exosomes, Figure 6 The potential of HA-modified rose exosomes. It can be seen that rose exosomes are vesicles with a diameter of about 160.7nm. The NTA results show that the concentration is 5.4E+10Particles / mL and the initial zeta potential is -13.67±0.85mV. The rose exosomes modified with HA are vesicles with a diameter of about 155.9nm. The NTA results show that the concentration is 4.6E+10Particles / mL and the zeta potential after modification is -38.7±0.17mV.

[0053] Effect verification 2:

[0054] Verification of the targeting of HA-modified rose exosomes:

[0055] The natural rose exosomes and the HA-modified rose exosomes obtained in Example 1 were fluorescently labeled with PKH67. RAW264.7 cells were revived, digested and counted when the cells grew to about 50%-60%, and inoculated into a well plate. After inoculation, they were cultured in an incubator at 37°C and 5% carbon dioxide for 24 hours. After treating the cells with the fluorescently labeled natural rose exosomes and the HA-modified rose exosomes obtained in Example 1 for 16 hours, they were fixed with paraformaldehyde and observed under a fluorescence microscope.

[0056] The results are as follows Figure 7 and Figure 8 As shown, after 16 hours of observation and statistics after treating macrophage Raw264.7 with 0.15 mg / mL rose exosomes and HA-modified rose exosomes, it was found that the targeted cell ratio of the HA-modified rose exosome treatment group was 11.83%, and that of the rose exosome group was 0.5%, with a 23.66-fold increase in the targeted ratio. The fluorescence intensity statistics showed that the fluorescence intensity of the HA-modified rose exosome group was significantly higher than that of the rose exosome group, with a 25.46-fold increase in fluorescence.

[0057] Effect verification 3: Verification of the anti-inflammatory effect of HA-modified rose exosomes:

[0058] When the RAW264.7 cells grew to about 50%-60%, they were digested and counted. 4 / well were inoculated in a 12-well plate and cultured in an incubator at 37°C and 5% carbon dioxide for 24 hours. After treating the cells with rose exosomes and HA-modified rose exosomes for 2 hours, 50 μL of normal culture medium was added to each well of the blank control group, and 50 μL of the prepared working solution containing LPS (final concentration: 1 μg / mL) was added to each well of the remaining groups, and then cultured in an incubator at 37°C and 5% carbon dioxide for 24 hours. After 24 hours, the cell supernatant was collected and centrifuged at 1000rpm-1200rpm for 3 minutes. The supernatant was aspirated and the inflammatory factor index was detected.

[0059] The results are as follows Fig. 9 As shown: Based on the 24-hour experimental results of the LPS-induced Raw264.7 cell inflammation model experiment, the expression level of the pro-inflammatory factor TNF-α in the model group was significantly increased compared with that in the control group, indicating that the model was successfully constructed.

[0060] After LPS induction of Raw264.7 cells, compared with the model group, the TNF-α contents in the 2.5% and 5% rose exosome groups decreased by 15.27% and 19.55%, respectively; the TNF-α contents in the 2.5% and 5% targeted rose exosome groups decreased by 15.30% and 16.98%, respectively.

[0061] Example 2: Application of HA-modified rose exosomes in the preparation of cosmetics for improving skin inflammatory aging

[0062] Example 1 has demonstrated that HA-modified rose exosomes can inhibit the release of inflammatory factors, and therefore can be used to prepare cosmetics that improve skin inflammatory aging, such as essences or creams, promote skin tissue repair, maintain tissue homeostasis, and delay skin aging.

[0063] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0064] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0065] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for targeted modification of rose exosomes, characterized in that: The following steps are involved: Dissolve hyaluronic acid in rose exosomes, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, react for 8h-16h, centrifuge, and resuspend the precipitate to obtain hyaluronic acid-modified rose exosomes; The volume mass ratio of the hyaluronic acid and rose exosome solution is 1 mg: 0.5 ml-1 ml, and the hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide are in an equal molar ratio.

2. The method for targeted modification of rose exosomes according to claim 1, characterized in that: The concentration of the rose exosomes is 0.8 mg / mL-1 mg / mL.

3. The method for targeted modification of rose exosomes according to claim 2, characterized in that: The preparation method of rose exosomes comprises the following steps: 1) Wash and dry fresh rose petals, mix with an equal amount of PBS solution and homogenize to obtain a homogenate; 2) Centrifuge the homogenate obtained in 1) at 3000g-6000g for 15-30min, and retain the upper wine-red transparent solution; 3) Centrifuge the wine red transparent solution in 2) at 8000g-12000g for 40min-60min, and retain the supernatant; 4) Centrifuge the supernatant from 3) at 140000g-150000g for 60min-80min, and retain the precipitate; 5) resuspending the precipitate obtained in 4) with PBS solution and centrifuging at 140000g-150000g for 60min-80min, and retaining the precipitate; 6) Resuspend the precipitate from 5) with PBS solution and centrifuge at 3800g-4000g for 8min-10min, discard the insoluble precipitate, retain the supernatant, obtain rose exosomes, and adjust the concentration to 0.8mg / mL-1mg / mL.

4. Hyaluronic acid-modified rose exosomes prepared by the method according to any one of claims 1 to 3.

5. Use of the rose exosomes according to claim 4 in the preparation of anti-inflammatory drugs.

6. The use of rose exosomes in the preparation of anti-inflammatory drugs according to claim 5, characterized in that: The drug targets inflammatory cells and inhibits the release of inflammatory factors.

7. The use of rose exosomes in the preparation of anti-inflammatory drugs according to claim 6, characterized in that: The inflammatory factors include TNF-α.

8. An anti-inflammatory drug, characterized in that: The drug uses the hyaluronic acid-modified rose exosomes as claimed in claim 4 as an active ingredient.

9. The anti-inflammatory drug according to claim 8, characterized in that The drug also includes pharmaceutically acceptable excipients.

10. The use of the rose exosomes according to claim 4 in the preparation of cosmetics for improving skin inflammatory aging, characterized in that: The cosmetic is essence or cream.

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