Plant source exosome, preparation method thereof and application of plant source exosome as photosensitizer
By extracting and preparing plant-derived exosomes with photosensitive activity, the problem of insufficient enrichment of traditional photosensitizers in tumor sites is solved, and the effect of efficient killing of tumor cells in photodynamic therapy and diagnosis is achieved. Due to its stability and autofluorescence ability, it has great application advantages.
Patent Information
- Application Number
- CN202311169577.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-11
- Publication Date
- 2025-03-11
AI Technical Summary
Traditional organic small molecule photosensitizers are quickly cleared by inflammatory phagocytosis cells in live photodynamic therapy, resulting in insufficient enrichment and retention of photosensitizers in the tumor site, resulting in inefficient photodynamic therapy. At the same time, the second-generation photosensitive drugs have problems such as low solubility, high production cost and poor solution stability, which limits their application in photodynamic therapy.
Exosomes with photosensitive activity were prepared by extracting plant-sourced exosomes from Forsythia and using PBS solution extraction, ultrafiltration concentration and mixing. The exosome has two main absorption spectral bands at 555 and 595 nm and produces singlet oxygen under 595 nm LED yellow light, which can be enriched at the tumor site.
As a photosensitive agent, the plant-derived exosome shows efficient ability to kill tumor cells in photodynamic therapy and photodynamic diagnosis. Due to its stable membrane structure and autofluorescence ability, it has greater advantages over existing photosensitizers and has good application prospects.
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Figure CN119592494A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of photosensitive materials, and specifically relates to a plant-derived exosome, a preparation method thereof, and use thereof as a photosensitizer. Background Art
[0002] The application of photodynamic therapy (PDT) requires three important components, including photosensitizer, molecular oxygen and light source. As a key part of PDT, photosensitizer transfers the absorbed energy to the surrounding oxygen and produces reactive oxygen species (ROS), which then further react with nearby biomacromolecules (such as lipids, proteins, DNA) to kill cancer cells and complete PDT. Therefore, the effective enrichment of photosensitizers at the tumor site directly determines the key factor of efficacy.
[0003] However, during in vivo photodynamic therapy, traditional organic small molecule photosensitizers are usually rapidly cleared from the bloodstream by inflammatory phagocytes, resulting in only a small amount of photosensitizers being enriched and retained at the tumor site, resulting in low efficiency of photodynamic therapy. In addition, the current second-generation photosensitizers have low solubility. The cost of synthesis and production is high, and the solution stability is poor to a certain extent. Hypericin, an extract of the plant Hypericum perforatum, is insoluble in water, which makes intravenous injection problematic and limits indications. The waiting time after injection of the hematoporphyrin derivatives currently used in clinical practice is too long (usually 40-50 hours after PDT), which causes great inconvenience to patients. This limits its medical application in photodynamic therapy. Therefore, it is very urgent to seek plant-derived exosome photosensitizers, which are a new choice for photodynamic therapy. Therefore, finding and developing new photosensitizers is imminent and of great significance for accurate and efficient PDT.
[0004] Exosomes are phospholipid bilayer vesicles actively secreted by cells, containing a variety of functional nucleic acids, proteins and lipids, and are important mediators of intercellular communication. Based on their natural characteristics, exosomes can not only retain the pharmacological effects of their source cells, but also serve as natural delivery carriers. Among them, plant-derived exosomes are considered to be natural medicines for disease treatment due to their rich sources, high yields, and low immunogenicity risks. They have the advantages of simplicity, safety, environmental protection, low cost, and low toxicity. The lipid bilayer of plant exosomes can be internalized by human and animal cells. Therefore, if a plant-derived exosome with photosensitivity that can be used for PDT can be found, it will have a good application prospect. However, there is still a lack of relevant research in the prior art, and no plant-derived exosomes with photosensitivity that can be used for PDT have been found. Summary of the invention
[0005] In view of the problems in the prior art, the object of the present invention is to provide a plant-derived exosome, a preparation method thereof and use thereof as a photosensitizer.
[0006] A plant-derived exosome, which is extracted according to the following method:
[0007] Step 1, extracting Hypericum perforatum with PBS solution;
[0008] Step 2, separating the extract and concentrating it through an ultrafiltration concentrator;
[0009] Step 3, add PBS solution to the concentrated solution and mix well to obtain pure exosomes.
[0010] Preferably, the Hypericum perforatum is Hypericumperforatum L.
[0011] Preferably, in step 1, the concentration of the PBS solution is 0.01M;
[0012] And / or, the amount of the PBS solution is 5-10 times the amount of Hypericum perforatum;
[0013] And / or, the extraction condition is immersion at 18-25° C. for 18-24 hours.
[0014] Preferably, in step 2, the separation step comprises the following steps: filtering, centrifuging the filtrate at 12000-15000 g for 5-10 min, centrifuging the supernatant at 10000-12000 g for 30-60 min, and filtering the supernatant through a 0.22 um filter;
[0015] And / or, the concentration factor is 3-5 times.
[0016] Preferably, in step 3, the volume ratio of the concentrate to the PBS solution is 1:(1-5).
[0017] Preferably, in step 3, after obtaining the exosomes, the following steps are further performed:
[0018] Step 4, concentrating the solution containing the exosomes through an ultrafiltration concentration tube; the concentration multiple is 2-4 times;
[0019] Step 5, after the concentrate is collected, it is centrifuged at 120000-150000g for 60-90 min at 4°C, and the solid obtained by centrifugation is resuspended with PBS solution.
[0020] Preferably, the absorption spectrum of the exosomes has two main bands at 555 nm and 595 nm; the fluorescence spectrum of the exosomes exhibits two main bands at 595 nm and 680 nm.
[0021] The present invention also provides a method for preparing the above-mentioned plant-derived exosomes, which is characterized by:
[0022] Step 1, extracting Hypericum perforatum with PBS solution;
[0023] Step 2, separating the extract and concentrating it through an ultrafiltration concentrator;
[0024] Step 3, add PBS solution to the concentrated solution and mix well to obtain pure exosomes.
[0025] The present invention also provides use of the plant-derived exosomes as a photosensitizer.
[0026] Preferably, the photosensitizer is used for photodynamic therapy and / or photodynamic diagnosis.
[0027] The present invention provides a plant-derived exosome with photosensitizing activity, the absorption spectrum of which has two main bands at 555 and 595 nm. The fluorescence spectrum shows two main bands near 595 and 680 nm. Under irradiation with 595 nm LED yellow light, tumor cells produce a large amount of singlet oxygen. Tail vein injection is enriched at the tumor site. Therefore, it can be used as a photosensitizer in photodynamic therapy (PDT) and photodynamic diagnosis (PDD). The exosomes of the present invention are more stable because they have a membrane structure and can spontaneously generate fluorescence in a PBS solution, which makes it have a greater advantage over the existing photosensitizer hypericin with solubility problems. Therefore, the present invention has a good application prospect.
[0028] It should be particularly noted that the optical properties of the exosomes provided by the present invention are different from those of hypericin, which indicates that the active ingredients that produce optical activity or their luminescence principles in the exosomes of the present invention are different from those of hypericin.
[0029] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.
[0030] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 The characterization results of the exosomes prepared in Example 1, wherein A: transmission electron microscopy observation of exosome samples; B: particle size and concentration information of exosomes.
[0032] Figure 2 UV-visible absorption spectra (A) and fluorescence emission spectra (B) of the exosomes and hypericin prepared in Example 1.
[0033] Figure 3This is the singlet oxygen curve of the exosomes prepared in Example 1, and the excitation light source is 590 nm.
[0034] Figure 4 In vivo imaging 4 hours after tail vein injection of exosomes showed the accumulation of exosomes in the tumor.
[0035] Figure 5 Microscopic images of human esophageal squamous cell carcinoma KYSE150 cells treated with PBS or exosomes after co-culture for 12 hours.
[0036] Figure 6 Results of CCK8 cell viability test of human melanoma cells WM-266-4 treated with PBS, exosomes or hypericin (HYP). DETAILED DESCRIPTION
[0037] In the following examples and experimental examples, all reagents and raw materials used unless otherwise specified are commercially available.
[0038] Example 1 Plant-derived exosomes and preparation method thereof
[0039] The plant-derived exosomes provided in this embodiment were prepared according to the following method:
[0040] Preparation:
[0041] 1. Soak the ultrafiltration concentration tube (content volume 15 ml) in 70% ethanol for 30 min, sterilize under UV light, and wash twice with sterile pure water (to prevent ethanol from forming salts with PBS).
[0042] 2. When the qEV extraction column is used for the first time, it is filled with sodium azide, so it is in a sterile state. After use, wash the internal PBS with sterile pure water, and then store it with 20% ethanol for antibacterial treatment. When using it again, wash it with sterile pure water and then inject the sample.
[0043] Exosome extraction:
[0044] 1. Use a traditional Chinese medicine grinder to grind the dry Hypericum perforatum plant into powder, take 50g and soak it in 500ml of 0.01M PBS at 18-25℃ and mix for 18h.
[0045] 2. Filter initially with gauze, discard the residue and collect the filtrate.
[0046] 3. Centrifuge the liquid at 12000g for 5 min, discard the precipitate and collect the supernatant.
[0047] 4. The supernatant was centrifuged at 12000g for 30min, the precipitate was discarded and the supernatant was collected.
[0048] 5. The collected supernatant is sterilized by 0.22um filtration in a clean bench, and all subsequent operations must ensure sterility.
[0049] 6. The sieved extract was concentrated using a sterile ultrafiltration concentration tube (Sartorius, Germany) to one third (167 ml).
[0050] 7. Add 167 ml of 0.01 M PBS and mix well to obtain pure exosomes.
[0051] 8. The extracted exosomes are then concentrated in a separately sterilized concentration tube (Sartorius, Germany) to obtain high-concentration sterile exosomes, which are concentrated to one third.
[0052] 9. After collection, ultra-high speed centrifugation was performed at 4°C, 150,000 g, 90 min, and resuspended (vortexed) in 50 ml of sterile 0.01 M PBS.
[0053] 10. The exosome protein content measured by BCA was 100-300 μg / mL.
[0054] The technical solution of the present invention is further illustrated by experiments below.
[0055] Experimental Example 1 Characterization of plant-derived exosomes
[0056] In this experimental example, the plant-derived exosomes prepared in Example 1 were characterized.
[0057] Electron microscopy and particle size of exosomes Figure 1 As shown, the exosome particle size of the light-treated group for 30 mins and the no-light-treated group were both within the normal range.
[0058] The characterization results of UV-visible absorption spectra and fluorescence emission spectra of exosomes and hypericin are as follows Figure 2 As shown, the exosome absorption spectrum has two main bands at 555 and 595 nm. The fluorescence spectrum shows two main bands near 595 and 680 nm. The absorption spectrum of hypericin is at 555 and 595 nm, while the fluorescence spectrum shows two main bands near 595 and 640 nm. It can be seen that the optical properties of the exosomes provided by the present invention are different from those of hypericin, which indicates that the active ingredients that produce optical activity or their luminescence principles in the exosomes of the present invention are different from those of hypericin.
[0059] Singlet oxygen was detected using SOSG probe, which can specifically recognize singlet oxygen and produce green fluorescence. 2 μl of SOSG probe (100 μg dissolved in 33 μl methanol solution to prepare a 5 mM stock solution) was added to 2 ml of exosomes, and the fluorescence spectrum was obtained by scanning at an excitation wavelength of 504 nm, an emission wavelength of 525 nm, and a wavelength range of 510-620 nm.
[0060] like Figure 3 As shown, using 25mW / cm 2 LED light, no obvious fluorescence emission was observed without laser irradiation (0min). With the increase of illumination time (1, 2, 4, 6, 8, 10, 15, 20min), the singlet oxygen produced by exosomes gradually increased. The results showed that the singlet oxygen produced by exosomes increased with the increase of irradiation energy, indicating that exosomes are a new and excellent type II photosensitizer.
[0061] Experimental Example 2: Enrichment of exosomes in tumors
[0062] In this experimental example, the enrichment of the plant-derived exosomes prepared in Example 1 in nude mice inoculated with human melanoma cells WM-266-4 was studied.
[0063] 1. Experimental Methods
[0064] Human melanoma cells WM-266-4 were grown in DMEM medium with 10% FBS. The cells were cultured in an incubator at 37°C and 5% CO2. The skin on the back of nude mice was disinfected with 75% alcohol on a clean bench. 8X10 7 250-300ul of WM-266-4 cell suspension was inoculated subcutaneously on the back of nude mice. After 28 days, exosomes were injected into the tail vein and in vivo imaging of small animals was performed 4 hours later.
[0065] 2. Experimental Results
[0066] The results are as follows Figure 4 As shown, human melanoma cells WM-266-4. The exosome tail vein injection 4 hours in vivo imaging shows that the exosomes accumulate in the tumor location. This shows that the exosomes provided by the present invention have the ability to be enriched in tumors.
[0067] Example 3 Effect of exosomes on photodynamic therapy
[0068] 1. Experimental Methods
[0069] 1. Effects of Photodynamic Therapy
[0070] Human esophageal squamous cell carcinoma KYSE150 cells were grown in 10% FBS medium 1640 to 80-90%. After co-culture with PBS or exosomes for 12 hours, the cells were observed under a microscope with or without 590 nm light treatment ( Figure 5 ).
[0071] 2. CCK8 cell viability test
[0072] Human melanoma cells WM-266-4 were seeded in 96-well plates (5×10^3 cells per well and incubated in 100μL DMEM). The cells were grown to a density of 80% and incubated with different concentrations of exosomes (0, 5, 10, 15, 20μg / ml) for 12 hours. They were then irradiated with a 590nm LED light (20mW / cm2) for 10 minutes. At the same time, under the same experimental conditions, exosomes were also studied in the absence of laser irradiation. Hypericin (HYP) was used as a positive control. After further incubation for 1 hour, the cells were incubated at 37°C for another 1 hour using a CCK-8 kit. The absorbance value was then measured at 450nm using a microplate reader to indicate cell viability.
[0073] 2. Experimental Results
[0074] like Figure 5 As shown, a large number of cells in the light-treated exosome group died, while no obvious changes were observed in the other three groups. This indicates that the exosomes provided by the present invention can be used as photosensitizers to achieve photodynamic therapy.
[0075] like Figure 6 As shown, the test group is a cell culture group treated with different concentrations of exosomes; the control group is a PBS group with the same volume. When different concentrations of exosomes were used under dark conditions, almost no cytotoxicity was observed, indicating that exosomes have good biocompatibility in the absence of radiation. In contrast, when cells were incubated with different concentrations of exosomes under 20mW / cm2 light irradiation (590nm) for 10Min, the growth of tumor cells was inhibited to varying degrees. These results indicate that exosomes exhibit high cytotoxicity under irradiation.
[0076] Through the above embodiments and experimental examples, it can be seen that the present invention prepares a plant-derived exosome, which has photosensitivity and can be used as a photosensitizer in photodynamic therapy and photodynamic diagnosis. The exosomes of the present invention are more stable due to their membrane structure and can spontaneously generate fluorescence in PBS solution, which makes them have a greater advantage over the existing photosensitizer hypericin with solubility problems, and thus have a good application prospect.
Claims
1. A plant-derived exosome, characterized in that: It is extracted as follows: Step 1, extracting Hypericum perforatum with PBS solution; Step 2, separating the extract and concentrating it through an ultrafiltration concentrator; Step 3, add PBS solution to the concentrated solution and mix well to obtain pure exosomes.
2. The plant-derived exosomes according to claim 1, characterized in that: The Hypericum perforatum is Hypericumperforatum L.
3. The plant-derived exosomes according to claim 1, characterized in that: In step 1, the concentration of the PBS solution is 0.01M; And / or, the amount of the PBS solution is 5-10 times the amount of Hypericum perforatum; And / or, the extraction condition is immersion at 18-25° C. for 18-24 hours.
4. The plant-derived exosomes according to claim 1, characterized in that: In step 2, the separation step includes the following steps: filtering, centrifuging the filtrate at 12000-15000g for 5-10 min, centrifuging the supernatant at 10000-12000g for 30-60 min, and filtering the supernatant through a 0.22 um filter; And / or, the concentration factor is 3-5 times.
5. The plant-derived exosomes according to claim 1, characterized in that: In step 3, the volume ratio of the concentrate to the PBS solution is 1:(1-5).
6. The plant-derived exosomes according to claim 1, characterized in that: In step 3, after obtaining the exosomes, the following steps are further performed: Step 4, concentrating the solution containing the exosomes through an ultrafiltration concentration tube; the concentration multiple is 2-4 times; Step 5, after the concentrate is collected, it is centrifuged at 120000-150000g for 60-90 min at 4°C, and the solid obtained by centrifugation is resuspended with PBS solution.
7. The plant-derived exosomes according to any one of claims 1 to 6, characterized in that: The absorption spectrum of the exosomes has two main bands at 555nm and 595nm; the fluorescence spectrum of the exosomes shows two main bands at 595nm and 680nm.
8. The method for preparing plant-derived exosomes according to any one of claims 1 to 7, characterized in that: Step 1, extracting Hypericum perforatum with PBS solution; Step 2, separating the extract and concentrating it through an ultrafiltration concentrator; Step 3, add PBS solution to the concentrated solution and mix well to obtain pure exosomes.
9. Use of the plant-derived exosomes according to any one of claims 1 to 7 as a photosensitizer.
10. The use according to claim 9, characterized in that: The photosensitizer is used for photodynamic therapy and / or photodynamic diagnosis.