Preparation method and application of turmeric extracellular vesicle targeted drug delivery system with colon cancer resisting effect

Abstract

The invention provides a preparation method and application of a turmeric extracellular vesicle targeted drug delivery system with an anti-colon cancer effect. The engineering system of modifying CELNs and loading DOX by the TCP-1 colon cancer targeting peptide is constructed by extracting curcuma longa extracellular vesicles (CELNs). The system of loading adriamycin through targeted modified turmeric extracellular vesicles has better anti-tumor activity and tumor targeting property, and HE staining and serum detection results show that the system has no significant influence on important tissues, visceral organs and liver functions of mice. And the biological safety is improved while a better treatment effect is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and molecular biology, and in particular, relates to a preparation method and application of a turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect. Background Art

[0002] Colon cancer refers to a malignant tumor of the digestive tract originating from the colon epithelium, which is prone to occur in the rectum and the junction of the rectum and the sigmoid colon. Colon cancer is a digestive tract disease that is difficult to detect in the early stages. Postoperative radiotherapy and chemotherapy are highly toxic, and patients have many adverse reactions. Therefore, there is an urgent need to develop highly effective and low-toxic anti-colon cancer drugs to change this situation.

[0003] Doxorubicin hydrochloride (DOX) is an anthracycline antibiotic discovered from a mutant strain of Streptomyces posse. It is a chemotherapeutic agent with a broad antitumor spectrum. It exerts strong antitumor activity against a variety of tumor cells by inhibiting the synthesis of tumor cell nucleic acids. It is suitable for the treatment of malignant tumors such as acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, and bronchial lung cancer. However, the drug has obvious cardiotoxicity because its affinity with the myocardium is significantly higher than that of other tissues, and it can damage myocardial cells through semiquinone metabolites. In addition, it also has toxic side effects such as hepatotoxicity and nephrotoxicity, which seriously limits the clinical application of the drug. Targeted drug delivery system (TDS) is a new drug delivery method that uses carriers to load drugs and selectively concentrates drugs in target organs, target tissues, and target cells through local administration or systemic blood circulation to improve efficacy and reduce toxic side effects.

[0004] Turmeric (Curcuma L.) is a perennial herbaceous plant of the genus Curcuma (Curcuma longa L.) of the Zingiberaceae family. It is grown in Fujian, Guangdong, Guangxi, Taiwan, Yunnan, Tibet and other places in China. As a representative drug of "medicine and food have the same origin", it has a long history of medicinal use in my country.

[0005] Plant extracellular vesicles (EVs.) are membrane vesicles secreted by plant cells, with a specific phospholipid bilayer structure, saucer-shaped or cup-shaped, with a particle size of 30 to 300 nm, and containing lipids, proteins, nucleic acids and other active ingredients. Plant extracellular vesicles are similar in structure and function to animal exosomes. Exosomes are messengers for information exchange between cells and participate in the physiological processes of various diseases in the human body. It has the advantages of good biocompatibility and low immunogenicity. It can not only cross biological barriers, but also reflect the physiological and pathological conditions of cells.

[0006] Based on this, the present invention provides a preparation method and application of a turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect. Summary of the invention

[0007] The present invention found that the synthetic peptide CTPSPFSHC (TCP-1) has good pharmacokinetic properties, good plasma stability, rapid clearance in plasma, and high affinity for colorectal cancer.

[0008] The present invention found that compared with EVs from other sources, turmeric extracellular vesicles contain curcumin, a medicinal substance from its homologous plants, and therefore have more prominent biological activity than EVs from ordinary plants. Its structure can also be further modified to achieve targeted purposes or to exert multi-component synergistic effects.

[0009] The purpose of the present invention is to provide a preparation method and application of a turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect.

[0010] To achieve the above object, the present invention adopts the following technical solution: The present invention constructs an engineering system of TCP-1 colon cancer targeting peptide-modified CELNs and loaded with DOX by extracting turmeric extracellular vesicles (CELNs). HCT-116 cells and BALB / c male mice are used as research objects, respectively, and the anti-tumor activity is explored and verified by cell activity determination, cell uptake analysis, cell cycle analysis, and pharmacodynamic tests of tumor growth in mice: vesicles are extracted from turmeric by differential centrifugation and ultracentrifugation, and identified by transmission electron microscopy, nanoparticle tracking analysis, etc., to explore its anti-colon cancer effect.

[0011] A turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect is disclosed. The turmeric extracellular vesicle targeted drug delivery system is CELNs-TCP-1-DOX, which uses turmeric extracellular vesicles CELNs as carriers to load the drug doxorubicin hydrochloride DOX, and modifies the turmeric extracellular vesicles CELNs with a targeting cyclic polypeptide TCP-1, thereby constructing the turmeric extracellular vesicle targeted drug delivery system CELNs-TCP-1-DOX.

[0012] Furthermore, the preparation method of turmeric extracellular vesicles CELNs comprises the following steps: adding turmeric to phosphate buffered saline (PBS), soaking for 5 hours, first using a juicer to squeeze and crush the juice, filtering the squeezed turmeric juice, centrifuging at 1000×g for 10 min, 3000×g for 20 min, and 10000×g for 30 min at 4°C, and then taking the supernatant; using an ultra-high speed centrifuge to centrifuge the supernatant at 4°C for 2 h, the speed is 100000×g, and the precipitate is taken; the precipitate is resuspended with PBS, and a 30wt% sucrose solution is added, and the precipitate is centrifuged at 100000×g at 4°C for 2h. The color of the centrifuge tube band is observed, and the sample layer is aspirated and placed in a centrifuge tube, diluted with PBS, and centrifuged at 100000×g at 4°C for 1.5h, the supernatant is discarded, and PBS is added again, and the precipitate is centrifuged at 100000×g at 4°C for 1.5h, the supernatant is discarded, and the precipitate is resuspended with PBS to obtain the purified turmeric extracellular vesicle CELNs suspension.

[0013] Furthermore, the dosage ratio of turmeric and phosphate buffer saline (PBS) was 1:10 g / mL.

[0014] Furthermore, the pH of phosphate buffered saline (PBS) is 7.4.

[0015] Furthermore, the preparation method of the turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect includes: mixing CELNs suspension and DOX solution in a volume ratio of 1:1, ultrasonicating the mixed system for 30 seconds, stagnating for 30 seconds, and cycling for a total of 6 times, and then incubating at 37°C for 1 hour; adding TCP-1 to the drug-loaded system and mixing, incubating again at 37°C for 1 hour, and removing free DOX by centrifugation to obtain CELNs-TCP-1-DOX.

[0016] Furthermore, the concentrations of CELNs suspension and DOX solution were the same.

[0017] Furthermore, the concentration of the CELNs suspension was 1 mg / mL.

[0018] Furthermore, the concentration of the DOX solution was 1 mg / mL.

[0019] Furthermore, the mass ratio of TCP-1 and CELNs was 1:1.

[0020] Furthermore, the drug loading of DOX was 23%.

[0021] Application: Application of the turmeric extracellular vesicle targeted drug delivery system in the preparation of anti-colon cancer drugs.

[0022] The advantages of the present invention are: The present invention has found that turmeric extracellular vesicles have a certain therapeutic effect on colitis. In order to continue to study turmeric vesicles as potential anti-colon cancer components, the present invention uses differential ultracentrifugation to extract turmeric extracellular vesicles, purify and characterize them. The study found that turmeric extracellular vesicles also have certain biological activity against colon cancer. The turmeric extracellular vesicle-TCP-1 (CELNs-TCP-1) targeting system was constructed by co-incubation method, and the anticancer drug doxorubicin hydrochloride was loaded. The tumor-bearing mouse model was constructed by subcutaneous injection of colon cancer cells (CT26.WT), and the tail vein injection was administered to explore its in vitro antitumor activity and in vivo biosafety. The morphology, particle size and lipidomics results of CELNs showed that the CELNs prepared by the present invention were consistent with the evaluation criteria of extracellular vesicles. Compared with other groups, the system of turmeric extracellular vesicles loaded with doxorubicin after targeted modification has better antitumor activity and tumor targeting. The results of HE staining and serum detection show that it has no significant effect on important tissues and organs and liver function of mice. While achieving better therapeutic effects, biosafety is improved. In addition to being a drug carrier, turmeric extracellular vesicles can also be used as an auxiliary treatment for colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a flow chart of the preparation of CELNs-TCP-1-DOX; Figure 2 This is a macroscopic photograph of turmeric extracellular vesicles; Figure 3 This is a transmission electron micrograph of extracellular vesicles of turmeric; Figure 4 For the particle size analysis of turmeric extracellular vesicles; Figure 5 For lipid composition analysis of extracellular vesicles of turmeric; Figure 6 IR spectra of TCP-1, CELNs and CELNs-TCP-1; Figure 7 The results are for DPPH free radical scavenging rate of CELNs; Figure 8 The results are for the antioxidant capacity of CELNs ABTS; Fig. 9 The results are for the hydroxyl radical scavenging rate of CELNs; Fig.10 The results are for superoxide anion clearance of CELNs; Fig.11 For cytotoxicity experiments; A: Dox, B: CELNs, C: CELNs-TCP-1, D: CELNs-Dox, E: CELNs-TCP-1-Dox; Fig.12Cell uptake experiment; A: Dox, B: CELNs-Dox, C: CELNs-TCP-1-Dox, D: CELNs, E: CELNs-TCP-1; Fig.13 For cell cycle experiments; A: Control, B: CELNs, C: CELNs-TCP-1, D: Dox, E: CELNs-Dox, F: CELNs-TCP-1-Dox; Fig.14 This is an animal experiment; A: tumor size of each group after drug administration, B: tumor weight of each group of mice, C: tumor volume of each group of mice, D: weight change of mice after seven days of drug administration; Fig.15 In vivo imaging of small animals; Fig.16 HE staining sections of tumor tissues in each group; Fig.17 A: HE-stained sections of visceral tissues of mice in each group; B: ALT and AST levels in serum of mice after administration. DETAILED DESCRIPTION

[0024] In order to make the above features and advantages of the present invention more clearly understood, the following embodiments are given for detailed description. The methods of the present invention are all conventional methods in the art unless otherwise specified.

[0025] 1 Extraction and identification of turmeric vesicles 1.1 Extraction experiment of turmeric vesicles 500 g of turmeric was added to phosphate buffered saline (PBS, pH = 7.4) precooled to 4°C at a ratio of 1:10 (g / mL). After soaking for 5 hours, the juice was first squeezed and crushed with a juicer. The squeezed turmeric juice was filtered and centrifuged at 4°C at 1000×g for 10 min, 3000×g for 20 min, and 10000×g for 30 min to obtain the supernatant. The supernatant was centrifuged at 4°C for 2 h at a speed of 100,000×g using an ultrahigh-speed centrifuge. Take 1.47 g of precipitate and resuspend it with 2 mL of PBS, add 30 mL of prepared 30 wt% sucrose solution, weigh and balance, centrifuge at 100,000 × g at 4 ° C for 2 h, observe the color of the centrifuge tube band, aspirate the sample layer and put it in a centrifuge tube, dilute with 35 mL of PBS, centrifuge at 100,000 × g at 4 ° C for 1.5 h, discard the supernatant, add 35 mL of PBS and centrifuge at 100,000 × g at 4 ° C for 1.5 h, discard the supernatant, and resuspend the precipitate with 10 mL of PBS to obtain the purified turmeric vesicle suspension.

[0026] 1.2 Identification of turmeric extracellular vesicles The turmeric vesicles obtained by centrifugation were identified by transmission electron microscopy. The specific experimental process is as follows: Use a pipette to transfer 10 μL of vesicle suspension onto the wax disk, take a copper mesh so that the surface with the support membrane is in contact with the surface of the suspension, let it stand for 5 minutes, take out the copper mesh, use filter paper to absorb excess droplets, dry it slightly, take a 3% phosphotungstic acid solution, drop a small drop on the wax disk, place the copper mesh with the sample adsorbed on the surface of the dye solution (the sample is in contact with the dye solution), and let it stand for 3-5 minutes. Take out the copper mesh, use filter paper to absorb excess droplets, dry it, and take pictures with an electron microscope.

[0027] Nanoparticle analysis of turmeric extracellular vesicles was performed as follows: The size of CELNs was analyzed using nanoparticle tracking analysis technology. After the extracted CELNs suspension was diluted to an appropriate concentration with PBS buffer, the particle number, particle size and potential information could be obtained.

[0028] 1.3 Lipid composition analysis of CELNs Ultra-high performance liquid chromatography-mass spectrometry was used to characterize the types and contents of lipids in CELNs. 200 μL of CELNs was added with 400 μL of methyl tert-butyl ether and 80 μL of methanol. The mixture was vortexed for 30 s and centrifuged at 3000 r / min for 15 min to separate the organic phase and the aqueous phase. The methyl tert-butyl ether layer was dried with nitrogen and redissolved in 100 μL of dichloromethane:methanol (1:1, v:v) solution for subsequent detection.

[0029] 1.4 Preparation of CELNs-TCP-1-DOX 1 mg / mL DOX solution was added to 1 mg / mL CELNs suspension (1:1, v:v), the mixed system was sonicated for 30 s, stagnated for 30 s, and cycled 6 times in total, and incubated at 37 °C for 1 h. After adding 1 mg TCP-1 to the drug-loaded system (1:1, m:m, TCP-1:CELNs), it was incubated at 37 °C for 1 h again, and CELNs-TCP-1-DOX was obtained after centrifugation to remove free DOX. The CELNs-TCP-1-DOX obtained by the above method can be evaluated by high performance liquid chromatography to evaluate the amount of DOX loaded on CELNs, and the drug loading is 23%.

[0030] At the same time, the connection between CELNs and TCP-1 was determined by infrared spectrometer. The operation steps are as follows: the sample to be tested is mixed with potassium bromide, ground and pressed into tablets, and then detected in infrared spectrometer.

[0031] 2. Determination of antioxidant activity of CELNs 2.1 DPPH free radical scavenging ability determination 5 mg DPPH was accurately weighed, diluted with methanol and fixed to a 50 ml volumetric flask, and stored in the dark for later use. 20 μL of turmeric EVs suspension of different concentrations (5, 10, 20, 30, 40, 50, 60 mg / ml) and 180 μL DPPH methanol solution were added to a 96-well plate, and three replicate wells were set up. Methanol was used instead of the suspension as a blank, and the plate was stored in the dark for 30 min. The absorbance of each well was measured at a wavelength of 517 nm using an ELISA reader to calculate the free radical scavenging rate.

[0032] Clearance rate (%) = (A blank - A sample) / A blank × 100% 2.2 Determination of ABTS cation radical scavenging ability The kit was used according to the instructions to prepare the ABTS working solution. 200 μL of ABTS working solution was added to each well of the 96-well plate, and 10 μL of purified water was added to the blank control well; 10 μL of Trolox standard solution of different concentrations (0.15, 0.3, 0.6, 0.9, 1.2, 1.5 mM) was added to the standard well; 10 μL of turmeric EVs suspension of different mass concentrations (10, 20, 30, 40, 50, 60, 70, 80 mg / ml) was added to the sample well and reacted at room temperature for 2-6 minutes for determination. The absorbance was measured at a wavelength of 734 nm using an ELISA reader, and the antioxidant capacity was calculated according to the instructions of the kit.

[0033] 2.3 Hydroxyl radical scavenging ability determination In the detection wells of a 96-well plate, 50 μL of turmeric EVs suspension of different concentrations (10, 20, 30, 40, 50, 60, 70 mg / ml), 50 μL of 6 mmol / L FeSO4 aqueous solution, and 100 μL of 6 mmol / L H2O2 aqueous solution were added respectively, shaken well, and left at room temperature for 10 min. 50 μL of 6 mmol / L salicylic acid-anhydrous ethanol solution was added to each well, shaken well, and left at room temperature for 30 min. Water was used instead of the suspension as a blank, and 3 replicate wells were set up. The absorbance was measured at a wavelength of 510 nm using an enzyme reader to calculate the hydroxyl radical scavenging rate.

[0034] Hydroxyl radical scavenging rate = (A blank - A sample) / A blank × 100%.

[0035] 2.4 Determination of superoxide anion scavenging ability In a 1.5 ml centrifuge tube, 900 μL of 0.05 mmol / L pH=7.4 phosphate buffer was added and incubated in a water bath at 25°C for 20 min. Then, 200 μL of turmeric EVs suspension of different concentrations (5, 10, 20, 30, 40, 50, 60, 70 mg / ml) and 80 μL of 25 mmol / L pyrogallol solution were added and mixed. The mixture was reacted in a water bath at 25°C for 5 min. Then, 200 μL of 80 mmol / L HCl was added to terminate the reaction. The mixture was shaken for 3 min and centrifuged at 7000 r / min for 3 min. Water was used instead of the sample as the blank, and water was used instead of pyrogallol as the corresponding sample solution A2. The absorbance was measured at a wavelength of 420 nm, and the superoxide anion clearance rate was calculated.

[0036] Clearance rate = [A blank - (A sample - A2)] / A blank × 100% 3 Evaluation of the inhibitory effect of CELNs on colon cancer 3.1 Cell assay 3.1.1 HCT116 cell culture HCT116 cells are grown in a culture medium with a ratio of DMEM high glucose medium: serum: penicillin-streptomycin double antibody of 100:10:1; they grow adherently at 37°C and 5% CO2. Generally, the culture dish can be covered within 48-72 hours, and then passage or subsequent experiments can be performed.

[0037] 3.1.2 Cytotoxicity assay Experimental group: cells were treated with 5 groups of drugs, namely DOX, CELNs, CELNs-DOX, CELNs-TCP-1, and CELNs-TCP-1-DOX, and drug-containing culture fluids of different mass concentrations were prepared.

[0038] Control group: cells were added with complete culture medium.

[0039] Blank group: no cells, only complete culture medium was added.

[0040] HCT-116 cells in the logarithmic growth phase were seeded in 96-well plates at a number of 2000 per well (n=3). After the cells adhered to the wall, five groups of drugs including DOX, CELNs, CELNs-DOX, CELNs-TCP-1, and CELNs-TCP-1-DOX at different concentrations were added and cultured for 24 h. Then, CCK-8 solution was added and incubated for 1 h. The absorbance (OD) was measured at a wavelength of 450 nm to analyze the cell survival rate under different conditions.

[0041] 3.1.3 Cellular uptake assay DiO was added to CELNs, CELNs-TCP-1, and CELNs-TCP-1-DOX to a final concentration of 5 μM (DiO was dissolved in DMSO), incubated at 37 °C for 30 min, and centrifuged at 3500 r / min for 10 min to remove the free DiO dye, thereby obtaining three samples labeled with DiO. HCT-116 cells were inoculated in a confocal culture dish, and after the cells adhered to the wall, the above five drugs were added to the cells and cultured in a cell culture incubator for 1 h. The cells were fixed with 1 mL of 4% paraformaldehyde for 30 min and then stained with DAPI for 30 min. Finally, they were washed with PBS and observed under a laser confocal microscope.

[0042] 3.1.4 Cell cycle assay The detection of cell cycle mainly adopts propidium iodide staining method. HCT-116 cells were inoculated in 6-well plates, and the above five different drugs were added and incubated for 1 hour. The cell culture medium was collected, digested with trypsin, and then added to the culture medium collected previously, centrifuged at 1000 g for 5 min, and the supernatant was removed and 1 mL of pre-cooled PBS buffer was added to resuspend the cells. 1 mL of pre-cooled 70% ethanol was added, and after being blown evenly, it was fixed at 4 ℃ for 12-24 h, and centrifuged at 1000 g for 5 min. 1 mL of pre-cooled PBS buffer was added again, the cells were resuspended, and the cells were centrifuged again. 0.5 mL of propidium iodide staining solution was added to each cell sample, and after being warmed at 37 ℃ in the dark for 30 min, it was filtered with a 70 μm pore size cell filter and analyzed by flow cytometry.

[0043] 3.2 Animal testing 3.2.1 Establishment of colon cancer model mice BALB / c male mice aged 6-8 weeks were prepared and divided into PBS group, DOX group, CELNs group, CELNs-DOX group, CELNs-TCP-1 group, and CELNs-TCP-1-DOX group. HCT-116 cells were injected subcutaneously, and colon cancer model mice were established when the tumor volume grew to about 50 mm.

[0044] 3.2.2 Pharmacodynamics of drug delivery system on tumor growth in mice PBS, DOX, CELNs, CELNs-DOX, CELNs-TCP-1, and CELNs-TCP-1-DOX were injected into mice via tail vein administration, and the dosage was determined according to the body surface area of ​​the animals.

[0045] Blood was collected from the ophthalmic vein of the mice and stored for blood testing; the mice were dissected, and the heart, liver, spleen, lung, kidney and tumor were removed and fixed in 4% paraformaldehyde solution; the tissue was removed from the paraformaldehyde, the formaldehyde solution was absorbed with absorbent paper, and the surface of the tissue was trimmed flat with a blade; the trimmed tissue was immersed in 70% (4~12h), 80% (4~12h), 90% (1~2h), and 100% (1~2h) ethanol solutions in turn to replace the formaldehyde solution in the tissue; the tissue was immersed in xylene solution to replace the ethanol therein, and the immersion was stopped when the edge of the tissue became translucent; the oven containing paraffin was opened in advance to melt the paraffin, the tissue was immersed in paraffin for 3 h, and then placed in another oven containing paraffin for 1 h to replace the xylene in the tissue. Pour the melted paraffin into the embedding frame, quickly remove the tissue from the paraffin and place it in the embedding frame, with the cut surface facing down, and mark it with label paper. Note that the paraffin should be kept in a melted state during the process; in order to obtain continuous and smooth slices during slicing, pre-cool the wax block containing the tissue sample in a -20°C refrigerator before slicing, fix the wax block on the slicer, and perform continuous slicing; carefully transfer the slices to 40°C warm water for spreading; pick up the slices with a slide, and then place them in an oven for baking. Baking can prevent the slices from falling off the slide during the staining process. After baking, staining can be performed. If the slices are not stained immediately after baking, they can be placed in a slice box after cooling at room temperature and stored in a -20°C refrigerator. They need to be baked again before staining.

[0046] The tissue sections were stained with a hematoxylin and eosin (HE) staining kit. After hematoxylin and eosin staining, the cell nucleus appeared blue and the cytoplasm appeared pink or red, so that the tissue or lesion could be observed. Sample processing: Dewax the paraffin sections in xylene for 5 to 10 minutes, replace with fresh xylene, dewax for 5 to 10 minutes, anhydrous ethanol for 5 minutes, 90% ethanol for 2 minutes, 80% ethanol for 2 minutes, 70% ethanol for 2 minutes, and distilled water for 2 minutes. Hematoxylin and eosin (HE) staining: For the samples treated above, stain with hematoxylin staining solution for 5 to 10 minutes, rinse off the excess staining solution in tap water for about 10 minutes, wash again with distilled water, and stain with eosin staining solution for 30 seconds to 2 minutes. At this time, if direct observation is required, wash twice with 70% ethanol. If direct observation is not required, dehydration, transparency and sealing are still required after washing with 70% ethanol. After staining, obtain pictures of each group of tissues under a microscope.

[0047] 4 Results and discussion 4.1 Extraction and Isolation of Curcuma Extracellular Vesicles like Figure 2As shown, the appearance results of turmeric extracellular vesicles: the extracted turmeric extracellular vesicles are a khaki suspension with good fluidity.

[0048] 4.2 Identification of Curcuma Extracellular Vesicles Transmission electron microscopy (TEM) like Figure 3 As shown in the figure, the morphological characterization results of turmeric extracellular vesicles showed that under electron microscopy, turmeric extracellular vesicles had obvious lipid bilayers, a saucer-shaped structure, and a diameter between 30 nm and 200 nm.

[0049] Nanoparticle Tracking Analysis (NTA) like Figure 4 As shown, the particle size analysis results of turmeric extracellular vesicles showed that the particle size distribution of turmeric extracellular vesicles was relatively uniform, with an average particle size of about 193 nm.

[0050] 4.3 Lipid composition analysis of CELNs like Figure 5 As shown in the figure, there are as many as 6 lipid components in CELNs that account for more than 5%, namely triglyceride (TG, 30%), diglyceride (DG, 12.7%), ceramide (Cer, 11.8%), sphingoid base (SPH, 9.1%), phosphatidylcholine (PC, 7.3%) and fatty acid (FA, 6.4%).

[0051] 4.4 IR spectra analysis of TCP-1, CELNs and CELNs-TCP-1 like Figure 6 As shown in the infrared spectrum of CELNs, 3367cm -1 2925cm is the stretching vibration absorption peak of OH or NH bond; -1 and 2854cm -1 The stretching vibration of the carbon-hydrogen bond (CH) at 1648 cm -1 The C=O stretching vibration peak of the amide group (-CONH-) in the peptide chain is 1416 cm -1 The CH bending vibration peaks corresponding to long-chain alkyl groups (CH3 and CH2) are at 1328 cm -1 The peak at 1076cm is the P=O stretching vibration peak, which comes from the phosphate group of the phospholipid head; -1 The COP bond is related to the stretching vibration of the COP bond, indicating the COP bond between the glycerol and phosphate groups in phospholipids; 779 cm -1 527cm is the out-of-plane bending vibration peak of the CH of the long-chain fatty acid alkyl part; -1 It is related to the bending vibration of the POC bond of the phospholipid head group.

[0052] From the infrared spectrum of TCP-1, we can see that 3367cm -1 The 2920cm-1 is the stretching vibration absorption peak of the OH or NH bond. The TCP-1 molecule contains multiple amine groups and hydroxyl groups. -1 and 2854cm -1 The position corresponds to the stretching vibration of the carbon-hydrogen bond (CH). TCP-1 molecules contain multiple methyl and methylene groups, so they show a strong absorption peak; 1739cm -1 The stretching vibration peak of the ester group (C=O) in the molecule should be at 1656cm -1 The C=O stretching vibration peak (amide I band) corresponding to the amide group (-CONH-) at 1536 cm -1 The NH bond bending vibration peak (amide II band) is at 1468 cm -1 The peaks at 1350cm correspond to the CH bending vibration peaks of the methyl and methylene groups (CH3 and CH2) in the molecule; -1 The stretching vibration peak of CN bond is at 1250cm -1 and 1110cm -1 The position is related to the stretching vibration of the CO bond, indicating the CO bond of the ester group and the hydroxyl group in the TCP-1 molecule; 950cm -1 and 842cm -1 The peak at corresponds to the out-of-plane bending vibration of CH on the aromatic ring, which comes from the indole ring structure in the TCP-1 molecule.

[0053] From the infrared spectrum of CELNs-TCP-1, we can see that 3425cm -1 The 2922cm-1 peak is the stretching vibration absorption peak of the OH or NH bond, which comes from the phospholipid head of the CELNs part and the amine and hydroxyl groups in TCP-1; -1 and 2854cm -1 1645 cm-1 corresponds to the stretching vibration of the carbon-hydrogen bond (CH), which comes from the long-chain fatty acids in the phospholipid bilayer and the alkyl groups in TCP-1; -1 The C=O stretching vibration peak (amide I band) corresponding to the amide group (-CONH-) is derived from the fatty acid esters in the CELNs part and the multiple amide bonds in TCP-1; 1405cm -1 The CH bending vibration peaks corresponding to the methyl and methylene (CH3 and CH2) in the long-chain alkyl group come from the long-chain fatty acids in the phospholipid bilayer and the alkyl group in TCP-1; 1153cm -1 The peak at 1076 cm is the P=O stretching vibration peak in the phosphate group, which comes from the phospholipid head of the CELNs part; -1The stretching vibrations of COP and ether bonds (COC) are related to the phospholipid head of CELNs and the COC bond in TCP-1; 950 cm -1 and 859cm -1 The peak at 544cm corresponds to the out-of-plane bending vibration of CH on the aromatic ring, which comes from the indole ring structure in the TCP-1 molecule; -1 It mainly corresponds to the bending vibration of the POC bond, and part of it comes from the characteristic absorption of the SS bond, resulting in a strong absorption peak here and a characteristic vibration mode.

[0054] Summarize: Through the above analysis, the characteristic peaks of TCP-1 do appear in the infrared spectrum of CELNs-TCP-1. These peaks include: 3425cm -1 : NH and OH stretching vibrations, indicating the presence of amine and hydroxyl groups in TCP-1.

[0055] 1645cm -1 : The C=O stretching vibration of the amide group (amide I band), indicating the existence of an amide bond in TCP-1.

[0056] 950cm -1 and 859cm -1 : The out-of-plane bending vibration peak of CH on the aromatic ring indicates the existence of the indole ring structure in TCP-1.

[0057] 544cm -1 :The characteristic absorption partly originates from the SS bond, confirming the existence of disulfide bonds in TCP-1.

[0058] In addition, the infrared spectrum of CELNs-TCP-1 also contains the characteristic peaks of CELNs, especially: 1153cm -1 and 1076cm -1 : The characteristic absorption peaks of P=O, COP and COC bonds of the phospholipid head group indicate that the phospholipid bilayer is successfully connected to TCP-1.

[0059] The infrared spectrum of CELNs-TCP-1 shows the successful connection between plant vesicles (phospholipid bilayer) and TCP-1, which is manifested by the co-existence of characteristic peaks of CELNs and TCP-1. These characteristic peaks indicate that CELNs and TCP-1 form a stable complex through chemical bonding, supporting the formation of plant vesicle modification targets (CELNs-TCP-1).

[0060] 5 Antioxidant capacity of vesicles at different concentrations 5.1 DPPH free radical scavenging rate results like Figure 7 As shown in the figure, the DPPH free radical scavenging rate of turmeric extracellular vesicles gradually increased with the increase of concentration. In the concentration range of 5-40 mg / ml, the DPPH free radical scavenging rate increased from 19.29% to 55.44%. 50 The value was 34.41 mg / ml, indicating that the extracellular vesicles of turmeric have a weak ability to scavenge DPPH free radicals.

[0061] 5.2 ABTS antioxidant capacity results like Figure 8 As shown in the figure, the scavenging rate of ABTS free radicals by turmeric extracellular vesicles gradually increased with the increase of concentration. In the concentration range of 2.5~60 mg / mL, the scavenging rate of ABTS free radicals increased from 16.53% to 66.83%. 50 The value is 23.64 mg / mL.

[0062] 5.3 Hydroxyl radical scavenging rate results like Fig. 9 As shown in the figure, the scavenging rate of turmeric extracellular vesicles for hydroxyl radicals gradually increased with the increase of concentration. In the concentration range of 10-50 mg / ml, the scavenging rate of hydroxyl radicals increased from 27.92% to 74.14%. 50 The value is 23.57 mg / ml.

[0063] 5.4 Superoxide anion clearance results like Fig.10 As shown in the figure, the superoxide anion clearance rate of turmeric extracellular vesicles gradually increased with the increase of concentration. In the concentration range of 10-50 mg / ml, the superoxide anion clearance rate increased from 32.32% to 78.67%. 50 The value is 20.23mg / ml.

[0064] 6 Evaluation of the effect of CELNs in inhibiting colon cancer 6.1 Cell assay 6.1.1 Evaluation of the inhibitory effect of CELNs-TCP-1-DOX on HCT-116 cell proliferation The experimental results showed that DOX, CELNs, CELNs-DOX, CELNs-TCP-1, and CELNs-TCP-1-DOX all showed different degrees of inhibitory effects on the proliferation of HCT-116 cells, and the inhibitory effects were concentration-dependent. Fig.11As shown, the IC50 values ​​were A (DOX): 793.6 ng / mL, B (CELNs): 608.9 µg / mL, C (CELNs-TCP-1): 355.8 µg / mL, D (CELNs-DOX): 130.9 µg / mL, and E (CELNs-TCP-1-DOX): 55.1 µg / mL. All five groups of samples have the effect of inhibiting cell proliferation. At the same concentration, the ability of CELNs, CELNs-TCP-1, CELNs-DOX, and CELNs-TCP-1-DOX to inhibit the proliferation of HCT-116 cells gradually increases; at the same concentration, CELNs, CELNs-TCP-1, and CELNs-TCP-1 have better ability to inhibit the proliferation of HCT-116 cells, indicating that the target-modified CELNs have better targeting; CELNs-DOX and CELNs-TCP-1-DOX have better ability to inhibit the proliferation of HCT-116 cells than CELNs-DOX at the same concentration, and the DOX content in the system is also less than CELNs-DOX. In summary, the CELNs-TCP-1-DOX system has better ability to inhibit the proliferation of HCT-116 cells and is targeted to colon cancer.

[0065] 6.1.2 Evaluation of CELNs-TCP-1-DOX uptake by HCT-116 cells like Fig.12 As shown, after the cells were treated with different drugs, red fluorescence and green fluorescence could be observed on the cells, indicating that the five groups of drugs could be engulfed by the cells to varying degrees. Among them, the red and green fluorescence of the CELNs-TCP-1-DOX group were the strongest.

[0066] 6.1.3 Cell cycle analysis like Fig.13 As shown, after being treated with the above five groups of drugs, the number of HCT-116 cells in the G1 / G0 phase increased to varying degrees, indicating that the effect of inhibiting cell proliferation ability was enhanced to varying degrees.

[0067] 6.2 Animal testing like Fig.14 As shown in the figure, the drug was injected into the tumor-bearing mice through the tail vein. After 7 consecutive days of treatment, the tumor size of each group after administration (A); the tumor weight of each group of mice (B); the tumor volume of each group of mice (C); the weight change of mice after 7 days of administration (D); the results showed that the tumor tissue volume and tumor weight of the group injected with CELNs-TCP-1-DOX through the tail vein were significantly reduced. Fig.14In Figure D, it can be seen that seven consecutive days of administration had no effect on body weight. In vivo imaging results of small animals showed that TCP-1-modified CELNs can actively target and accumulate at tumor sites ( Fig.15 ).

[0068] like Fig.16 As shown in the figure, HE staining results showed that compared with the PBS treatment group, the tumor tissue in the CELNs-TCP-1-DOX treatment group showed severe necrosis, cell vacuolar changes, and inflammatory cell infiltration ( Fig.16 ) In order to evaluate the safety of TCP-1 modified CELNs and loaded with DOX in therapeutic applications, the main organs of the mice including heart, liver, spleen, lung and kidney as well as serum were collected for safety evaluation after treatment. Fig.17 As shown in the figure, HE staining results showed that the DOX group, CELNs group, CELNs-TCP-1 group, CELNs-DOX group, and CELNs-TCP-1-DOX group had no significant effect on the histopathological structure of the main tissues and organs of the mice, such as the heart, liver, spleen, lung, and kidney ( Fig.17 In addition, serum tests found that the five intervention groups had no significant effect on the liver function indicators AST and ALT of mice ( Fig.17 (middle B).

[0069] Plant-derived extracellular vesicles can not only play a pharmacological role as an active ingredient, but also play a unique advantage as a drug carrier. The present invention found that turmeric extracellular vesicles have a certain therapeutic effect on colitis. In order to continue to study turmeric vesicles as a potential anti-colon cancer component, the present invention uses differential ultracentrifugation to extract turmeric extracellular vesicles, purify and characterize them. The turmeric extracellular vesicle-TCP-1 (CELNs-TCP-1) targeting system was constructed by co-incubation method, and the anticancer drug doxorubicin hydrochloride was loaded. The tumor-bearing mouse model was constructed by subcutaneous injection of colon cancer cells (CT26.WT), and the tail vein injection was administered to explore its in vitro antitumor activity and in vivo biosafety. The morphology, particle size and lipidomics results of CELNs showed that the CELNs prepared by the present invention were consistent with the evaluation criteria of extracellular vesicles. Compared with other groups, the system of turmeric extracellular vesicles loaded with doxorubicin after targeted modification has better antitumor activity and tumor targeting. The results of HE staining and serum detection showed that it had no significant effect on important tissues and organs and liver function of mice. It achieves better therapeutic effects while improving biosafety.

[0070] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect, characterized in that: The turmeric extracellular vesicle targeted drug delivery system is CELNs-TCP-1-DOX, which uses turmeric extracellular vesicles CELNs as carriers, loads the drug doxorubicin hydrochloride DOX, and modifies the turmeric extracellular vesicles CELNs with a targeting cyclic polypeptide TCP-1, thereby constructing the turmeric extracellular vesicle targeted drug delivery system CELNs-TCP-1-DOX.

2. The turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect according to claim 1, characterized in that: The preparation method of turmeric extracellular vesicles CELNs comprises the following steps: adding turmeric to phosphate buffered saline (PBS), soaking for 5 hours, firstly squeezing and crushing the juice with a juicer, filtering the squeezed turmeric juice, centrifuging at 1000×g for 10 min, 3000×g for 20 min, and 10000×g for 30 min at 4°C, and then taking the supernatant; using an ultra-high speed centrifuge to centrifuge the supernatant at 4°C for 2 h, the speed is 100000×g, and the precipitate is taken; the precipitate is resuspended with PBS, and a 30wt% sucrose solution is added, and the precipitate is centrifuged at 100000×g at 4°C for 2h. The color of the centrifuge tube band is observed, and the sample layer is aspirated and placed in a centrifuge tube, diluted with PBS, and centrifuged at 100000×g at 4°C for 1.5h, the supernatant is discarded, and PBS is added again, and the precipitate is centrifuged at 100000×g at 4°C for 1.5h, the supernatant is discarded, and the precipitate is resuspended with PBS to obtain the purified turmeric extracellular vesicle CELNs suspension.

3. The turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect according to claim 2, characterized in that: The usage ratio of turmeric and phosphate buffer saline (PBS) was 1:10 g / mL.

4. The turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect according to claim 2, characterized in that: Phosphate buffered saline (PBS) has a pH of 7.

4.

5. The method for preparing the turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect according to claim 1, characterized in that: The CELNs suspension and DOX solution were mixed in a volume ratio of 1:1, and the mixed system was sonicated for 30 s, stagnated for 30 s, and cycled for a total of 6 times, and then incubated at 37 °C for 1 h; TCP-1 was added to the drug-loaded system and mixed, and then incubated again at 37 °C for 1 h. After centrifugation to remove free DOX, CELNs-TCP-1-DOX was obtained.

6. The method for preparing the turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect according to claim 5, characterized in that: The concentration of CELNs suspension was 1 mg / mL.

7. The method for preparing the turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect according to claim 5, characterized in that: The concentration of the DOX solution was 1 mg / mL.

8. The method for preparing the turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect according to claim 5, characterized in that: The mass ratio of TCP-1 and CELNs was 1:

1.

9. The method for preparing the turmeric extracellular vesicle targeted drug delivery system with anti-colon cancer effect according to claim 5, characterized in that: The drug loading of DOX was 23%.

10. Use of the turmeric extracellular vesicle targeted drug delivery system according to claim 1 in the preparation of anti-colon cancer drugs.

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