A urea compound and its exosome loading method and application

Abstract

The present invention discloses a urea compound and its exosome loading method and application, belonging to the field of synthetic technology of drug delivery materials. The technical solution of the present invention is as follows: the molecular structure of the urea compound is: wherein R 1 is methoxy or bromine, R 2 is a hydrogen atom, a halogen, or a methoxy group; R 3 The present invention has discovered a method for efficiently preparing urea-linked triazole structures; the compound molecule can inhibit the IDO1 target and enhance the body's immunity; and a method for loading exosomes has been discovered, which is simple and efficient and can be used for drug delivery.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of drug delivery materials, and specifically relates to a urea compound and an exosome loading method and application thereof. Background Art

[0002] Urea is an organic compound composed of carbon, nitrogen, oxygen, and hydrogen. In the 18th century, humans first isolated urea from urine. In the 19th century, scientists discovered that urea could be synthesized from the inorganic ammonium cyanate, overturning the traditional theory of "vitality" and opening a new chapter in organic chemistry. Urea structural fragments are important backbones for drug development and are widely used in the medical field. For example, melanin-concentrating hormone (MCH) in the human body is involved in many physiological functions, especially playing an important role in food intake and energy balance. Compound G, which has a urea structure, can act as an MCH antagonist and can help reduce weight. Kotakadi reported that compound H, which contains a urea structure, has an excellent inhibitory effect on the proliferation of Staphylococcus aureus. Khan et al. found that compound I can effectively inhibit pancreatic cancer cells and laryngeal squamous cell carcinoma cells. Lipton's laboratory found that compound J exhibits moderate inhibition of HIV-1 protease. Currently marketed anti-tumor drugs containing urea structures include Lenvatinib, a tyrosine kinase inhibitor used to treat renal or thyroid cancer, which can effectively slow or prevent tumor cell growth; and sorafenib, a novel multi-targeted oral cancer treatment. It is used to treat gastrointestinal stromal tumors and metastatic renal cells that are unresponsive or intolerant to standard therapies. It selectively targets receptors for certain proteins, which are believed to act as molecular switches in tumor growth. It has received Fast Track designation from the FDA for these indications in the United States.

[0003] IDO1 is considered an immunomodulator, playing a role in pathogenic inflammation and promoting immune tolerance to tumor antigens. It primarily achieves immune tolerance by promoting tryptophan consumption and producing a series of toxic kynurenine metabolites, which in turn activate the GCN2 pathway, inhibit the mTOR pathway, inhibit the toxic effects of kynurenine, and promote the differentiation of regulatory T cells (Tregs). IDO1 has been found to be overexpressed in various tumor tissues, making it a potential target for tumor immunosuppression. Several IDO1 inhibitors, such as epacadostat, indoximod, navoximod, EOS-200271, and BMS-986205, are currently in clinical trials. To identify novel and more effective IDO1 inhibitors, we linked diphenylurea compounds with different substituents with triazole compounds with different substituents via methyl or isopropyl structures through a click reaction, resulting in five novel diphenylurea-linked triazole derivatives. We hope that these new compounds will combine the biological activities of both and have a certain effect on anti-IDO1 activity. We are also studying ways to load these compounds into exosomes so that they can effectively enter cells. Summary of the Invention

[0004] The urea compound and its exosome loading method and application described in the present invention are characterized in that the molecular structure of the urea compound is: where R 1 is methoxy or bromine, R 2 is a hydrogen atom, a halogen, or a methoxy group; R 3 is a hydrogen atom, a halogen, or a methoxy group.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solution, a urea compound and an exosome loading method thereof, characterized by the specific steps of:

[0006] (1): In a reaction flask with stirring, at 0°C, a dichloromethane solution containing a certain amount of phenyl isocyanate compounds is slowly added dropwise to a dichloromethane solution containing 3-amino-3-methyl-1-butyne and triethylamine. The mixture is stirred at 0°C for a period of time, tert-butyl alcohol and water are added, and the mixture is heated to room temperature. Then, a certain amount of benzyl azide compounds and cuprous iodide are added. The reaction is continued at room temperature for a period of time. Water is added to the reaction system, stirred, and filtered to separate the organic phase. The aqueous phase is then extracted with dichloromethane for several times. The organic phases are combined and dried over anhydrous magnesium sulfate and concentrated. Finally, the mixture is passed through silica gel. The product is separated by column chromatography; the molar ratio of the phenyl isocyanate compound to 3-amino-3-methyl-1-butyne and triethylamine is 1:1-1.1:1-1.1; the molar ratio of the phenyl isocyanate compound to the benzyl azide compound is 1:1-1.1; the mass ratio of the benzyl azide compound to cuprous iodide is 10:1; the phenyl isocyanate compound is 4-methoxyphenyl isocyanate or 4-bromophenyl isocyanate; and the benzyl azide compound is 3-bromobenzyl azide or 3-bromobenzyl azide or 3-methoxybenzyl azide.

[0007] (2): Using a silicon carbide microchannel reactor, a dichloromethane solution containing a certain amount of 3-amino-3-methyl-1-butyne was prepared and marked as A, and a tert-butyl alcohol solution containing a certain amount of benzyl azide compounds and cuprous iodide was prepared, and a mixed solution of dichloromethane and water was marked as B. Solution A and solution B were respectively connected to the two feed pumps of the reactor and simultaneously entered the reactor at a certain speed. The reactor temperature was set to 40°C. The two reaction solutions were mixed and reacted in the reactor and then flowed into dichloromethane containing isocyanate phenyl compounds and triethylamine through the discharge port. Stirring was performed while adding. When the solution in the microchannel reactor was completely discharged, Continue stirring for a period of time, filter the reaction solution, separate the organic phase, extract the aqueous phase with dichloromethane for multiple times, combine the organic phases, dry with anhydrous magnesium sulfate, concentrate, and finally dry to obtain the product; the molar ratio of the 3-amino-3-methyl-1-butyne to the benzyl azide compound is 1:1; the mass ratio of the benzyl azide compound to cuprous iodide is 10:0.4-0.5; the molar ratio of the 3-amino-3-methyl-1-butyne to the isocyanate phenyl compound is 1:1; the benzyl azide compound is 3-bromobenzyl azide, 2-bromobenzyl azide, or 3-methoxybenzyl azide.

[0008] (3): The supernatant of the laboratory-cultured Dunaliella cells was collected and ultracentrifuged in an ultrahigh-speed centrifuge. The exosome precipitate was resuspended in phosphate buffer and the exosomes were co-incubated with the obtained compounds by ultrasound. The specific conditions were as follows: 1000 μg of purified exosomes were gently mixed with 1 mL of the compound at concentrations of 30 μM, 60 μM, 90 μM, and 120 μM, respectively. After incubation at 37° for 1 hour, the ultrasonic probe was immersed in the mixture and ultrasound was performed for 10 cycles (500 V, 2 KHZ, 4 s pulse / 2 s pause). The whole process was carried out in an ice bath. After the ultrasound was completed, the mixture was centrifuged in a high-speed centrifuge (10,000 × g, 10 min) to preliminarily remove the drug that was not encapsulated in the exosomes. Finally, the above mixture was added to the exosome purification column and centrifuged at low speed (750 × g, 10 min) to completely remove the excess compound to obtain the loaded exosomes.

[0009] (4): The supernatant of Dunaliella cells cultured in the laboratory was collected, ultracentrifuged in an ultrahigh-speed centrifuge, and the exosome precipitate was resuspended with phosphate buffer, and the obtained exosomes were incubated with the obtained compound in supercritical carbon dioxide. The specific conditions were as follows: 2000 μg of purified exosomes and 2 mL of the compound with a concentration of 90 μM and 120 μM were added to a 20 mL carbon dioxide supercritical reaction vessel, the carbon dioxide cylinder was opened, the air inlet valve was opened, the carbon dioxide pump was turned on, carbon dioxide was introduced, and the pressure of the reactor was observed to reach 7.2 Mpa, at this time, liquid appears inside the reaction vessel. Maintain this pressure condition and pay attention to the changes in temperature and pressure in the reactor during the reaction. After slow stirring for 0.5h, slowly release the carbon dioxide in the reactor. After the pressure is completely released, open the reactor and centrifuge the mixture in a high-speed centrifuge (10000xg, 10min) to preliminarily remove the drug that is not encapsulated into the exosomes. Finally, the above mixture is added to the exosome purification column and centrifuged at low speed (750xg, 10min) to completely remove the excess compounds to obtain the loaded exosomes.

[0010] Technical advantages: The present invention designed and obtained a novel class of urea-linked triazole structure compound molecules, which have a certain rigid structure, can inhibit the activity of IDO1, and can enhance the body's immunity; and discovered an efficient preparation method of urea-linked triazole structure through a microchannel reactor; discovered a way to load urea-linked triazole structure compounds with exosomes, which is not only efficient, but also simple and clean in preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the NMR image of the product prepared in Example 1.

[0012] Figure 2This is the NMR image of the product prepared in Example 2.

[0013] Figure 3 This is the NMR image of the product prepared in Example 4.

[0014] Figure 4 This is the NMR image of the product prepared in Example 5.

[0015] Figure 5 This is the NMR image of the product prepared in Example 7. DETAILED DESCRIPTION

[0016] The above contents of the present invention are further described in detail below through examples, but this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0017] Example 1

[0018]

[0019] In a stirred reaction flask, at 0°C, 200 mL of a dichloromethane solution containing 15 g of 4-methoxyphenylisocyanate was slowly added dropwise to 200 mL of dichloromethane containing 8.4 g of 3-amino-3-methyl-1-butyne and 10 g of triethylamine. The reaction was stirred at 0°C for 1 h. 100 mL of tert-butanol and 100 mL of water were added, and the temperature was warmed to room temperature. Then, 21 g of 2-bromobenzyl azide and 2 g of cuprous iodide were added. The reaction was continued at room temperature for 2 h. 300 mL of water was added to the reaction system, stirred, and filtered to separate the organic phase. The aqueous phase was then extracted several times with 100 mL of dichloromethane. The combined organic phases were dried over 50 g of anhydrous magnesium sulfate and concentrated. Finally, 29.01 g of the product was separated by silica gel column chromatography. 1 H NMR (600MHz, DMSO-d6) δ8.23(s,1H),7.99(s,1H),7.68(d,J=12.0Hz,1H),7.38(t,J1=12.0Hz,J2=12.0Hz,1H),7.30(t,J1=12.0Hz,J 2=12.0Hz,1H),7.23-7.20(m,2H),7.06(d,J=6.0Hz,1H),6.78(d,J=12.0Hz,2H),6.40(s,1H),5.65(s,2H),3.68(s,3H),1.64(s,6H).

[0020] Example 2

[0021]

[0022] In a stirred reaction flask, at 0°C, 200 mL of a dichloromethane solution containing 15 g of 4-methoxyphenylisocyanate was slowly added dropwise to 200 mL of dichloromethane containing 8.4 g of 3-amino-3-methyl-1-butyne and 10 g of triethylamine. The reaction was stirred at 0°C for 1 hour. 100 mL of tert-butanol and 100 mL of water were added, and the temperature was warmed to room temperature. Then, 21 g of 3-bromobenzyl azide and 2 g of cuprous iodide were added. The reaction was continued at room temperature for 2 hours. 300 mL of water was added to the reaction system, stirred, and filtered to separate the organic phase. The aqueous phase was then extracted several times with 100 mL of dichloromethane. The organic phases were combined, dried over 50 g of anhydrous magnesium sulfate, and concentrated. Finally, 24.57 g of the product was separated by silica gel column chromatography. 1 H NMR(600MHz,DMSO-d6)δ8.22(s,1H),8.05(s,1H),7.53(s,2H),7.36-7.29(m,2H),7.23- 7.20(m,2H),6.78(d,J=18.0Hz,2H),6.39(s,1H),5.57(s,2H),3.67(s,3H),1.62(s,6H).

[0023] Example 3

[0024]

[0025] A silicon carbide microchannel reactor from Chemttrix, Germany, was used to prepare a 10 mL dichloromethane solution containing 0.85 g of 3-amino-3-methyl-1-butyne, labeled A. A mixed solution of 4 mL of tert-butanol, 3 mL of dichloromethane, and 3 mL of water, containing 2.1 g of 3-bromobenzyl azide and 0.1 g of cuprous iodide, was labeled B. Solution A and solution B were respectively connected to the two feed pumps of the reactor and simultaneously entered the reactor at a rate of 1 mL / min. The reactor temperature was set to 40°C. The two reaction solutions were mixed and reacted in the reactor and then flowed through the discharge port into 12 mL of dichloromethane containing 1.5 g of 4-methoxyphenylisocyanate and 1 g of triethylamine, stirred while adding. When the solution in the microchannel reactor flowed out completely, stirring was continued for 10 min, the reaction solution was filtered, the organic phase was separated, the aqueous phase was extracted several times with 5 mL of dichloromethane, the organic phases were combined, dried over 3 g of anhydrous magnesium sulfate, and concentrated, and finally 4.16 g of product was obtained after drying. 1 H NMR(600MHz,DMSO-d6)δ8.22(s,1H),8.05(s,1H),7.53(s,2H),7.36-7.29(m,2H),7.23- 7.20(m,2H),6.78(d,J=18.0Hz,2H),6.39(s,1H),5.57(s,2H),3.67(s,3H),1.62(s,6H).

[0026] Example 4

[0027]

[0028] In a stirred reaction flask, at 0°C, 200 mL of a dichloromethane solution containing 15 g of 4-methoxyphenylisocyanate was slowly added dropwise to 8.4 g of 3-amino-3-methyl-1-butyne and 10 g of triethylamine in 200 mL of dichloromethane. The mixture was stirred at 0°C for 1 h. 100 mL of tert-butanol and 100 mL of water were added, and the temperature was raised to room temperature. Then, 17 g of 3-methoxybenzyl azide and 2 g of cuprous iodide were added. The reaction was continued at room temperature for 2 h. 300 mL of water was added to the reaction system, stirred, and filtered to separate the organic phase. The aqueous phase was extracted several times with 100 mL of dichloromethane. The organic phases were combined, dried over 50 g of anhydrous magnesium sulfate, and concentrated. Finally, 31.82 g of the product was separated by silica gel column chromatography. 1 H NMR(600MHz,DMSO-d6)δ8.23(s,1H),8.01(s,1H),7.29-7.20(m,3H),6.89-6 .77(m,5H),6.39(s,1H),5.52(s,2H),3.72(s,3H),3.68(s,3H),1.63(s,6H).

[0029] Example 5

[0030]

[0031] In a stirred reaction flask, at 0°C, 200 mL of a dichloromethane solution containing 20 g of 4-bromoisocyanatophenyl ester was slowly added dropwise to 200 mL of a solution containing 8.4 g of 3-amino-3-methyl-1-butyne and 10 g of triethylamine. The mixture was stirred at 0°C for 1 h. 100 mL of tert-butanol and 100 mL of water were added, and the temperature was raised to room temperature. 21 g of 2-bromobenzyl azide and 2 g of cuprous iodide were then added. The reaction was continued at room temperature for 2 h. 300 mL of water was added to the reaction system, and the mixture was stirred and filtered to separate the organic phase. The aqueous phase was extracted several times with 100 mL of dichloromethane. The organic phases were combined, dried over 50 g of anhydrous magnesium sulfate, and concentrated. Finally, 27.74 g of the product was separated by silica gel column chromatography. 1 H NMR(600MHz,DMSO-d6)δ8.58(s,1H),8.01(s,1H),7.68(d,J=12.0Hz,1H),7.4 0-7.28(m,6H),7.05(d,J=12.0Hz,1H),6.57(s,1H),5.65(s,2H),1.64(s,6H).

[0032] Example 6

[0033]

[0034] A silicon carbide microchannel reactor from Chemttrix, Germany, was prepared, and 10 mL of a dichloromethane solution containing 0.85 g of 3-amino-3-methyl-1-butyne was prepared, labeled A. A mixed solution of 4 mL of tert-butanol, 3 mL of dichloromethane, and 3 mL of water was prepared, labeled B. Solution A and solution B were respectively connected to the two feed pumps of the reactor and simultaneously entered the reactor at a rate of 1 mL / min. The reactor temperature was set to 40°C, and the two reaction solutions were mixed and reacted in the reactor. The mixture then flowed through the discharge port into 20 mL of dichloromethane containing 2 g of 4-bromoisocyanatophenyl ester and 1 g of triethylamine, stirred while adding, and when the solution in the microchannel reactor flowed out completely, stirring was continued for 30 min, the reaction solution was filtered, the organic phase was separated, the aqueous phase was extracted several times with 5 mL of dichloromethane, the organic phases were combined, dried over 3 g of anhydrous magnesium sulfate, and concentrated, and finally 4.59 g of product was obtained after drying. 1 H NMR(600MHz,DMSO-d6)δ8.58(s,1H),8.01(s,1H),7.68(d,J=12.0Hz,1H),7.4 0-7.28(m,6H),7.05(d,J=12.0Hz,1H),6.57(s,1H),5.65(s,2H),1.64(s,6H).

[0035] Example 7

[0036]

[0037] In a stirred reaction flask, at 0°C, 200 mL of a dichloromethane solution containing 20 g of 4-bromoisocyanatophenyl ester was slowly added dropwise to 200 mL of a solution containing 8.4 g of 3-amino-3-methyl-1-butyne and 10 g of triethylamine. The mixture was stirred at 0°C for 1 h. 100 mL of tert-butanol and 100 mL of water were added, and the temperature was raised to room temperature. 21 g of 3-bromobenzyl azide and 2 g of cuprous iodide were then added. The reaction was continued at room temperature for 2 h. 300 mL of water was added to the reaction system, and the mixture was stirred and filtered to separate the organic phase. The aqueous phase was extracted several times with 100 mL of dichloromethane. The organic phases were combined, dried over 50 g of anhydrous magnesium sulfate, and concentrated. Finally, 26.94 g of the product was separated by silica gel column chromatography. 1H NMR(600MHz,DMSO-d6)δ8.58(s,1H),8.07(s,1H),7.54-7.52(m,2H),7.46-7.43(m ,2H),7.36-7.34(m,2H),7.31-7.28(m,2H),6.57(s,1H),5.58(s,2H),1.63(s,6H).

[0038] Example 8

[0039]

[0040] A silicon carbide microchannel reactor from Chemttrix, Germany, was prepared. 10 mL of a dichloromethane solution containing 0.85 g of 3-amino-3-methyl-1-butyne was prepared, labeled A. 4 mL of tert-butanol, 3 mL of dichloromethane, and 3 mL of water were prepared, labeled B. Solution A and solution B were respectively connected to the two feed pumps of the reactor and simultaneously entered the reactor at a rate of 1 mL / min. The reactor temperature was set to 40°C. The two reaction solutions were mixed and reacted in the reactor and then flowed through the discharge port into 20 mL of dichloromethane containing 2 g of 4-bromoisocyanatophenyl ester and 1 g of triethylamine. Stirring was performed while adding. When the solution in the microchannel reactor flowed out completely, stirring was continued for 15 min. The reaction solution was filtered, the organic phase was separated, and the aqueous phase was extracted several times with 5 mL of dichloromethane. The organic phases were combined and dried over 3 g of anhydrous magnesium sulfate and concentrated. Finally, 4.68 g of product was obtained after drying. 1 H NMR(600MHz,DMSO-d6)δ8.58(s,1H),8.07(s,1H),7.54-7.52(m,2H),7.46-7.43(m ,2H),7.36-7.34(m,2H),7.31-7.28(m,2H),6.57(s,1H),5.58(s,2H),1.63(s,6H).

[0041] Example 9

[0042] Remove a viable human cervical cancer HeLa cell culture dish from the CO2 incubator and perform the following operations: perform aseptic operations next to an alcohol lamp, open the dish lid, aspirate the culture medium into a waste container, wash the culture medium in the culture bottle twice with 2 mL of PBS, digest with 0.25% trypsin, and terminate digestion when the intercellular spaces increase and the cells become small circles. Use a pipette to blow on the bottom of the culture bottle to remove the cells, transfer the resulting cell suspension to a sterile centrifuge tube, set the centrifuge to 1000 rpm, centrifuge for 3 minutes, then slowly pour out the supernatant in the centrifuge tube, add 2 to 5 mL of culture medium, and count the cells under an inverted microscope. Based on the count results, viable human cervical cancer HeLa cells in logarithmic growth phase were plated at 50,000 cells per well in a 96-well cell culture plate and cultured in RPMI1640 supplemented with 10% fetal bovine serum for 5-6 hours. 100 μL of the test compound (concentrations of 0.1 μM, 1.0 μM, 10.0 μM, 0.3 μM, 3.0 μM, and 30.0 μM) and recombinant human interferon-γ (final concentration of 100 ng / mg) diluted in culture medium were added to activate IDO1 expression in the HeLa cells. The 96-well cell culture plate was then incubated for 18 hours in a 37°C cell culture incubator containing 5% carbon dioxide. The reaction was terminated with a volume of 6.1N trichloroacetic acid and then incubated at 50°C for 30 minutes. After precipitation, the supernatant was developed with p-dimethylaminobenzaldehyde and the absorbance at 480 nm was measured using a multifunctional microplate reader. The group treated with IFNγ medium without drug was taken as 100% (At), and the group treated with 0.1% DMSO medium was taken as blank control (Ab). The absorbance under different treatment conditions was calculated according to the following formula: Absorbance % = (A-Ab) / (At-Ab), A: drug treatment + 100 ng / mL IFNγ, Ab: blank control, At: no drug, only 100 ng / mL IFNγ. IC values ​​were generated using Graph Pad Prism 8.0 software. 50 The inhibition curve of the values ​​was plotted. Preliminary IDO1 inhibition experiments revealed that some compounds have a certain inhibitory effect on IDO1, which can be further studied.

[0043] Drug Information <![CDATA[IDO1(IC 50 ,μM)]]> The compound obtained in Example 1 >10 The compound obtained in Example 2 1.73±0.97 Compound obtained in Example 4 5.82±2.65 Compound obtained in Example 5 >10 Compound obtained in Example 7 >10

[0044] Example 10

[0045] In order to better enable the compound to enter the cell, we tried to use exosomes for loading. Exosomes are a kind of vesicles secreted by cells. They can easily penetrate the cell membrane and enter the cell, and are excellent drug carriers. The supernatant of Dunaliella cells cultured in the laboratory was collected, ultracentrifuged by ultracentrifugation, and the exosome precipitate was resuspended with phosphate buffer. The obtained exosomes were ultrasonically co-incubated with the compound obtained in Example 2. The specific conditions were as follows: 1000 μg of purified exosomes were gently mixed with 1 mL of the compound at concentrations of 30 μM, 60 μM, 90 μM, and 120 μM, respectively. After incubation at 37° on a constant temperature shaker for 1 hour, the ultrasonic probe was immersed in the mixed solution, and the ultrasound was divided into 10 cycles (500 V, 2 KHZ, 4s pulse / 2s The whole process was carried out on an ice bath. After the ultrasonication, the mixture was centrifuged in a high-speed centrifuge (10,000 × g, 10 min) to preliminarily remove the drug that was not encapsulated into the exosomes. Finally, the above mixture was added to the exosome purification column and centrifuged at low speed (750 × g, 10 min) to completely remove the excess compounds. The exosomes were added to 1 mL of PBS and stirred evenly. After 100 μL was aspirated, the absorbance was measured at 300 nm using a microplate reader.

[0046] 1 μg / μL 0.5 μg / μL 0.25 μg / μL 0.125 μg / μL 0.0625 μg / μL Duplicate hole 1 0.599 0.5 0.416 0.393 0.354 Duplicate hole 2 0.518 0.481 0.425 0.315 0.366

[0047] After the compound obtained in Example 2 was loaded onto exosomes, the absorbance was found to be higher than before loading, reaching a maximum at 90 μM. Continuing to increase the compound concentration, the loading amount was not obvious, indicating that the maximum loading concentration under this condition was 90 μM using this method.

[0048]

[0049]

[0050] Example 11

[0051] The supernatant of the laboratory-cultured Dunaliella cells was collected and ultracentrifuged in an ultracentrifuge. The exosome precipitate was resuspended in phosphate buffer, and the obtained exosomes were incubated with supercritical carbon dioxide with the compound obtained in Example 2. The specific conditions were as follows: 2000 μg of purified exosomes and 2 mL of the compound with a concentration of 90 μM and 120 μM were added to a 20 mL supercritical carbon dioxide reaction vessel. The carbon dioxide cylinder was opened, the air inlet valve was opened, the carbon dioxide pump was turned on, and carbon dioxide was introduced. When the pressure of the reactor reached 7.2 MPa, liquid appeared inside the reaction vessel. The pressure condition was maintained, and the temperature and pressure in the reactor were carefully observed during the reaction. After slow stirring for 0.5 h, the carbon dioxide in the reactor was slowly released. After the pressure was completely released, the reactor was opened and the mixture was centrifuged in a high-speed centrifuge (10,000 × g, 10 min) to preliminarily remove the drugs not encapsulated in the exosomes. Finally, the above mixture was added to the exosome purification column and centrifuged at low speed (750 × g, 10 min) to completely remove the excess compounds. The exosomes were added to 1 mL of PBS and stirred evenly. After absorbing 100 μL, the absorbance was measured at 300 nm using a microplate reader. The absorbances of the duplicate wells at a concentration of 90 μM were 1.242 and 1.164, respectively, and the absorbances of the duplicate wells at a concentration of 120 μM were 1.317 and 1.378, respectively.

[0052] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing urea-loaded Dunaliella exosomes, characterized in that: The specific steps are as follows: collecting the supernatant of laboratory-cultured Dunaliella cells, ultracentrifuging them in an ultrahigh-speed centrifuge, and resuspending the exosome precipitate in phosphate buffer. The obtained exosomes are then ultrasonically incubated with urea compounds. The specific conditions are as follows: 1000 μg of purified exosomes are gently mixed with 1 mL of compounds at concentrations of 30 μM, 60 μM, 90 μM, and 120 μM, respectively. After incubation at 37° on a constant temperature shaker for 1 hour, the ultrasonic probe is immersed in the mixture, and the ultrasound is divided into 10 cycles. The entire process is carried out on an ice bath. After the ultrasound is completed, the mixture is centrifuged in a high-speed centrifuge at 10,000×g for 10 minutes to preliminarily remove the drug that is not encapsulated into the exosomes. Finally, the above mixture is added to the exosome purification column and centrifuged at 750×g for 10 minutes to completely remove the excess compound to obtain the loaded exosomes. The molecular structure of the urea compound is: .

2. Use of the urea compound-loaded Dunaliella exosomes obtained by the preparation method according to claim 1 in preparing a product that inhibits IDO1 enzyme activity.

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