Drug delivery system based on dendritic cell exosome and preparation method and application thereof

Abstract

The invention relates to a drug delivery system based on dendritic cell exosomes as well as a preparation method and application of the drug delivery system. The drug delivery system comprises a dendritic cell-derived exosome, an indoleamine 2, 3-dioxygenase inhibitor wrapped in the exosome, and a death receptor monoclonal antibody modified on the surface of the exosome. The death receptor monoclonal antibody is connected with a coupling agent to modify the surface of the exosome. The drug delivery system carries the indoleamine 2, 3-dioxygenase inhibitor to inhibit the conversion of tryptophan and relieve the malignant melanoma immunosuppression microenvironment, and specifically targets a death receptor on the surface of a malignant melanoma cell by loading the death receptor monoclonal antibody, so that the targeting property of the delivery system is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a drug delivery system based on dendritic cell exosomes, and a preparation method and application thereof. Background Art

[0002] Malignant melanoma (MM) is a malignant tumor originating from melanocytes. MM accounts for approximately 90% of all skin cancer mortality and is one of the most aggressive and metastatic malignancies. It can metastasize from a relatively small primary lesion to multiple sites, including the lungs, liver, brain, lymph nodes, and bones. The five-year survival rate for patients with metastatic MM is only approximately 10%.

[0003] In recent years, immunotherapy and targeted therapy have made significant progress and are becoming important treatment options for multiple myeloma. Immunotherapy activates or enhances the patient's own immune system to identify and attack cancer cells, while targeted therapy precisely targets specific molecules or genetic mutations within cancer cells. Both approaches have demonstrated promising clinical efficacy, providing new treatment options for patients.

[0004] Studies have found that one of the immune escape mechanisms of MM is through a metabolic pathway mediated by indoleamine 2,3-dioxygenase (IDO). IDO catalyzes the conversion of tryptophan to kynurenine, directly inhibiting T cell function while enhancing the immunosuppressive effects of Treg cells, thereby forming an immunosuppressive microenvironment.

[0005] Previous studies have shown that IDO inhibitors such as Indoximod, Epacadostat, and NLG919 have significant anti-tumor effects. However, several clinical trials have failed to achieve the expected therapeutic effects. This may be related to the difficulty in maintaining effective local drug concentrations in the tumor due to their oral administration method. Although IDO inhibitors have low toxicity, they may still cause immune-related adverse reactions, such as fatigue and gastrointestinal discomfort.

[0006] Therefore, developing more effective drug delivery systems to improve the therapeutic efficacy and targeting ability of IDO inhibitors has become a technical problem that needs to be solved urgently. Summary of the Invention

[0007] To address the shortcomings of the existing technology, the present invention aims to provide a dendritic cell exosome-based drug delivery system, its preparation method, and its application. This drug delivery system incorporates an IDO inhibitor to inhibit tryptophan conversion, thereby relieving the immunosuppressive microenvironment of multiple myeloma (MM). Furthermore, the system is loaded with a death receptor monoclonal antibody to specifically target death receptors on the surface of MM cells, enhancing the targeting of the delivery system.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a dendritic cell exosome-based drug delivery system, which comprises dendritic cell-derived exosomes, an indoleamine 2,3-dioxygenase inhibitor encapsulated in the exosomes, and a death receptor monoclonal antibody modified on the surface of the exosomes.

[0010] The death receptor monoclonal antibody is modified on the surface of the exosomes by connecting with a coupling agent.

[0011] Exosomes derived from dendritic cells (DCs) retain the molecular characteristics of DCs, containing the genetic information, functional proteins, and lipid components of the source cells. This unique composition not only makes exosomes promising drug delivery vehicles, but also inherits the immunomodulatory functions of DCs. Death receptors are highly expressed on the surface of MM cells and can specifically bind to tumor necrosis factor-related apoptosis-inducing ligands to transmit apoptotic signals, inducing apoptosis without significant toxicity to normal cells.

[0012] This invention uses dendritic cell-derived exosomes as nano-delivery vehicles to activate the specific immune response of cytotoxic T lymphocytes, thereby enhancing the body's immune system's ability to recognize and eliminate tumor cells. Furthermore, the exosomes encapsulate an indoleamine 2,3-dioxygenase inhibitor (IDO inhibitor) to inhibit the conversion of tryptophan, blocking the immunosuppressive pathway from tryptophan to kynurenine, thereby relieving the MM immunosuppressive microenvironment. Simultaneously, by loading the exosomes with death receptor monoclonal antibodies to specifically target death receptors on the surface of MM cells, the targeted delivery is enhanced, thus constructing a novel drug delivery system that activates and reverses the tumor immunosuppressive microenvironment through immune response.

[0013] Preferably, the indoleamine 2,3-dioxygenase inhibitor comprises NLG919.

[0014] Preferably, the death receptor monoclonal antibody comprises a death receptor 5 monoclonal antibody (DR5mAb).

[0015] Preferably, the mass ratio of the exosomes, the indoleamine 2,3-dioxygenase inhibitor and the death receptor monoclonal antibody is 1:(0.2-2):(2-10).

[0016] Among them, the specific point values in 0.2-2 can be selected from 0.2, 0.5, 0.8, 1.1, 1.4, 1.7, 2, etc., and the specific point values in 2-10 can be selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0017] The present invention controls the mass ratio of exosomes, indoleamine 2,3-dioxygenase inhibitors and death receptor monoclonal antibodies to 1:(0.2-2):(2-10), thereby increasing the encapsulation efficiency and drug loading of indoleamine 2,3-dioxygenase inhibitors by exosomes, thereby improving the therapeutic effect of the delivery system.

[0018] Preferably, the coupling agent comprises phospholipid-polyethylene glycol-maleimide.

[0019] Preferably, the phospholipid comprises distearoylphosphatidylethanolamine.

[0020] Preferably, the molecular weight of the polyethylene glycol is 1000-4000 Da, for example, 1000 Da, 2000 Da, 3000 Da, 4000 Da, etc.

[0021] In a second aspect, the present invention provides a method for preparing the drug delivery system according to the first aspect, the preparation method comprising the following steps:

[0022] (1) Exosomes derived from dendritic cells and an indoleamine 2,3-dioxygenase inhibitor are mixed and incubated to obtain exosomes encapsulating the indoleamine 2,3-dioxygenase inhibitor; and a death receptor monoclonal antibody and a coupling agent are mixed and reacted to obtain a monoclonal antibody modified with the coupling agent.

[0023] (2) The exosomes encapsulating the indoleamine 2,3-dioxygenase inhibitor are mixed and incubated with the monoclonal antibody modified with the coupling agent to obtain the drug delivery system.

[0024] Preferably, the method for preparing dendritic cell-derived exosomes in step (1) comprises the following steps:

[0025] (I) The supernatant of the dendritic cell suspension is collected and concentrated by ultrafiltration to obtain a concentrate.

[0026] (II) The concentrated solution is mixed with an exosome extraction reagent, centrifuged, and the precipitate is collected to obtain dendritic cell-derived exosomes.

[0027] Preferably, the temperature of the mixed incubation in step (1) is 30-45° C., and the time of the mixed incubation is 1-3 h.

[0028] Preferably, the mass ratio of the death receptor monoclonal antibody to the coupling agent in step (1) is 1:(5-20).

[0029] Preferably, the temperature of the mixing reaction in step (1) is 30-45° C., and the mixing reaction time is 1-3 h.

[0030] Among them, the specific point values in 30-45℃ can be selected from 30℃, 35℃, 40℃, 45℃, etc., the specific point values in 1-3h can be selected from 1h, 1.5h, 2h, 2.5h, 3h, etc., and the specific point values in 1:(5-20) can be selected from 1:5, 1:10, 1:15, 1:20, etc.

[0031] Preferably, the temperature of the mixed incubation in step (2) is 30-45° C., and the time of the mixed incubation is 1-3 h.

[0032] Among them, the specific point values in 30-45℃ can be selected from 30℃, 35℃, 40℃, 45℃, etc., and the specific point values in 1-3h can be selected from 1h, 1.5h, 2h, 2.5h, 3h, etc.

[0033] In a third aspect, the present invention provides a use of the drug delivery system as described in the first aspect in preparing a drug for treating malignant tumors.

[0034] Preferably, the malignant tumor comprises malignant melanoma.

[0035] In a fourth aspect, the present invention provides a use of the drug delivery system as described in the first aspect in the preparation of a tumor cell proliferation inhibitor.

[0036] Preferably, the malignant tumor comprises malignant melanoma.

[0037] The numerical range described in the present invention includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] In the dendritic cell exosome-based drug delivery system constructed by the present invention, the dendritic cell-derived exosomes retain the molecular characteristics of DC cells, and contain the genetic information, functional proteins and lipid components of the source cells. This unique composition makes the dendritic cell-derived exosomes not only have the potential to serve as drug carriers, but also inherit the immunomodulatory function of DC cells, by activating the specific immune response of cytotoxic T lymphocytes, thereby enhancing the body's immune system's ability to recognize and eliminate tumor cells.

[0040] Furthermore, the exosomes described in this invention inhibit the conversion of tryptophan by encapsulating an indoleamine 2,3-dioxygenase inhibitor (IDO inhibitor), blocking the immunosuppressive pathway from tryptophan to kynurenine, thereby relieving the immunosuppressive microenvironment of MM. Simultaneously, the exosomes are loaded with a death receptor monoclonal antibody to specifically target the death receptors on the surface of MM cells, enhancing the targeted delivery.

[0041] Furthermore, the present invention controls the mass ratio of exosomes, indoleamine 2,3-dioxygenase inhibitors and death receptor monoclonal antibodies to 1:(0.2-2):(2-10), which can improve the encapsulation efficiency and drug loading capacity of exosomes for indoleamine 2,3-dioxygenase inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the standard curve of exosome concentration.

[0043] Figure 2 These are the particle size and potential characterization diagrams of the exosomes and drug delivery system in Verification Example 1, where A is the particle size distribution diagram of mDexo, B is the Zeta potential diagram of mDexo, C is the particle size distribution diagram of DR5mAb-mDexo-NLG919, and D is the Zeta potential diagram of DR5mAb-mDexo-NLG919.

[0044] Figure 3 This is a stability test chart of DR5mAb-mDexo-NLG919 in Verification Example 1.

[0045] Figure 4 This is a transmission electron microscopy characterization image of exosomes in Verification Example 2.

[0046] Figure 5 This is the result of characterizing the characteristic proteins of exosomes in Verification Example 2.

[0047] Figure 6 This is a diagram showing the coupling verification results of exosomes and DR5mAb in Verification Example 4.

[0048] Figure 7 This is a test chart of the killing ability of DR5mAb-mDexo-NLG919 on A375 cells in Test Example 1.

[0049] Figure 8 This is a test chart of the killing ability of different experimental groups on A375 cells in Test Example 1.

[0050] Figure 9 This is a confocal microscopy image of the drug delivery system for verifying the uptake ability of A375 cells in Test Example 2.

[0051] Figure 10 This is a flow cytometry verification diagram of the drug delivery system uptake capability verification of A375 cells in Test Example 2.

[0052] Figure 11 This is a graph showing the apoptosis ability test of A375 cells by different experimental groups in Test Example 3.

[0053] Figure 12This is a test chart of the IDO inhibition ability of different experimental groups in Test Example 4. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0055] The exosome concentration involved in the following specific implementation method was determined using a BCA kit, and the specific steps are as follows:

[0056] Add an appropriate amount of exosome solution to a certain amount of PMSF, mix thoroughly, then add RIPA lysis buffer and lyse thoroughly on ice for 30 minutes. After lysis, centrifuge the mixture at 12,000 rpm at 4°C for 10 minutes. Discard the precipitate and aspirate the supernatant (exosome supernatant solution) for later use. Take 20 μL of a 25 mg / mL protein standard solution and add 980 μL of PBS solution to dilute the protein standard to a concentration of 0.5 mg / mL.

[0057] The protein standard was added to a 96-well plate at 0, 1, 2, 4, 8, 12, 16, and 20 μL, and PBS solution was added to make up the volume of each well to 20 μL. 200 μL of BCA working solution (BCA reagent A solution: BCA reagent B solution = 50:1) was added to each well, incubated in a 37°C incubator for 30 minutes, and the absorbance value was measured by a microplate reader. A standard curve was drawn with protein concentration as the horizontal axis and absorbance as the vertical axis. The results are shown in Figure 2. Figure 1 shown.

[0058] A certain amount of exosome supernatant solution was diluted appropriately and added to each well of a 96-well plate. 20 μL of the solution was then added to each well of the plate. 200 μL of BCA working solution was then added to each well. The plates were incubated at 37°C for 30 minutes. The absorbance was measured using a microplate reader, and the exosome protein concentration was calculated using the standard curve.

[0059] Preparation Example 1

[0060] This preparation example provides exosomes derived from dendritic cells, and the preparation method is as follows:

[0061] (1) Induction of maturation of dendritic cells:

[0062] Dendritic cells were cultured in RPMI 1640 medium. When the cells grew to about 50% confluence, the medium was discarded, and the cells were washed three times with PBS. RPMI 1640 medium containing 10 μg / mL lipopolysaccharide was added and cultured for a further 48 h.

[0063] (2) Extraction and separation of exosomes from dendritic cells:

[0064] (2.1) Aseptically collect DC cell supernatant after 48 h of culture. Centrifuge the collected supernatant at 4°C, 4000g for 30 min to remove large cell debris. Collect the supernatant after centrifugation. Filter the supernatant through a 0.22 μm pore size sterile filter membrane to remove large extracellular vesicles and cell debris. Transfer the filtered supernatant to a 100 kDa ultrafiltration tube and ultrafiltration and centrifugation at 4°C, 4000g for 30 min to concentrate the sample. Add PBS buffer to the concentrate and ultrafiltration and centrifuge to clarify the concentrate.

[0065] (2.2) Add the exosome extraction reagent (SBI product number 220906-002) to the concentrate at a volume ratio of 5:1. Mix thoroughly and let stand overnight at 4°C. Centrifuge the mixture at 1500g for 30 minutes at 4°C. Remove the supernatant and add sterile water for injection to mix thoroughly. Centrifuge the mixture at 1500g for 30 minutes at 4°C. Remove the supernatant. The white precipitate at the bottom is mature dendritic cell-derived exosomes (hereinafter referred to as mDexo). Add an appropriate amount of PBS buffer solution to mix thoroughly and store at -80°C until ready for use.

[0066] Example 1

[0067] This embodiment provides a drug delivery system based on dendritic cell exosomes, and the preparation method thereof is as follows:

[0068] (1) Preparation of exosomes encapsulating NLG919:

[0069] Weigh 10 mg of NLG919 and place it in a volumetric flask. Add 100 μL of DMSO solution to prepare a 100 mg / mL NLG919 stock solution for use. The NLG919 stock solution was aspirated and diluted with PBS. The exosome solution obtained in Preparation Example 1 and the NLG919 stock solution were mixed at a mass ratio of 2:1 between the exosomes and NLG919. After mixing evenly, the mixture was incubated for 2 hours at 37 ° C and 200 rpm in an air bath constant temperature oscillator. After incubation, the mixture was placed in a 10 kDa ultrafiltration tube and centrifuged at 4 ° C and 3000 g for 30 minutes to remove NLG919 that was not encapsulated into the exosomes, thereby preparing exosomes encapsulating NLG919 (hereinafter referred to as mDexo-NLG919).

[0070] (2) Preparation of drug delivery system based on dendritic cell exosomes:

[0071] Accurately weigh 10 mg of DSPE-PEG-Mal into a 10 mL volumetric flask. Add PBS to the mark and mix thoroughly to prepare a 1 mg / mL DSPE-PEG-Mal solution. Add 10.6 μL of a 9.44 mg / mL solution of death receptor 5 monoclonal antibody (DR5mAb) and 1 mL of DSPE-PEG-Mal solution to the flask and mix thoroughly. Stir on a magnetic stirrer for 2 hours to allow the free sulfhydryl groups in DR5mAb to react with the maleimide groups in DSPE-PEG-Mal to form stable thioether bonds. After the reaction, transfer the mixture to a 10 kDa ultrafiltration tube and centrifuge at 3000 g for 30 minutes at 4°C to remove free, unreacted DSPE-PEG-Mal. Collect the resulting solution to obtain DSPE-PEG-Mal-DR5mAb micelles. DSPE-PEG-Mal-DR5mAb micelles were mixed with the mDexo-NLG919 solution obtained in step (1) at a mass ratio of 1:3 between exosomes and DR5mAb, and incubated on a magnetic stirrer for 1 h to allow the DSPE end to insert into the bilayer phospholipid membrane of the exosomes. After the reaction, a drug delivery system based on dendritic cell exosomes (hereinafter referred to as DR5mAb-mDexo-NLG919) was obtained.

[0072] Comparative Example 1

[0073] This comparative example provides an exosome modified with DR5mAb, which differs from Example 1 in that the NLG919 encapsulation treatment in step (1) is not performed, and the mass of the mDexo-NLG919 solution obtained in step (1) in step (2) is replaced with the mDexo obtained in Preparation Example 1. The remaining steps are consistent with Example 1 to prepare exosomes modified with DR5mAb (hereinafter referred to as DR5mAb-mDexo).

[0074] Verification Example 1

[0075] Particle size and potentiometric analysis of exosomes and drug delivery systems:

[0076] (1) The exosome solution mDexo obtained in Preparation Example 1 and the DR5mAb-mDexo-NLG919 obtained in Example 1 were respectively diluted to 2 mL with PBS solution, filtered with a 0.22 μm filter membrane, placed in a cuvette, and the particle size and potential of the exosomes were measured using a nanoparticle size analyzer and a Zeta potential analyzer. The measurement was repeated three times. The results were as follows: Figure 2Figure 1 shows the particle size distribution of mDexo (A), the zeta potential of mDexo (B), the zeta potential of DR5mAb-mDexo-NLG919 (C), and the zeta potential of DR5mAb-mDexo-NLG919 (D). The average particle size of mDexo is 77.74 ± 2.1 nm, the polydispersity index (PDI) is 0.259 ± 0.017, and the average zeta potential is -9.4 ± 1.3 mV. The particle size of DR5mAb-mDexo-NLG919 is approximately 129.14 ± 2.5 nm, with a uniform particle size distribution and a zeta potential of -6.17 ± 0.21 mV.

[0077] (2) Investigation of particle size and potential stability:

[0078] The DR5mAb-mDexo-NLG919 obtained in Example 1 was stored in PBS at 4°C for 7 days, and its stability was analyzed by measuring the particle size and potential within 7 days. The results are as follows: Figure 3 As shown, the particle size and potential results showed that the particle size and potential of the drug delivery system in PBS did not change significantly, and no particle aggregation and precipitation occurred, reflecting good storage stability and application stability.

[0079] Verification Example 2

[0080] Characterization of exosome morphology and characteristic proteins:

[0081] The morphology of the exosomes obtained in Preparation Example 1 was characterized using transmission electron microscopy (TEM). Figure 4 As shown, it can be seen that the separated mDexo presents a spherical or saucer-like structure, which is consistent with the typical structural characteristics of exosomes.

[0082] The expression of characteristic proteins CD9, TSG101, and CD81 in the exosomes obtained in Preparation Example 1 was verified by Western blotting. The results are as follows: Figure 5 As shown, the exosomes contained the expression of exosome characteristic proteins CD9, TSG101, and CD81, proving that the exosomes were successfully extracted.

[0083] Verification Example 3

[0084] Investigation of encapsulation efficiency and drug loading of exosomes encapsulating NLG919:

[0085] Referring to the method in step (1) of Example 1, the exosome solution obtained in Preparation Example 1 and the NLG919 stock solution were mixed in accordance with the mass ratio of exosomes to NLG919 of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, and 1:2, respectively. The mixture was incubated in an air bath constant temperature oscillator at 37 ° C and 200 rpm for 2 h, and the encapsulation efficiency and drug loading of NLG919 were determined by HPLC. The test results of the encapsulation efficiency and drug loading are shown in Table 1.

[0086] Table 1

[0087] Exosomes:NLG919(w / w) Encapsulation efficiency / % Drug loading% 10:1 33.85±0.39 3.27±0.04 8:1 32.49±0.40 3.90±0.04 6:1 37.85±0.51 5.93±0.08 4:1 52.39±1.53 11.58±0.30 2:1 54.27±0.72 21.34±0.23 1:1 49.74±1.26 33.22±0.56 1:2 41.73±0.82 45.49±0.49

[0088] The data in Table 1 show that when the mass ratio of exosomes to NLG919 ranges from 10:1 to 2:1, the encapsulation efficiency and drug loading gradually increase with increasing drug dosage. When the mass ratio of exosomes to NLG919 is 2:1, the encapsulation efficiency of NLG919 reaches its highest, at 54.27±0.72%. However, as the drug dosage continues to increase, the encapsulation efficiency decreases rapidly. This may be because the increase in drug concentration exceeds the carrying capacity of the exosome membrane, or that high drug concentration reduces the fluidity of the exosome membrane, resulting in a decrease in the encapsulation efficiency and more drug adherence to the exosome membrane.

[0089] Verification Example 4

[0090] Conjugation validation of exosomes and DR5mAb:

[0091] (1) The exosomes obtained in Preparation Example 1 were stained with the lipophilic dye Dil. Dil dye was added at a volume ratio of 1:10 to the exosomes, mixed evenly, and stirred in the dark on a magnetic stirrer at room temperature for 30 minutes. After incubation, the mixture was placed in a 10 kDa ultrafiltration tube and centrifuged at 3000 g for 30 minutes at 4°C to remove excess dye. Using the same method, DSPE-PEG-Mal-DR5mAb was labeled with FITC to obtain FITC-labeled DSPE-PEG-Mal-DR5mAb.

[0092] (2) Referring to the method in Example 1, FITC-labeled DSPE-PEG-Mal-DR5mAb and Dil-labeled mDexo-NLG919 were mixed and incubated, and the results were observed under a confocal microscope. Figure 6 As shown in the figure, FITC-labeled DSPE-PEG-Mal-DR5mAb showed green fluorescence, and Dil-labeled mDexo-NLG919 showed red fluorescence. Co-localization of red and green fluorescence was observed, indicating that the exosomes were successfully coupled to DR5mAb.

[0093] Test Example 1

[0094] The cell killing ability of DR5mAb-mDexo-NLG919 prepared in Example 1 was tested:

[0095] (1) Study on the toxicity of NLG919 to A375 cells:

[0096] The IC value of NLG919 on the inhibition of A375 cell proliferation was investigated by MTT assay. 50 The specific experimental steps are as follows:

[0097] (1.1) Experimental grouping method:

[0098] ① Experimental group: RPMI 1640 medium containing a series of concentration gradients of NLG919 (9.375 μg / mL, 18.75 μg / mL, 37.5 μg / mL, 75 μg / mL, 150 μg / mL, and 300 μg / mL, respectively);

[0099] ②Control group: Add the same volume of culture medium without NLG919 as that of the experimental group.

[0100] (1.2) Experimental methods:

[0101] Human malignant melanoma A375 cells were harvested, the culture medium discarded, and the cells were washed three times with PBS solution. An appropriate amount of trypsin was added for digestion, and the cells were centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in culture medium by pipetting to a cell suspension. The cells were counted and the concentration was adjusted to 5×10 4 / mL, plate a 96-well plate at a density of 5000 cells per well, add 100 μL per well, and place the culture plate in a 37°C, 5% CO2 incubator for overnight culture. Discard the original culture medium, add the culture medium of each experimental group and control group respectively, and place the culture plate in a 37°C, 5% CO2 incubator for further culture for 48 hours. Take out the culture plate, discard the medium containing the drug, add 20 μL of MTT solution to each well, and place the culture plate in a 37°C incubator for further incubation for 4 hours. After the incubation is completed, take out the culture plate, discard the MTT solution, add 150 μL of DMSO solution to each well, and place the culture plate in a shaker for incubation for 10 minutes. Use a microplate reader to measure the OD value at a wavelength of 570 nm to calculate the cell viability. The calculation formula is as follows:

[0102] Cell survival rate = (A a / A b )×100%; Where: A a is the OD value of the experimental group, A b is the OD value of the control group.

[0103] IC of NLG919 on the inhibition of A375 cell proliferation50 The result is as follows Figure 7 As shown in the figure, NLG919 has a significant inhibitory effect on the proliferation of A375 cells, and the inhibitory effect is concentration-dependent. According to the calculation, the IC 50 The value is 155.5μg / mL.

[0104] (2) Investigation of the toxicity of drug delivery system to A375 cells:

[0105] (2.1) Experimental grouping method:

[0106] ①NLG919 group: RPMI 1640 medium containing NLG919 at a series of concentration gradients (0.15 μg / mL, 0.3 μg / mL, 0.6 μg / mL, 1.2 μg / mL, 2.4 μg / mL, and 4.8 μg / mL, respectively);

[0107] ②mDexo group: RPMI 1640 medium containing a series of concentration gradients of mDexo prepared in Preparation Example 1 (0.3 μg / mL, 0.6 μg / mL, 1.2 μg / mL, 2.4 μg / mL, 4.8 μg / mL, and 9.6 μg / mL, respectively);

[0108] ③DR5mAb group: RPMI 1640 medium containing a series of concentration gradients of DR5mAb (0.9 μg / mL, 1.8 μg / mL, 3.6 μg / mL, 7.2 μg / mL, 14.4 μg / mL, and 28.8 μg / mL);

[0109] ④ DR5mAb-mDexo group: RPMI 1640 medium containing a series of concentration gradients of DR5mAb-mDexo prepared in Comparative Example 1 (the DR5mAb contents in DR5mAb-mDexo were 0.9 μg / mL, 1.8 μg / mL, 3.6 μg / mL, 7.2 g / mL, 14.4 μg / mL, and 28.8 μg / mL, and the mDexo contents were 0.3 μg / mL, 0.6 μg / mL, 1.2 μg / mL, 2.4 μg / mL, 4.8 μg / mL, and 9.6 μg / mL, respectively);

[0110] ⑤mDexo-NLG919 group: a series of concentration gradients containing RPMI 1640 culture medium containing mDexo-NLG919 prepared in step (1) of Example 1 (the mDexo contents in mDexo-NLG919 were 0.3 μg / mL, 0.6 μg / mL, 1.2 μg / mL, 2.4 μg / mL, 4.8 μg / mL, and 9.6 μg / mL, and the NLG919 contents were 0.15 μg / mL, 0.3 μg / mL, 0.6 μg / mL, 1.2 μg / mL, 2.4 μg / mL, and 4.8 μg / mL, respectively).

[0111] ⑥DR5mAb-mDexo-NLG919 group: a series of concentration gradients containing DR5mAb-mDexo-NLG919 prepared in Example 1 (the mDexo contents in DR5mAb-mDexo-NLG919 were 0.3 μg / mL, 0.6 μg / mL, 1.2 μg / mL, 2.4 μg / mL, 4.8 μg / mL, and 9.6 μg / mL, respectively; the NLG919 contents were 0.15 μg / mL, 0.3 μg / mL, 0.6 μg / mL, 1.2 μg / mL, 2.4 μg / mL, and 4.8 μg / mL, respectively; and the DR5mAb contents were 0.9 μg / mL, 1.8 μg / mL, 3.6 μg / mL, 7.2 g / mL, 14.4 μg / mL, and 28.8 μg / mL, respectively).

[0112] ⑦Control group: Add the same volume of RPMI 1640 medium without drug as that of groups ①-⑥.

[0113] (2.2) Experimental methods:

[0114] The experimental method refers to step (1.2), and the culture medium of each experimental group and control group in step (1.2) is replaced with the culture medium of the above groups ①-⑦, and the cell survival rate of each experimental group is tested after the experiment.

[0115] The cell survival rates of each experimental group are shown in Figure 8 As shown by Figure 8The results show that the blank mDexo (mDexo group) has no obvious inhibitory effect on A375 cells, indicating that the activation of the immune function of mDexo alone has limited killing effect on tumor cells. In addition, it can be seen from the figure that each preparation group showed a stronger inhibitory effect than the free NLG919 group. At the same concentration, compared with the mDexo-NLG919 group and the DR5mAb-mDexo group, the cell survival rate of the DR5mAb-mDexo-NLG919 group was lower, and the proliferation of A375 cells was better inhibited. The inhibitory effect showed obvious concentration dependence, indicating that the multimodal immunotherapy of DR5mAb-mDexo-NLG919 has strong tumor cell toxicity and anti-tumor cell proliferation effects.

[0116] Test Example 2

[0117] This test example tests the drug delivery system uptake capacity of A375 cells:

[0118] (1) Experimental groups:

[0119] ① DR5mAb-mDexo-NLG919 group: Referring to the method of Verification Example 4, the DR5mAb-mDexo-NLG919 prepared in Example 1 was fluorescently labeled with DiI.

[0120] ②DR5mAb + DR5mAb-mDexo-NLG919 group: Referring to the method of Verification Example 4, the DR5mAb-mDexo-NLG919 prepared in Example 1 was fluorescently labeled with DiI and mixed with DR5mAb (the concentration ratio of DR5mAb and DR5mAb in DR5mAb-mDexo-NLG919 was controlled to be 2:1).

[0121] (2) Experimental methods:

[0122] A375 cells were obtained, digested with trypsin, and centrifuged to prepare a 1×10 5 Use a pipette to draw an appropriate amount of PBS solution into a 6-well plate to completely wet the 6-well plate. Place a pre-sterilized cell slide into the 6-well plate to ensure that the slide fits tightly against the bottom of the 6-well plate. 5 The cells were plated in a 6-well plate at a density of 100 μl, with 2 mL per well. The culture plate was gently shaken to disperse the cells evenly, and then placed in a 37°C, 5% CO2 incubator for overnight culture.

[0123] (3) Test indicators

[0124] (3.1) Confocal experiment investigation:

[0125] Remove the cell culture plates, discard the culture medium, and sequentially add 2 mL of each of the above preparations for each experimental group. Incubate the plates for Groups 1 and 2 in a 37°C, 5% CO2 incubator for 0.5, 2, 4, and 8 hours. After incubation, discard the culture medium, rinse three times with appropriate amounts of PBS, and add 2 mL of 4% paraformaldehyde solution to each well. Fix the plates at room temperature for 20 minutes. Discard the fixative, rinse three times with PBS, and add 0.1 mL of DAPI dye to each well to stain the cell nuclei. Incubate at room temperature in the dark for 5 minutes. Discard the dye, rinse three times with PBS, and gently remove the cell slides with forceps, place them on glass slides, and mount the slides with nail polish. Observe and record the uptake of DR5mAb-mDexo-NLG919 by A375 cells under confocal microscopy.

[0126] The results are as follows Figure 9 As shown in the figure, the blue is the DAPI-labeled cell nucleus, the red is the Dil-labeled mDexo, and the overlay is the superposition of blue and red fluorescence. The results showed that different degrees of red fluorescence could be observed at different times. At 8 hours, the red fluorescence was the strongest, indicating that the most DR5mAb-mDexo-NLG919 entered the cells, and compared with the DR5mAb+DR5mAb-mDexo-NLG919 group, the DR5mAb-mDexo-NLG919 group was able to be taken up by cells more. It is speculated that the binding of DR5mAb-mDexo-NLG919 to A375 cells may be competitively inhibited by free DR5mAb, indicating that after modification with DR5mAb, DR5mAb-mDexo-NLG919 can specifically bind to the DR5 receptor highly expressed on the surface of A375 cells, thereby making the drug delivery system DR5mAb-mDexo-NLG919 more easily taken up by target cells.

[0127] (3.2) Flow cytometry investigation:

[0128] The cell culture plates were removed, the culture medium discarded, and 2 mL of the preparations from Groups 1 and 2 were added, respectively, and incubation continued. Blank cells (A375 cells without the drug preparation) served as the control group. The plates for Groups 1, 2, and the control group were placed in a 37°C, 5% CO2 incubator and incubated for an additional 8 h. After incubation, the culture medium was discarded, the cells were washed twice with PBS, and 100 μL of trypsin was added to each well. After digestion, the cell suspension was collected and centrifuged at 1000 rpm at 4°C for 5 min. The supernatant was discarded, the cells were washed twice with an appropriate amount of PBS, and the cells were resuspended in 500 μL of PBS. The cells were placed on ice and assayed for DR5mAb-mDexo-NLG919 uptake by A375 cells using flow cytometry.

[0129] Flow cytometry test results and statistical results are as follows Figure 10As shown in the figure, compared with the control group, the fluorescence of A375 cells treated with DR5mAb-mDexo-NLG919 in the histogram shifted significantly to the right, demonstrating that DR5mAb-mDexo-NLG919 can be effectively taken up by A375 cells. In DR5mAb-mDexo-NLG919 containing the same DR5mAb concentration, a competitive analysis was performed using free DR5mAb (i.e., DR5mAb+DR5mAb-mDexo-NLG919 group). The results showed that compared with DR5mAb-mDexo-NLG919, the fluorescence shift to the right in the histogram of cells treated with DR5mAb+DR5mAb-mDexo-NLG919 was smaller, indicating that the binding of DR5mAb-mDexo-NLG919 to A375 cells was competitively inhibited by DR5mAb, indicating that DR5mAb-mDexo-NLG919 was mainly internalized through a DR5mAb-mediated mechanism, proving that the connection of the DR5mAb target head can increase A375's uptake of the drug delivery system.

[0130] Test Example 3

[0131] This test example tests the apoptosis ability of the drug delivery system on A375 cells:

[0132] (1) Experimental groups:

[0133] ①NLG919 group: RPMI 1640 medium containing 3 μg / mL NLG919;

[0134] ②mDexo group: RPMI 1640 medium containing 6 μg / mL mDexo;

[0135] ③DR5mAb group: RPMI 1640 medium containing 18 μg / mL DR5mAb;

[0136] ④ DR5mAb-mDexo group: RPMI 1640 medium containing DR5mAb-mDexo prepared in Comparative Example 1 (DR5mAb content in DR5mAb-mDexo is 18 μg / mL, and mDexo content is 6 μg / mL);

[0137] ⑤mDexo-NLG919 group: RPMI 1640 culture medium containing mDexo-NLG919 prepared in step (1) of Example 1 (the mDexo content in mDexo-NLG919 is 6 μg / mL, and the NLG919 content is 3 μg / mL).

[0138] ⑥DR5mAb-mDexo-NLG919 group: RPMI 1640 medium containing DR5mAb-mDexo-NLG919 prepared in Example 1 (the mDexo content in DR5mAb-mDexo-NLG919 is 6 μg / mL, the NLG919 content is 3 μg / mL, and the DR5mAb content is 18 μg / mL).

[0139] ⑦Control group: Add the same volume of RPMI 1640 medium without drug as that of groups ①-⑥.

[0140] (2) Experimental methods:

[0141] Take A375 cells, discard the culture medium, add appropriate amount of PBS solution to wash 3 times, add trypsin to digest and centrifuge, discard the supernatant, add appropriate amount of culture medium to resuspend, and count the cells. Adjust the cell concentration to 1×10 5 / mL, according to 1×10 5 Plate cells at a density of 1 mL / well in a 12-well plate. Gently shake to evenly distribute the cells and incubate the plate in a 37°C, 5% CO2 incubator overnight. Discard the culture medium, rinse twice with an appropriate amount of PBS solution, then add the culture medium of each experimental group and continue incubation in a 37°C, 5% CO2 incubator for 48 hours. Collect the old culture medium, rinse twice with PBS solution, and add 100 μL of trypsin to each well. Terminate digestion by adding the old culture medium. Collect the cell suspension and place it in an EP tube. Centrifuge at 4°C, 1000 rpm for 5 minutes, discard the supernatant, and rinse once with PBS. The supernatant was discarded, and 195 μL of Annexin-FITC binding solution was added according to the kit instructions. The cells were gently blown evenly. 5 μL of Annexin-FITC and 10 μL of propidium iodide (PI) staining solution were added to the above experimental groups ①-⑦, respectively, and mixed evenly. Single staining group 1 (Annexin-FITC staining) and single staining group 2 (PI staining) were set up to add 5 μL of Annexin-FITC or 10 μL of propidium iodide (PI) staining solution, respectively. The cells were incubated on ice in the dark for 15 min. After the incubation, the cells were detected by flow cytometry.

[0142] The results of cell apoptosis ability test are as follows Figure 11As shown, the results showed that the mDexo group, NLG919 group, and DR5mAb group basically did not cause apoptosis in A375 cells, and there was no significant difference in the apoptosis produced by the control group. Compared with the mDexo-NLG919 group, the DR5mAb-mDexo group and the DR5mAb-mDexo-NLG919 group both had significant apoptosis ability, and the DR5mAb-mDexo-NLG919 group had the highest apoptosis rate. Compared with mDExo-NLG919 or DR5mAb-mDexo alone, DR5mAb-mDexo-NLG919 significantly enhanced the early and late apoptosis and necrosis of A375 cells, further demonstrating the potential of DR5mAb-mDexo-NLG919 combined immunotherapy for anti-MM.

[0143] Test Example 4

[0144] This test case tests the inhibitory effect of the drug delivery system on IDO:

[0145] (1) Experimental groups:

[0146] ①NLG919 group: RPMI 1640 medium containing a series of concentration gradients of NLG919 (0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1.6 μg / mL, and 3.2 μg / mL, respectively).

[0147] ②mDexo-NLG919 group: a series of concentration gradients containing mDexo-NLG919 prepared in step (1) of Example 1 (the mDexo contents in mDexo-NLG919 were 0.2 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1.6 μg / mL, 3.2 μg / mL, and 6.4 μg / mL, and the NLG919 contents were 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1.6 μg / mL, and 3.2 μg / mL, respectively).

[0148] ③DR5mAb-mDexo-NLG919: A series of concentration gradients contained DR5mAb-mDexo-NLG919 prepared in Example 1 (the mDexo contents in mDexo-NLG919 were 0.2 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1.6 μg / mL, 3.2 μg / mL, and 6.4 μg / mL, respectively; the NLG919 contents were 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1.6 μg / mL, and 3.2 μg / mL, respectively; and the DR5mAb contents were 0.6 μg / mL, 1.2 μg / mL, 2.4 μg / mL, 4.8 g / mL, 9.6 μg / mL, and 19.2 μg / mL, respectively).

[0149] (2) Experimental methods:

[0150] Remove A375 cells, discard the culture medium, wash three times with PBS, add an appropriate amount of trypsin, digest, centrifuge, discard the supernatant, add an appropriate amount of culture medium to resuspend into a uniform cell suspension, and count the cells. Plate 20,000 cells per well in a 96-well plate, add 100 μL of culture medium to each well, gently shake the plate to evenly distribute the cells, and incubate the plate in a 37°C, 5% CO2 incubator overnight. Discard the culture medium, add IFN-γ to a final concentration of 80 ng / mL per well, and incubate in the incubator for another 12 hours. Discard the IFN-γ, add the culture medium containing the different drug preparations described above for each experimental group, and incubate the plate in the incubator for another 24 hours. Remove the plate, aspirate 150 μL of cell supernatant from each group, mix with 75 μL of 30% trichloroacetic acid solution, and incubate in a 50°C water bath for 30 minutes. After incubation, centrifuge at 3000 rpm for 10 minutes, take 100 μL of supernatant from each well, mix evenly with an equal amount of Ehrlich reagent (glacial acetic acid solution containing 2% p-dimethylaminobenzaldehyde), and incubate at room temperature in the dark for 10 minutes. After incubation, measure the OD value at 490 nm using a microplate reader.

[0151] The results are as follows Figure 12 As shown in the data, after treatment with NLG919, mDexo-NLG919, and DR5mAb-mDexo-NLG919, the degree of IDO inhibition increased with increasing concentration and eventually tended to be flat. Compared with NLG919, mDexo-NLG919 and DR5mAb-mDexo-NLG919 showed stronger IDO inhibitory activity, and DR5mAb-mDexo-NLG919 had a stronger IDO inhibitory effect, indicating that DR5mAb-mDexo-NLG919 has the IDO inhibitory activity brought by NLG919 and can improve the stability of NLG919 to a greater extent.

[0152] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0153] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0154] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A drug delivery system based on dendritic cell exosomes, characterized in that: The drug delivery system includes dendritic cell-derived exosomes, an indoleamine 2,3-dioxygenase inhibitor encapsulated in the exosomes, and a death receptor monoclonal antibody modified on the surface of the exosomes; The death receptor monoclonal antibody is modified on the surface of the exosomes by connecting with a coupling agent.

2. The drug delivery system according to claim 1, wherein The indoleamine 2,3-dioxygenase inhibitors include NLG919; Preferably, the death receptor monoclonal antibody comprises a death receptor 5 monoclonal antibody.

3. The drug delivery system according to claim 1 or 2, characterized in that The mass ratio of the exosomes, the indoleamine 2,3-dioxygenase inhibitor and the death receptor monoclonal antibody is 1:(0.2-2):(2-10).

4. The drug delivery system according to any one of claims 1 to 3, characterized in that The coupling agent includes phospholipid-polyethylene glycol-maleimide; Preferably, the phospholipid comprises distearoylphosphatidylethanolamine; Preferably, the molecular weight of the polyethylene glycol is 1000-4000 Da.

5. A method for preparing a drug delivery system according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) exosomes derived from dendritic cells and an indoleamine 2,3-dioxygenase inhibitor are mixed and incubated to obtain exosomes encapsulating the indoleamine 2,3-dioxygenase inhibitor; a death receptor monoclonal antibody and a coupling agent are mixed and reacted to obtain a monoclonal antibody modified with the coupling agent; (2) The exosomes encapsulating the indoleamine 2,3-dioxygenase inhibitor are mixed and incubated with the monoclonal antibody modified with the coupling agent to obtain the drug delivery system.

6. The method for preparing the drug delivery system according to claim 5, wherein: The method for preparing dendritic cell-derived exosomes in step (1) comprises the following steps: (I) collecting the supernatant of the dendritic cell suspension and concentrating it by ultrafiltration to obtain a concentrate; (II) The concentrated solution is mixed with an exosome extraction reagent, centrifuged, and the precipitate is collected to obtain dendritic cell-derived exosomes.

7. The method for preparing the drug delivery system according to claim 5 or 6, wherein: The temperature of the mixed incubation in step (1) is 30-45° C., and the mixed incubation time is 1-3 hours; Preferably, the mass ratio of the death receptor monoclonal antibody to the coupling agent in step (1) is 1:(5-20); Preferably, the temperature of the mixing reaction in step (1) is 30-45° C., and the mixing reaction time is 1-3 h.

8. The method for preparing a drug delivery system according to any one of claims 5 to 7, wherein: The temperature of the mixed incubation in step (2) is 30-45° C., and the time of the mixed incubation is 1-3 h.

9. Use of the drug delivery system according to any one of claims 1 to 4 in the preparation of a drug for treating malignant tumors; Preferably, the malignant tumor comprises malignant melanoma.

10. Use of the drug delivery system according to any one of claims 1 to 4 in the preparation of a tumor cell proliferation inhibitor; Preferably, the malignant tumor comprises malignant melanoma.

Images