Selenium peptide conjugate for specifically activating NK (Natural Killer) cells as well as preparation method and application of selenium peptide conjugate

Abstract

The invention discloses a selenopeptide conjugate for specifically activating NK (Natural Killer) cells as well as a preparation method and application of the selenopeptide conjugate, the selenopeptide conjugate comprises three parts, namely novel indocyanine green (IR820), a self-assembled polypeptide structure and selenomethionine (MSe), and the structure is IR820-Ser-Phe-Met (Se)-Phe-Met (Se)-Ser. The conjugate can induce activation of the NK cells and improve the toxicity of the NK cells to tumor cells, and shows good anti-tumor activity. In addition, the nano structure formed by the selenium peptide conjugate can effectively improve the photo-thermal treatment effect of IR820. The invention provides a novel NK cell activation and photothermal therapy combined molecule, and a new strategy is provided for tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a seleno-peptide conjugate for specifically activating NK cells, and a preparation method and application thereof. Background Art

[0002] In the past decade, cancer immunotherapy has been shown to have great prospects in clinical treatment, among which T cells have been at the forefront of immunotherapy research. Although T cell-based immunotherapy has been successfully applied to a wide range of cancers, overall, only a small number of patients benefit. Natural killer (NK) cells are a type of cytotoxic lymphocyte in the innate immune system that is different from T cells and B cells. They are characterized by the ability to spontaneously detect and kill infected or malignant cells, and play anti-tumor and anti-viral roles in the body. In addition to T cells, NK cell-mediated tumor immunotherapy has the advantages of safety, controllability and few transplantation restrictions, so it has received widespread attention. However, in the tumor microenvironment, tumor cells escape the surveillance and killing of NK cells, resulting in the inhibition of NK cell function. Therefore, how to effectively activate NK cells and enhance their anti-tumor activity is the key to tumor immunotherapy.

[0003] Selenium (Se) is an essential trace element for the human body and a central component of selenoproteins, such as the antioxidants glutathione peroxidase and thioredoxin reductase. It plays a key role in redox regulation in the human body, protects the body from damage by ROS, regulates thyroid metabolism and the immune system, and plays a role in the activation of immune function. Due to the redox reaction under mild conditions, Se-containing nanomaterials have shown great potential in biomedical applications in cancer chemotherapy and immunotherapy. Previous studies have shown that Se can activate NK cell-based immunotherapy and enhance NK cell cytotoxicity, but the mechanism is still unclear. In recent years, self-assembling peptide nanomedicines have attracted widespread attention due to their biocompatibility and activatable biological effects, such as enhanced tumor tissue retention and on-demand therapeutic activity. So far, nature-inspired secondary biomacromolecules, such as selenopeptides and selenoproteins, have rarely been reported as nanocarriers for disease treatment. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a selenopeptide conjugate that specifically activates NK cells and a preparation method and application thereof, providing a new solution for tumor immunotherapy.

[0005] The present invention is achieved by a seleno-peptide conjugate (hereinafter referred to as IR820-SFM) that specifically activates NK cells. Se FM Se S, including new indocyanine green (IR820), self-assembling peptide structure and selenomethionine (M Se) three parts, and its structure is: IR820-Ser-Phe-Met(Se)-Phe-Met(Se)-Ser.

[0006] The method for preparing the seleno-peptide conjugate that specifically activates NK cells is a solid phase synthesis method.

[0007] The selenopeptide conjugate is formed into nanoparticles with uniform size by ultrasonic standing.

[0008] The selenopeptide conjugate that specifically activates NK cells and the use of the selenopeptide conjugate that specifically activates NK cells prepared by the above preparation method in the preparation of anti-tumor drugs.

[0009] The anti-tumor drug is a photothermal therapy drug.

[0010] The advantages and technical effects of the present invention are as follows: the seleno-peptide conjugate for specifically activating NK cells of the present invention comprises a novel indocyanine green (IR820), a self-assembling polypeptide structure and selenomethionine (M Se ), formed by covalent bond connection. The seleno-peptide conjugate can be assembled to form nanoparticles, induce NK cell activation, enhance NK cell-mediated cytotoxicity, and enhance the photothermal therapy effect, thereby showing good anti-tumor activity and providing a new solution for tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the structural formula of the IR820-selenopeptide conjugate of the present invention.

[0012] Figure 2 The SFM of the present invention Se FM Se S and IR820-SFM Se FM Se Transmission electron microscopy (TEM) images of S (a) SFM Se FM Se TEM image of S, (b) IR820-SFM Se FM Se TEM image of S).

[0013] Figure 3 The SFM of the present invention Se FM Se S and IR820-SFM Se FM Se NK cell activation diagram of S ((a) is the secretion level of TNF-α, (b) is the secretion level of IFN-γ).

[0014] Figure 4 The SFM of the present invention Se FM SeS and IR820-SFM Se FM Se The toxicity of S to tumor cells MDA-MB-231 ((a) is SFM Se FM Se Toxicity of S to MDA-MB-231 cells, (b) IR820-SFM Se FM Se S toxicity to MDA-MB-231 cells).

[0015] Figure 5 For IR820-SFM Se FM Se Toxicity of S to tumor cells MDA-MB-231 under 808nm laser irradiation. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0017] A seleno-peptide conjugate (hereinafter referred to as IR820-SFM) that specifically activates NK cells of the present invention Se FM Se S, including new indocyanine green (IR820), self-assembling peptide structure and selenomethionine (M Se ) three parts, and its structure is: IR820-Ser-Phe-Met(Se)-Phe-Met(Se)-Ser.

[0018] The method for preparing the seleno-peptide conjugate that specifically activates NK cells is a solid phase synthesis method.

[0019] The selenopeptide conjugate is formed into nanoparticles with uniform size by ultrasonic standing.

[0020] The selenopeptide conjugate that specifically activates NK cells and the use of the selenopeptide conjugate that specifically activates NK cells prepared by the above preparation method in the preparation of anti-tumor drugs.

[0021] The anti-tumor drug is a photothermal therapy drug.

[0022] IR820-SFM Se FM Se S Its structure can be seen Figure 1 .

[0023] Example 1 This embodiment provides a self-assembling selenopeptide as shown below, and its preparation method is as follows:

[0024] (1) Weigh 400 mg of dichlororesin (amino acid loading rate 0.5 mmol / g) into a 10 mL peptide synthesis tube, add 5 mL of dichloromethane (DCM), shake on a shaker for 30 min (220 rpm) to fully swell the resin, and filter out the DCM.

[0025] (2) Weigh 153.38 mg of Fmoc-Ser(tBu)-OH (S) into a 10 mL centrifuge tube, add 5 mL of N,N-dimethylformamide (DMF) to dissolve, and add 200 μL of N,N-diisopropylethylamine (DIEA) to mix. Add the mixed solution to the peptide synthesis tube, place it on a shaker at room temperature for 5 hours, and filter out the liquid in the peptide synthesis tube. Use DCM and DMF to rinse the resin alternately for 3 times.

[0026] (3) Filter the liquid in the peptide synthesis tube cleanly, add 5 mL DMF solution containing 200 μL methanol (MeOH) to cap the tube, place it on a shaker for 20 min, filter the liquid in the peptide synthesis tube cleanly, and rinse it three times alternately with DCM and DMF.

[0027] (4) Filter out the liquid in the synthesis tube, add 5 mL of 20% piperidine solution, place on a shaker for 20 min to remove Fmoc protection, filter out the piperidine solution, rinse the peptide synthesis tube three times with DCM and DMF alternately, and finally filter out the liquid in the peptide synthesis tube.

[0028] (5) Take a 0.2 mL centrifuge tube and add a drop of ninhydrin and a drop of phenol. Use a capillary to pick up a small amount of resin and add it to the tube and stir well. Heat it in a metal bath at 100°C for three minutes. If the resin turns purple-black, it means that the Fmoc deprotection is successful and the next step can be carried out. If there is no obvious color change, the deprotection needs to be repeated. Use DCM and DMF to rinse three times alternately.

[0029] (6) Take a 10 mL centrifuge tube and weigh 167.34 mg of Fmoc-Selenomethionine (M Se ), 151.70 mg benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), add 5 mL DMF to dissolve, add 200 μL DIEA to the solution and mix well. Add the mixed solution to the synthesis tube and place it on a shaker for 10 h. After sufficient reaction, filter out the liquid in the peptide synthesis tube and rinse it alternately with DCM and DMF 3 times.

[0030] (7) Add one drop of ninhydrin and one drop of phenol to a 0.2 mL centrifuge tube, then add a little resin and stir evenly. Heat in a metal bath at 100°C for three minutes. If the resin does not change color, the coupling is successful.

[0031] (8) Repeat the above Fmoc deprotection step and deprotection detection, and continue the amino acid coupling step to convert Fmoc-Phe-OH (F), Fmoc-Selenomethionine (M Se ), Fmoc-Phe-OH (F), and Fmoc-Ser(tBu)-OH (S) were coupled to the resin one by one.

[0032] (9) After all coupling reactions are completed, rinse with DCM and DMF alternately for 3 times, add 5 mL of 20% piperidine solution, place on a shaker for 20 min to remove Fmoc protection, filter out the piperidine solution, and rinse the peptide synthesis tube with DCM and DMF alternately for 3 times. Check whether Fmoc is successfully deprotected.

[0033] (10) After removing the Fmoc protection, rinse the peptide synthesis tube three times with DCM and DMF alternately, filter out the liquid in the peptide synthesis tube, rinse the resin three times with MeOH, and vacuum for 30 min.

[0034] (11) Add 5 mL of lysis buffer (2.5 mL of trifluoroacetic acid, 2.5 mL of dichloromethane) to the peptide synthesis tube and shake on a shaker for 2 h to decompose the resin. Collect the solution and spin the sample until it is completely dry.

[0035] (12) Add icy ether dropwise to the spin-dried sample and perform sonication. Transfer the solid-liquid mixture to a centrifuge tube and centrifuge (12000 r / min, 5 min, 4°C). Discard the supernatant to obtain SFM. Se FM Se S selenopeptide.

[0036] Example 2 This embodiment provides the following self-assembled IR820-selenopeptide conjugate, and the preparation method thereof is as follows:

[0037] (1) After coupling the amino acid using a method similar to Example 1 (1-8), remove the Fmoc protection.

[0038] (2) Weigh 186.88 mg of new indocyanine green (IR820), 23.35 mg of 3-mercaptopropionic acid (19.14 μL), and 85.30 mg of DIEA (114.96 μL), dissolve in 1-2 mL of anhydrous DMF, add to a reaction bottle, and react at 40 °C for 3 h. The conversion rate detected by HPLC should be above 90% (the mobile phase is pure water and chromatographic acetonitrile containing 1‰ trifluoroacetic acid, the method is gradient elution, 0-42 min, 90% water-10% water, detection at 680 nm).

[0039] (3) Add the reaction solution to the peptide synthesis tube. Weigh 83.43 mg of HBTU and dissolve it in 4 mL of DMF. Add it to the peptide synthesis tube and place it on a shaker to react for 10 h. After sufficient reaction, filter out the liquid in the peptide synthesis tube and rinse it alternately with DCM and DMF for 3 times.

[0040] (4) Rinse the resin repeatedly with MeOH three times and apply vacuum for 30 min.

[0041] (5) Repeat steps 11-12 of Example 1 and discard the supernatant to obtain IR820-SFM Se FM Se S-selenopeptide conjugates.

[0042] Example 3 This example provides the preparation and characterization of selenopeptides and IR820-selenopeptide conjugate assemblies (1) Prepare 20 mmol / L SFM Se FM Se S and IR820-SFM Se FM Se Take a certain volume of the DMSO stock solution of S, dilute it to 400 μmol / L with PBS, vortex to dissolve, and assemble by ultrasonication. Take 10 μL of 400 μmol / L SFM Se FM Se S and IR820-SFM Se FM Se The S solution was added dropwise to the surface of the copper mesh, allowed to settle for 10 min, discarded, washed once with 10 μL of pure water, and dried naturally. A Hitachi HT7700 transmission electron microscope (TEM) was used to obtain transmission electron images to characterize the SFM Se FM Se S and IR820-SFM Se FM Se The shape of S.

[0043] (2) TEM images showing SFM Se FM Se S can be assembled to form nanofiber structures ( Figure 2 a), IR820-SFM Se FM Se S can form an amphiphilic structure and its morphology changes to nanoparticles ( Figure 2 b).

[0044] Example 4 This example verifies the activation of NK cells by selenopeptides and IR820-selenopeptide conjugates, and the steps are as follows: (1) Digestion of MDA-MB-231 cells in the logarithmic growth phase at 5×10 3 The cells were plated at a density of 10 cells / well in a 96-well plate and incubated overnight in an incubator containing 5% carbon dioxide at 37°C. Se FM Se S (100 µmol / L) or IR820-SFM Se FM Se After incubation for 24 h with fresh medium containing S (100 µmol / L), the old medium was discarded, the cells were washed once with PBS, and fresh medium containing NK cells (E / T = 1:10) was added and incubated for another 24 h.

[0045] (2) Collect NK cells (5e 5 ~1e 6 ), BD Golgi Plug (brefeldin A) was added at 1:1000 to inhibit Golgi transport, centrifuged at 4000 rpm / min at room temperature for 3 min, discarded the supernatant and washed with 1 mL wash buffer (1×PBS+1%FBS), discarded the supernatant; resuspended in 100 μL BD Fixation / Permeabilization Buffer, fixed at 4°C in the dark for 20 min; washed 1~2 times with 1 mL 1×BD Perm / Wash Buffer, diluted cytokine (1:50) flow cytometry antibody with 1×BD Perm / Wash Buffer, resuspended with 50 μL diluted antibody, stained at 4°C in the dark for 30 min, washed once with 1 mL1×BD Perm / Wash Buffer, washed with 1 mL wash buffer, discarded the supernatant and resuspended with wash buffer to about 2e 6 / mL, transfer to a 5 mL flow tube, resuspend using a vortexer and then load onto the machine.

[0046] (3) If Figure 3 As shown, flow cytometry was used to analyze the secretion of TNF-α and IFN-γ by NK cells co-incubated with MDA-MB-231. The results showed that SFM Se FMSe S and IR820-SFM Se FM Se S can increase the secretion of TNF-α and IFN-γ in NK cells, among which IR820-SFM se FM se S significantly enhanced the secretion level of IFN-γ by NK cells. This shows that IR820-selenopeptide conjugate can specifically recognize and bind to NK cells, secrete TNF-α and IFN-γ, and has good NK cell activation properties.

[0047] Example 5 Toxicity of selenopeptides and IR820-selenopeptide conjugates to MDA-MB-231 tumor cells (1) Digest MDA-MB-231 cells in the logarithmic growth phase and add 3×10 3 The cells were plated at a density of 10 cells / well in a 96-well plate, incubated overnight, the old culture medium was discarded, and SFM containing different concentrations was added. Se FM Se SorIR820-SFM Se FM Se The cells were incubated with fresh medium of S for 24 h, the old medium was discarded, the cells were washed once with PBS, 100 µL of fresh medium and 10 µL of MTT (5 mg / mL) were added, and the cells were incubated for another 4 h in an incubator at 37°C containing 5% carbon dioxide. Finally, the medium was discarded, 100 µL of DMSO was added to dissolve the formazan, and the optical density of the solution was measured at 570 nm using a microplate reader. The cell viability was expressed as a percentage of the control culture value and was calculated using the following formula. Cell viability (%) = (OD drug - OD blank) / (OD control - OD blank) × 100%.

[0048] SFM Se FM Se S concentration: 25, 50, 100, 200, 300, 400, 500 µmol / L IR820-SFM Se FM Se S concentration: 1, 2.5, 5, 10, 25, 50 µmol / L.

[0049] (2) If Figure 4 As shown, SFM Se FM Se S-selenopeptide itself did not show significant killing effect on 231 cells ( Figure 4 a), while the seleno-peptide conjugate IR820-SFM Se FM Se S has a significant killing effect on 231 cells ( Figure 4b), and showed concentration dependence.

[0050] Example 6 Toxicity of IR820-selenopeptide conjugate to MDA-MB-231 tumor cells under laser irradiation (1) The IR820 derivative synthesized in Example 2 (2) is referred to as IR820-COOH and IR820-SFM. Se FM Se The cytotoxicity of the two compounds under 808 nm laser irradiation was compared.

[0051] (2) Digestion of MDA-MB-231 cells in the logarithmic growth phase at 3×10 3 The cells were plated in 96-well plates at a density of 10 cells / well and incubated overnight. The old culture medium was discarded and different concentrations of IR820-COOH or IR820-SFM were added. Se FM Se S was cultured in fresh medium for 24 h and irradiated with 808 nm laser for 5 min (0.5 W / cm 2 ), continue incubation for 24 h, discard the old culture medium, wash once with PBS, add 100 µL of fresh culture medium and 10 µL of MTT (5 mg / mL), and continue incubation for 4 h in an incubator at 37°C containing 5% carbon dioxide. Finally, discard the culture medium, add 100 µL of DMSO to dissolve formazan, and measure the optical density of the solution at 570 nm using a microplate reader. Cell viability is expressed as a percentage of the control culture value and is calculated using the following formula. Cell viability (%) = (OD drug-OD blank) / (OD control-OD blank) × 100%.

[0052] Concentration of IR820-COOH: 1, 2.5, 5, 10, 25, 50 µmol / L IR820-SFM Se FM Se S concentration: 1, 2.5, 5, 10, 25, 50 µmol / L.

[0053] (3) If Figure 5 As shown in , IR820-COOH exhibited a certain degree of cytotoxicity at high concentrations (above 25 µmol / L), but by comparison, the phototoxicity of IR820 to MDA-MB-231 was significantly enhanced after coupling with selenopeptide.

[0054] In summary, the IR820-selenopeptide conjugate of the present invention can specifically activate NK cells, secrete cytokines TNF-α and IFN-γ, and has good NK cell activation properties and photothermal therapy effects.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A seleno-peptide conjugate for specifically activating NK cells, characterized in that: Including new indocyanine green IR820, self-assembling peptide structure and selenomethionine M Se There are three parts, and the structure is: IR820-Ser-Phe-Met(Se)-Phe-Met(Se)-Ser.

2. The method for preparing a selenopeptide conjugate for specifically activating NK cells according to claim 1, characterized in that: It is a solid phase synthesis method.

3. The method for preparing a selenopeptide conjugate for specifically activating NK cells according to claim 2, characterized in that: The selenopeptide conjugate is formed into nanoparticles with uniform size by ultrasonic standing.

4. Use of the selenopeptide conjugate for specifically activating NK cells as claimed in claim 1 or the selenopeptide conjugate for specifically activating NK cells obtained by the preparation method according to any one of claims 2 to 3 in the preparation of anti-tumor drugs.

5. The use of the selenopeptide conjugate for specifically activating NK cells according to claim 4 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is a photothermal therapy drug.

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