A functional drug-carrying small molecule peptide for immune regulation

Abstract

The present invention discloses a functional drug-carrying small molecule peptide for immunomodulation, and belongs to the technical field of the establishment and preparation of small molecule peptide nano drug carrier systems. The functional drug-carrying small molecule peptide has MMP-2 responsiveness and can be used as a drug carrier, and the structural formula is shown in Figure 1. The functional drug-carrying small molecule peptide drug-carrying system MC / Pep1 (experimental peptide carrying mitoxantrone and cinnamaldehyde) of the present invention can target tumor cells, and can increase the cytotoxicity to tumor cells and reduce the toxic side effects to normal cells. It co-loads Mit and CA, which increases the retention time of the combined drug in tumor cells. More importantly, MC / Pep1 can effectively enhance tumor immunity and alleviate the tumor immunosuppressive microenvironment. As a drug carrier, a functional drug-carrying polypeptide that responds to the tumor microenvironment and adjusts tumor immunity can be used as a promising tool in tumor treatment.

Description

Technical Field

[0001] The invention relates to the technical field of establishment and preparation of a small molecule peptide nano drug carrier system, and in particular to a functional drug-carrying small molecule peptide for immunomodulation. Background Art

[0002] Immunotherapy has attracted great interest in tumor treatment. Chemotherapy is one of the most common interventions in cancer treatment, but tumor chemotherapy may cause temporary or long-term immune function decline. An ideal drug delivery system can not only promote the uptake and accumulation of drugs in tumors, but also improve the therapeutic efficiency of immunotherapy. Monoclonal antibodies, small molecule inhibitors, immune checkpoint inhibitors and immunomodulatory drugs have been introduced into tumor immunotherapy. Although there are drug carriers that achieve immunotherapy functions by delivering these immunostimulatory molecules, the cost of immunomodulatory drugs is high. There is an urgent need to develop drug delivery systems that do not utilize immune checkpoint inhibitors but have immunomodulatory functions and simple preparation processes. Peptides have attracted widespread attention due to their good biocompatibility, easy synthesis and adjustable functionalized structure. However, disappointingly, the immune response induced by single peptide vaccines is weak, which has always been a problem in cancer immunotherapy.

[0003] The morphological controllability of self-assembling peptides in response to environmental stimuli has attracted great interest. Self-assembling peptides are flexible and reactive, allowing controlled drug release through tumor microenvironmental reactions. Some peptides are targeted or cell-penetrating, allowing targeted drug delivery and easy uptake by tumor cells. For peptides, enzymes in the physiological environment are important catalysts. Matrix metalloproteinase 2 (MMP-2) is one of the main enzymes that degrade the extracellular matrix skeleton protein type IV collagen. Studies have found that MMP-2 is closely related to tumor invasion and metastasis, autoimmune diseases, and angiogenesis in tumor tissues. The morphological transformation induced by MMP-2 is expected to induce the in situ release of chemotherapeutic drugs while achieving more biological functions.

[0004] Some chemotherapeutic drugs, such as mitoxantrone (Mit), can trigger immunogenic cell death (ICD), transforming immunosuppressive "cold" tumors into immunoactive "hot" tumors by releasing tumor-associated antigens and danger-associated molecular patterns (DAMPs). In addition, reactive oxygen species (ROS)-induced ER oxidative stress is an important prerequisite for the occurrence of ICD. Therefore, increasing intracellular ROS using drugs is expected to amplify ICD. Using morphologically transformable peptides is expected to enhance the ICD-inducing effect of loaded drugs. Summary of the invention

[0005] In order to overcome the above defects, the present invention provides a functional drug-carrying small molecule peptide for immunomodulation, which can target tumor cells, increase the cytotoxicity to tumor cells and reduce the toxic side effects to normal cells.

[0006] The present invention is achieved by the following technical solutions:

[0007] A functional drug-carrying small molecule peptide for immunomodulation, which has MMP-2 responsiveness and can be used as a drug carrier, has the structural formula:

[0008] .

[0009] The present invention also provides an application of a functional drug-carrying small molecule peptide for immunomodulation as a drug carrier, wherein the functional drug-carrying small molecule peptide is the functional drug-carrying small molecule peptide, and the functional drug-carrying small molecule peptide encapsulates chemotherapy drugs and self-assembles into spherical nanoparticles.

[0010] Preferably, the chemotherapy drug includes: any one of mitoxantrone (Mit) and cinnamaldehyde (CA) or a combination thereof.

[0011] The present invention also provides a functional drug-carrying small molecule peptide drug-carrying carrier for immunomodulation, wherein the functional drug-carrying small molecule peptide drug-carrying carrier comprises the functional drug-carrying small molecule peptide forming spherical nanoparticles which can be used as a drug-carrying carrier.

[0012] Preferably, the functional drug-carrying small molecule peptide drug-carrying carrier responds to matrix metalloproteinase 2 (MMP-2) in the tumor microenvironment, and the self-assembly morphology is transformed into nanofiber drug release, and the drug retention time in tumor cells is prolonged and the drug concentration is higher.

[0013] The present invention also provides a method for preparing a functional drug-carrying small molecule peptide for immunomodulation that encapsulates a chemotherapeutic drug and self-assembles into spherical nanoparticles, comprising the following steps: dissolving the functional drug-carrying small molecule peptide and the chemotherapeutic drug in dimethyl sulfoxide, then adding HEPES buffer, and ultrasonicating to obtain the drug-carrying functional drug-carrying small molecule peptide spherical nanoparticles.

[0014] Preferably, the pH value of the HEPES buffer is 7.4.

[0015] Preferably, the method comprises the following steps: dissolving 2 mg of the functional drug-carrying small molecule peptide, 0.4 mg Mit and 0.4 mg CA in 10 μL dimethyl sulfoxide, adding 990 μL HEPES buffer having a pH of 7.4, and ultrasonically treating for 3 min to obtain a functional drug-carrying small molecule peptide solution containing Mit and CA, and placing the solution at room temperature for later use.

[0016] The present invention also provides a method for preparing an enzyme-responsive functional drug-carrying small molecule peptide for immune regulation that encapsulates chemotherapy drugs and self-assembles into spherical nanoparticles, comprising the following steps: adding MMP-2, CaCl2, and NaCl to the obtained peptide solution, and incubating at 37° C. to obtain an enzyme-responsive peptide solution.

[0017] Preferably, 10 μL of 10 μg / mL MMP-2, 10 μL of 1 M CaCl2, and 10 μL of 15 M NaCl are added to the obtained peptide solution, and the mixture is incubated at 37° C. to obtain an enzyme-responsive peptide solution.

[0018] Beneficial effects of the present invention:

[0019] The functional drug-carrying small molecule peptide drug-carrying system MC / Pep1 (experimental peptide carrying mitoxantrone and cinnamaldehyde) of the present invention can target tumor cells, increase the cytotoxicity to tumor cells and reduce the toxic side effects to normal cells. MC / Pep1 undergoes a morphological transformation in the tumor microenvironment, and it co-carries Mit and CA, which increases the retention time of the combined drug in tumor cells. Compared with MC / Pep2 (control peptide carrying mitoxantrone and cinnamaldehyde) that cannot be transformed in morphology, MC / Pep1 more effectively inhibits tumor growth. More importantly, MC / Pep1 can effectively enhance tumor immunity and alleviate the tumor immunosuppressive microenvironment. As a drug carrier, a functional drug-carrying polypeptide that responds to the tumor microenvironment and adjusts tumor immunity can be used as a promising tool in tumor treatment, which provides a theoretical and experimental basis for the development of a new drug delivery system. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the structural formula of the functional drug-carrying small molecule peptide Pep1 of the present invention;

[0021] Figure 2 is the structural formula of the control peptide Pep2 of the present invention;

[0022] Figure 3 is the cumulative release of Mit and CA before and after the addition of MMP-2, where A is the cumulative release rate of Mit (%), and B is the cumulative release rate of CA (%);

[0023] Figure 4 is the cell survival rate (%) of MB49 and 3T3 cells after incubation with Pep1 and Pep2 for 12 h and 24 h, respectively;

[0024] Figure 5Cell uptake and intracellular retention of coumarin 6 (C6)-loaded peptides, where A is the CLSM image of MB49 cells incubated with free C6, C6 / Pep2 and C6 / Pep1 for 0.25 h, and B is the CLSM image of MB49 cells incubated with free C6, C6 / Pep2 and C6 / Pep1 for 2 h, blue is the cell nucleus; green is coumarin 6; scale bar: 50 μm;

[0025] Figure 6 The mean fluorescence intensity analysis of MB49 cells uptake of coumarin 6-loaded peptide after 0.25 h and 2 h respectively;

[0026] Figure 7 CLSM images of MB49 cells co-incubated with free C6, C6 / Pep2 and C6 / Pep1, where A is the CLSM image of co-incubation for 96 h, and B is the CLSM image of co-incubation for 120 h, the scale bar is 50 μm, blue is the cell nucleus, and green is coumarin 6;

[0027] Figure 8 CLSM images of MB49 cells co-incubated with Saline, Mit, MC, MC / Pep2, and MC / Pep2 for 24 h CRT;

[0028] Fig. 9 Images for in vitro maturation analysis of DC cells. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to specific embodiments, but this does not limit the protection scope of the present invention.

[0030] Example 1

[0031] A functional drug-carrying small molecule peptide for immunomodulation, which has MMP-2 responsiveness and can be used as a drug carrier. The structural formula is shown in Figure 1 Abbreviated as Pep1, as a control peptide, an amphiphilic small molecule peptide Pep2 without HER2 targeting peptide was designed, the structural formula is shown in Figure 2 .

[0032] Example 2

[0033] Preparation of functional drug-loaded small molecule peptides for immunomodulation that encapsulate chemotherapy drugs and self-assemble into spherical nanoparticles

[0034] Pep1 (2 mg) was dissolved in dimethyl sulfoxide (DMSO; Sigma, 10 μL), 990 μL of HEPES buffer was added, and sonicated for 3 min to produce a Mit-loaded peptide solution, abbreviated as Mi / Pep1, which was left at room temperature for later use.

[0035] Pep1 (2 mg) and CA (0.4 mg) were dissolved in dimethyl sulfoxide (DMSO; Sigma, 10 μL), and then 990 μL of HEPES buffer was added and sonicated for 3 min to prepare a CA-loaded peptide solution, abbreviated as CA / Pep1, which was left at room temperature for later use.

[0036] Pep1 (2 mg) was dissolved in dimethyl sulfoxide (DMSO; Sigma, 10 μL) with Mit (0.4 mg) and CA (0.4 mg), and then 990 μL of HEPES buffer was added and sonicated for 3 min to prepare a peptide solution loaded with Mit and CA, abbreviated as MC / Pep1, which was left at room temperature for later use.

[0037] Similarly, control peptide Mi / Pep2, CA / Pep2 solution, and MC / Pep2 solution were prepared.

[0038] Example 3

[0039] Preparation of enzyme-responsive functional small molecule peptides for immunomodulation that encapsulate chemotherapy drugs and self-assemble into spherical nanoparticles

[0040] To 1 mL of the peptide solution obtained in Example 2, 10 μL of 10 μg / mL MMP-2, 10 μL of 1M CaCl2, and 10 μL of 15M NaCl were added, and the mixture was incubated at 37° C. to obtain an enzyme-responsive peptide solution.

[0041] Example 4

[0042] In vitro drug release

[0043] The drug-loaded peptide solutions Mi / Pep1 and CA / Pep1 obtained in Example 2 were placed at room temperature for 12 hours, added to a dialysis bag with a molecular weight of 1000Da, and dialyzed in 2L of distilled water for 1 hour. Then, 10μL of 10μg / mL MMP-2, 10μL of 1M CaCl2, and 10μL of 15M NaCl were added to the drug-loaded peptide solution. The dialysis bags were soaked in 25mL PBS and placed in a 37°C shaker. At certain time intervals, 2ml of liquid was taken out, 2ml of fresh liquid was added, and the solution was detected with an ultraviolet spectrophotometer (680nm and 256nm). The polypeptide solution without MMP-2 was used as a control. The results are shown in Figure 3 .

[0044] Conclusion: When the time was extended to 72 hours, the drug released by Mi / Pep1 with the addition of MMP-2 reached 66.0%, while the release rate (Mit) of the control group without the addition of MMP-2 was only about 35.0% ( Figure 3 A). After adding MMP-2 to CA / Pep1, the cumulative release rate of CA reached about 70.0% at 72h, while the cumulative release rate of CA in the group without adding MMP-2 was only 30.0% ( Figure 3 B). This indicates that after being encapsulated by the carrier material, Mit and CA are released in small amounts under physiological conditions, which is beneficial to reducing the toxic side effects of anti-tumor drugs on non-target organs. In tumor tissues where MMP-2 is overexpressed, a large amount of anti-tumor drugs can be released, thereby improving the anti-tumor effect.

[0045] Example 5

[0046] Cytotoxicity assay

[0047] The CCK-8 method was used to detect the toxic side effects of functional drug-loaded small molecule peptides Pep2 and Pep1 on MB49 and 3T3 cells. 3000-5000 cells were added to each well of a 96-well plate and cultured overnight in an incubator to wait for adhesion. After adhesion, the liquid in the well was removed, and a 1:1 (serum-free culture medium: HEPES buffer or peptide solution) volume of reagent was added in groups. After reacting for 12h or 24h, 10μL (per 100μL liquid) of CCK-8 reagent was added to the well, incubated in a 37℃ oven for 2h, and the optical density (OD) of the sample was measured at 450nm using an enzyme reader. The results are shown in Figure 4 .

[0048] Conclusion: At 12h, the survival rate of MB49 cells in the Pep1 group was reduced compared with the blank group and the Pep2 group, while the survival rate of 3T3 cells remained basically unchanged. When the co-incubation time was extended to 24h, the survival rate of MB49 cells was further reduced compared with the survival rate at 12h. The cytotoxic effect of Pep1 on MB49 cells gradually increased over time. Matrix metalloproteinase 2 overexpressed in tumor cells can catalyze the conversion of polypeptide nanoparticles into nanofibers, have membrane destruction ability, and can penetrate the plasma membrane to induce cell death. This indicates that Pep1 can partially inhibit the growth of tumor cells in vitro and has low toxicity to normal cells.

[0049] Example 6

[0050] Cellular uptake assay

[0051] Coumarin (Cou6) was abbreviated as C6 to replace Mit and CA. Fluorescently labeled drug-loaded peptides were prepared using the method of Example 2. The products were abbreviated as C6 / Pep2 and C6 / Pep1. The centrifuged MB49 cells were cultured in an 8-well plate at 37°C for 24 hours for cell attachment. After attachment, free C6, C6 / Pep2, and C6 / Pep1 were mixed with serum-free culture medium at a volume of 1 / 20 and added to different culture wells. After incubation for 0.25 hours and 2 hours, the cells were fixed and stained with 0.1 μg / mL DAPI. After the cells were washed with PBS, the distribution of different preparations in tumor cells was observed and compared using a confocal laser scanning microscope (CLSM). The CLSM image results are shown in Figure 5 .

[0052] Conclusion: After co-culture with C6 for 0.25 h, there was weak green fluorescence in MB49 cells, and the green fluorescence in MB49 cells in the C6 / Pep1 group was slightly stronger than that in MB49 cells in the C6 / Pep2 group. As the incubation time was extended to 2 hours, the intracellular drug uptake increased, and the fluorescence in the C6 group was still very weak. In contrast, a large amount of green fluorescence appeared in MB49 cells in the C6 / Pep1 group, and it was significantly stronger than that in the C6 / Pep2 group.

[0053] Digest the cells with EDTA-free trypsin, centrifuge the suspension, and resuspend in PBS (beat slowly to prevent cell breakage). Repeat twice, then pass 500 μl of PBS cell suspension through a membrane and place it in a glass tube for quantitative analysis using a flow cytometer. Figure 6 .

[0054] Conclusion: Compared with free C6 and 6 / Pep2 groups, the drug uptake of cells in the C6 / Pep1 group was significantly increased, which was consistent with the results of laser confocal microscopy observation ( Figure 5 ), indicating that Pep1 can increase cellular uptake.

[0055] Example 7

[0056] Cell retention

[0057] To analyze the retention time of different preparations in the tumor MB49 cells, we used laser confocal imaging for observation. The centrifuged MB49 cells were cultured in an 8-well plate at 37°C for 24 hours for cell adhesion. After adhesion, free C6, C6 / Pep2, and C6 / Pep1 were mixed with serum-free culture medium at a volume of 1 / 20 and added to different culture wells. After incubation for 2 hours, the liquid in the wells was removed, and fresh complete culture medium was added to continue culturing. After incubation for 96 hours and 120 hours, the cells were fixed and stained with 0.1μg / mL DAPI. After washing the cells with PBS, laser confocal imaging was used to observe and compare the distribution of different preparations in tumor cells. Laser confocal microscopy (CLSM) was used to observe the cells co-cultured with free C6, C6 / Pep2, and C6 / Pep1 for 96 hours and 120 hours. The results are shown in Figure 7 .

[0058] Conclusion: Almost no green fluorescence was observed in MB49 cells co-cultured with free DOX at 96 hours and 120 hours. In addition, a large amount of green fluorescence was observed in MB49 cells co-cultured with C6 / Pep2 and C6 / Pep1, and the C6 / Pep1 group was significantly stronger than the C6 / Pep2 group. Compared with 96 hours, the fluorescence of the C6 / Pep1 group at 120 hours decreased, but more fluorescence could still be observed, while the fluorescence of the C6 / Pep2 group had become weak.

[0059] Example 8

[0060] CRT testing

[0061] The mixed solution of Mit and CA is abbreviated as MC.

[0062] MB49 cells were cultured in 8-well plates at 37°C for 24 hours for cell attachment. After attachment, saline, mitoxantrone (Mit), mitoxantrone and cinnamaldehyde (MC), MC / Pep2, and MC / Pep1 were mixed with serum-free culture medium at a volume of 1 / 200 and added to different culture wells for 24 hours. After paraformaldehyde fixation, BSA blocking for 2 hours, CRT primary antibody was added for overnight incubation at 4°C, FITC-coupled secondary antibody was incubated for 2 hours, PBS was washed, and CLSM was used for observation. Results are shown in Figure 8 .

[0063] Conclusion: CRT exposure is a characteristic of ICD, and CRT exposure on the cell membrane triggers DC to phagocytize released antigens, thereby initiating a tumor immune cycle. CLSM images showed that the exposure level of cells treated with Mit alone to CRT was negligible, while the red fluorescence intensity of the MC / Pep1-treated group was the strongest, indicating that MC / Pep1 can strongly induce the transport of CRT from the endoplasmic reticulum to the cell membrane.

[0064] Example 9

[0065] In vitro maturation assay of DCs

[0066] Bone marrow-derived dendritic cells (BMDCs) were extracted from mouse leg bones and cultured in a medium containing granulocyte colony-stimulating factor (GMSF) for 7 days. MB49 was co-cultured with saline, Mit, MC, MC / Pep2, and MC / Pep1 for 12 hours to induce apoptosis. BMDCs were inoculated into the lower chamber of the Transwell chamber, and MB49 was added to the upper chamber of the Transwell chamber for 24 hours. After induction, BMDCs in the 24-well plate were centrifuged, stained with antigen differentiation cluster (CD) 80, CD86, and CD11c antibodies for 30 minutes, resuspended in PBS, and the content of mature dendritic cells in different experimental groups was analyzed by flow cytometry. See Fig. 9 .

[0067] Conclusion: Normal saline, Mit, MC, MC / Pep2, and MC / Pep1 were co-cultured with MB49 cells to induce cell apoptosis, and then used to induce BMDCs maturation. The maturation rates of BMDCs in each group were 10.1%, 17.6%, 20.3%, 20.9%, and 45.8%, respectively. Compared with free drug and drug-loaded control peptide, MC / Pep1 significantly induced the production of ROS and ICD effect. ROS further triggered the endoplasmic reticulum stress response, and dying tumor cells released "eat me" signals, activated the antigen presentation process, effectively promoted the maturation of BMDCs, induced subsequent effector tumor-specific T cell immune response, and effectively regulated tumor immunity.

Claims

1. A functional drug-carrying small molecule peptide for immunomodulation, characterized in that: The functional drug-carrying small molecule peptide has MMP-2 responsiveness and can be used as a drug-carrying carrier. The structural formula is: 。 2. An application of a functional drug-carrying small molecule peptide for immunomodulation as a drug-carrying carrier, characterized in that: The functional drug-carrying small molecule peptide is the functional drug-carrying small molecule peptide according to claim 1, and the functional drug-carrying small molecule peptide encapsulates the chemotherapeutic drug and self-assembles into spherical nanoparticles.

3. The use of the functional drug-carrying small molecule peptide according to claim 2 as a drug-carrying carrier, characterized in that: The chemotherapy drugs include: any one of mitoxantrone and cinnamaldehyde or a combination thereof.

4. A functional small molecule peptide drug carrier for immunomodulation, characterized in that: The functional drug-carrying small molecule peptide drug carrier comprises the functional drug-carrying small molecule peptide that can be used as a drug carrier as described in claim 1.

5. The functional drug-carrying small molecule peptide drug-carrying carrier according to claim 4, characterized in that: The functional drug-carrying small molecule peptide drug-carrying carrier responds to matrix metalloproteinase 2 in the tumor microenvironment, and the self-assembly morphology is transformed into nanofiber drug release, and the drug retention time in tumor cells is prolonged, and the drug concentration is higher.

6. A method for preparing functional drug-carrying small molecule peptides for immunomodulation that encapsulate chemotherapy drugs and self-assemble into spherical nanoparticles, characterized in that: The method comprises the following steps: dissolving the functional drug-carrying small molecule peptide and chemotherapy drugs described in claim 1 in dimethyl sulfoxide, then adding HEPES buffer, and ultrasonicating to obtain drug-carrying functional drug-carrying small molecule peptide spherical nanoparticles.

7. The method for preparing functional drug-carrying small molecule peptides encapsulating chemotherapy drugs and self-assembling into spherical nanoparticles according to claim 6, characterized in that: The pH value of the HEPES buffer is 7.

4.

8. The method for preparing a functional drug-carrying small molecule peptide encapsulating a chemotherapeutic drug and self-assembling into spherical nanoparticles according to any one of claims 6 and 7, characterized in that: The method comprises the following steps: dissolving 2 mg of the functional drug-carrying small molecule peptide according to claim 1, 0.4 mg of Mit and 0.4 mg of CA in 10 μL of dimethyl sulfoxide, adding 990 μL of HEPES buffer having a pH of 7.4, and ultrasonically treating for 3 minutes to obtain a peptide solution carrying Mit and CA, which is placed at room temperature for later use.

9. A method for preparing enzyme-responsive functional drug-carrying small molecule peptides for immunomodulation that encapsulate chemotherapy drugs and self-assemble into spherical nanoparticles, characterized in that: The method comprises the following steps: adding MMP-2, CaCl2 and NaCl to the peptide solution obtained in claim 8, and incubating at 37° C. to obtain an enzyme-responsive peptide solution.

10. The method for preparing functional drug-carrying small molecule peptides encapsulating chemotherapy drugs and self-assembling into spherical nanoparticles according to claim 9, characterized in that: The method comprises the following steps: adding 10 μL of 10 μg / mL MMP-2, 10 μL of 1M CaCl2, and 10 μL of 15M NaCl to the peptide solution obtained in claim 8, and incubating at 37°C to obtain an enzyme-responsive peptide solution.

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