Composite micelle co-loaded with immune checkpoint blocking agent as well as preparation method and application of composite micelle

Abstract

The invention discloses a composite micelle co-loaded with an immune checkpoint blocking agent as well as a preparation method and application of the composite micelle. The preparation method comprises the following steps: mixing PCL3K-TK-NHS, CVRARTR and a solvent, stirring, dialyzing, and drying to obtain PCL3K-TK-CVRARTR; the preparation method comprises the following steps: carrying out amidation on PCL3K-NH2 and NHS-PEG5K-RGD, so as to obtain PCL3K-PEG5K-RGD; the preparation method comprises the following steps: mixing PCL3k-TK-CVRARTR, PCL3K-PEG5K-RGD, 1-MT and a solvent, adding the mixture into PBS (Phosphate Buffer Solution) to form a micelle, dialyzing, and removing the solvent to obtain the composite micelle. The preparation method disclosed by the invention is simple and has the advantages of low cost, low toxicity and high efficiency, and the prepared composite micelle can simultaneously transport the IDO blocking agent 1-MT and the antagonistic PD-L1 polypeptide to a tumor site, so that effective accumulation of the IDO blocking agent 1-MT and the antagonistic PD-L1 polypeptide at the tumor site is realized, an immune checkpoint is cooperatively blocked, and the anti-tumor effect of an immune system is recovered.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a composite micelle co-loaded with an immune checkpoint blocker, and a preparation method and application thereof. Background Art

[0002] After a century of research, cancer immunotherapy has revolutionized oncology and provided new treatment options for many types of cancer. Cancer immunotherapy aims to train host immune cells in lymphoid tissues and anti-tumor immune cells in the tumor microenvironment to find and destroy tumor cells. The anti-tumor immune response initiated by immunotherapy can promote systemic immune surveillance and eliminate local and disseminated metastatic tumors. In addition, immunotherapy may establish long-term immune memory and mediate immune protection against tumor recurrence. Immune checkpoint blockade therapy, represented by programmed cell death ligand-1 and its receptor (programmed cell death 1 / programmed cell death ligand 1, PD-1 / PD-L1), is an emerging treatment method in recent years. It has been widely and deeply studied and is also one of the hot spots in tumor immunotherapy. In clinical treatment, although PD-1 / PD-L1 therapy can play a good therapeutic role in a variety of solid tumors and prolong the median survival of patients, only a few cancer patients benefit from PD-1 / PD-L1 therapy because other immunosuppressive molecules are also triggered during the immune escape of tumor cells, such as indoleamine 2,3-dioxygenase (IDO). IDO is the rate-limiting enzyme of intracellular tryptophan along the kynurenine metabolic pathway. Overexpression of IDO can accelerate the catalysis of intracellular tryptophan metabolism to produce excessive kynurenine, thereby leading to the depletion of tryptophan that is beneficial to T cells and the accumulation of kynurenine that is toxic to T cells. At the same time, studies have shown that kynurenine, a metabolite of tryptophan, can inactivate effector T cells and inhibit the immune function of dendritic cells. At the same time, overexpression of IDO in tumor cells can increase tumor migration. 1-Methyl-DL-tryptophan (1-MT) is an analog of tryptophan and an IDO inhibitor. It can compete with tryptophan for the overexpressed IDO in tumor cells in the form of substrate competition, inhibit tryptophan metabolism, and reduce the production of kynurenine. On this basis, it is speculated that the combination of IDO inhibitors and PD-L1 antagonists can synergistically enhance the effect of PD-1 / PD-L1 immune checkpoint blockade therapy. However, traditional drug carriers have the following disadvantages: (1) It is difficult to synergistically deliver two immune checkpoint blockers; (2) The drugs are easily metabolized and degraded in the body; (3) The drugs cannot form effective accumulation at the tumor site; (4) The biocompatibility and biosafety of drug-loaded micelles in the body are poor, which limits their application. Summary of the invention

[0003] The technical problem to be solved by the present invention is how to deliver immune checkpoint blockers.

[0004] The present invention solves the above technical problems through the following technical means:

[0005] A method for preparing composite micelles co-loaded with immune checkpoint blockers comprises the following steps:

[0006] S1. PCL 3K -TK-NHS, CVRARTR and solvent are mixed, stirred, dialyzed and dried to obtain PCL 3K -TK-CVRARTR;

[0007] S2, PCL 3K -NH2 and NHS-PEG 5K -RGD is amidated to give PCL 3K -PEG 5K -RGD;

[0008] S3, the obtained PCL 3k -TK-CVRARTR, PCL 3K -PEG 5K -RGD, 1-MT and a solvent are mixed, added to a PBS buffer to form micelles, dialyzed, and the solvent is removed to obtain the composite micelles co-loaded with the immune checkpoint blocker.

[0009] Preferably, in S1, the solvent is DMF, and the stirring is stirring at room temperature for 3-5 h; in S3, the solvent is DMSO.

[0010] Preferably, in S1, the PCL 3K -The mass ratio of TK-NHS and CVRARTR is 20-50:10-18.

[0011] Preferably, in S1, the PCL 3K -The dosage ratio of TK-NHS to solvent is 10 mg: 1-2 ml.

[0012] Preferably, in S2, the solvent used in the reaction process is DMF, and TEA is used as a catalyst.

[0013] Preferably, in S2, the PCL 3K The dosage ratio of -NH2 to solvent is 4-7mg:1ml.

[0014] Preferably, in S2, the PCL 3K -NH2 and NHS-PEG 5K -RGD mass ratio is 20-40:15-30.

[0015] Preferably, in S3, the PCL 3k -TK-CVRARTR, PCL3K -PEG 5K The mass ratio of -RGD and 1-MT is 3-5:5-7:1.

[0016] Preferably, in S3, the usage ratio of 1-MT to solvent is 1 mg:3 mL.

[0017] The present invention also provides a composite micelle co-loaded with an immune checkpoint blocker, which is prepared by using the preparation method of the composite micelle co-loaded with an immune checkpoint blocker.

[0018] The present invention also proposes the use of a composite micelle co-loaded with an immune checkpoint blocker in the preparation of an anti-tumor drug.

[0019] The advantages of the present invention are:

[0020] RGD is an emerging tumor targeting peptide that can bind to the ανβ3 receptor on the surface of the tumor, thereby targeting tumor cells and causing the drug to accumulate in large quantities at the tumor site. The purpose of treating tumors is achieved. The present invention constructs a composite micelle with a double-chain "core-shell" structure. Through reasonable modification, the PD-L1 antagonist polypeptide is encapsulated in the "shell", and the "core" is loaded with small molecule chemical drugs that are water-insoluble IDO inhibitors. PEG-RGD is then connected to another chain to achieve the "drug / carrier" complex after drug encapsulation. It remains inert in the blood circulation and normal tissues, prevents the polypeptide from being rapidly degraded in the body, and actively targets the tumor to form an effective accumulation at the tumor site, thereby achieving the purpose of tumor treatment.

[0021] The preparation method of the invention is simple and has the advantages of low cost, low toxicity and high efficiency.

[0022] The composite micelles prepared by the above-mentioned ratio of monomers and preparation method can simultaneously transport the IDO blocker 1-MT and the antagonist PD-L1 polypeptide to the tumor site, achieve effective accumulation in the tumor site, synergistically block the immune checkpoints, and restore the anti-tumor effect of the immune system. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the preparation and in vivo delivery mechanism of the composite micelles in Example 1 of the present invention;

[0024] Figure 2 PCL in Example 1 of the present invention 3K -PEG 5K -H-NMR spectrum of RGD;

[0025] Figure 3 PCL in Example 1 of the present invention 3K -TK-CVRARTR H NMR spectrum;

[0026] Figure 4 The DSC (differential scanning calorimeter) result diagram of the blank micelle and the drug-loaded micelle in Example 3 of the present invention;

[0027] Figure 5 is a transmission electron microscopy image of the composite micelle in Example 1 of the present invention;

[0028] Figure 6 is a particle size diagram of the composite micelle in Example 1 of the present invention;

[0029] Figure 7 The toxicity test results of the composite micelles and free 1-MT on 4T1 cells in Example 1 of the present invention (the horizontal axis is the concentration of the drug 1-MT);

[0030] Figure 8 The enrichment of the composite micelles with or without RGD peptide in the tumor site in the embodiment of the present invention, and the enrichment of the composite micelles with or without RGD peptide encapsulated Cy7 in various organs of mice at different time points;

[0031] Fig. 9 The figures show the effects of different drugs in the present invention on the inhibition of tumor growth and the changes in the weight of mice;

[0032] Fig.10 The results of histochemical analysis of tumor sites by different drugs in the embodiments of the present invention;

[0033] Fig.11 The toxicity test results of the composite micelles and free 1-MT on 3T3 cells in Example 1 of the present invention (the horizontal axis is the concentration of the drug 1-MT). DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0036] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0037] Example 1

[0038] A method for preparing composite micelles co-loaded with immune checkpoint blockers comprises the following steps:

[0039] (1) PCL 3K -TK-CVRARTR preparation: 50 mg of PCL 3K -TK-NHS and 17.76 mg of CVRARTR were added to 5 mL of DMF and stirred at room temperature for 4 h at 500 r / min. The solution was then dialyzed in ultrapure water for 12 h (MWCO: 1000 Da). The dialyzed liquid was vacuum freeze-dried for 24 h to obtain PCL as a white solid powder. 3K -TK-CVRARTR.

[0040] (2) PCL 3K -PEG 5K Preparation of -RGD:PCL 3K -PEG 5K -RGD is through PCL 3K -NH2 and NHS-PEG 5K Briefly, 30 mg of PCL 3K -NH2 and 25 mg NHS-PEG 5K -RGD was dissolved in 5 mL of DMF, and 2.235 mg of TEA was added thereto as a catalyst, stirred at 500 r / min for 4 h at room temperature, and then dialyzed (MWCO: 10000 Da) for 24 h. The dialyzed product was vacuum freeze-dried to obtain PCL 3K -PEG 5K -RGD white powder.

[0041] (3) Preparation of composite micelles: PCL obtained in steps (1) and (2) 3k -TK-CVRARTR and PCL 3K -PEG 5K -RGD were added to a 3mL sterile EP tube, and 1mg of 1-MT was weighed and added to the tube, and then 3mL of DMSO was added to the tube and stirred to dissolve. The dissolved solution was slowly added to 7mL of PBS (pH7.4, 10mM) to form micelles, and the addition process was stirred at room temperature. After the addition was completed, the formed micelles were dialyzed with ultrapure water for 12h to remove the organic solvent DMSO to obtain a clear and transparent composite micelle solution, that is, the composite micelle 1-MT@CTP / RPP co-loaded with immune checkpoint blockers.

[0042] Example 2

[0043] A method for preparing composite micelles co-loaded with immune checkpoint blockers comprises the following steps:

[0044] (1) PCL 3K Preparation of -TK-CVRARTR: 20 mg of PCL 3K -TK-NHS and 10 mg of CVRARTR were added to 3 ml of DMF and stirred at room temperature for 3 h at 500 r / min. The solution was then dialyzed in ultrapure water for 12 h (MWCO: 1000 Da). The dialyzed liquid was vacuum freeze-dried for 24 h to obtain PCL as a white solid powder. 3K -TK-CVRARTR.

[0045] (2) PCL 3K -PEG 5K Preparation of -RGD:PCL 3K -PEG 5K -RGD is through PCL 3K -NH2 and NHS-PEG 5K Briefly, 20 mg of PCL 3K -NH2 and 15 mg NHS-PEG 5K -RGD was dissolved in 5 mL of DMF, and 2.235 mg of TEA was added thereto as a catalyst, stirred at 500 r / min for 3 h at room temperature, and then dialyzed (MWCO: 10000 Da) for 24 h. The dialyzed product was vacuum freeze-dried to obtain PCL 3K -PEG 5K -RGD white powder.

[0046] (3) Preparation of composite micelles: PCL obtained in steps (1) and (2) 3k -TK-CVRARTR and PCL 3K -PEG 5K -RGD was taken 4mg and 6mg respectively and added to a 3mL sterile EP tube, and 1mg of 1-MT was also weighed and added to the tube, and then 3mL of DMSO was added to the tube and stirred to dissolve. The dissolved solution was slowly added to 7mL of PBS (pH 7.4, 10mM) to form micelles, and the addition process was stirred at room temperature. After the addition was completed, the formed micelles were dialyzed with ultrapure water for 12h to remove the organic solvent DMSO to obtain a clear and transparent composite micelle solution, that is, the composite micelle 1-MT@CTP / RPP co-loaded with immune checkpoint blockers.

[0047] Example 3

[0048] A method for preparing composite micelles co-loaded with immune checkpoint blockers comprises the following steps:

[0049] (1) PCL 3K -TK-CVRARTR preparation: 30 mg of PCL3K -TK-NHS and 10 mg of CVRARTR were added to 5 ml of DMF and stirred at 400 r / min at room temperature for 5 h. The solution was then dialyzed in ultrapure water for 12 h (MWCO: 1000 Da). The dialyzed liquid was vacuum freeze-dried for 24 h to obtain PCL as a white solid powder. 3K -TK-CVRARTR.

[0050] (2) PCL 3K -PEG 5K Preparation of -RGD:PCL 3K -PEG 5K -RGD is through PCL 3K -NH2 and NHS-PEG 5K Briefly, 40 mg of PCL 3K -NH2 and 30 mg NHS-PEG 5K -RGD was dissolved in 6 mL of DMF, and 2.235 mg of TEA catalyst was added thereto, stirred at 500 r / min for 6 h at room temperature, and then dialyzed (MWCO: 10000 Da) for 24 h. The dialyzed product was vacuum freeze-dried to obtain PCL 3K -PEG 5K -RGD white powder.

[0051] (3) Preparation of composite micelles: PCL obtained in steps (1) and (2) 3k -TK-CVRARTR and PCL 3K -PEG 5K -RGD was taken 3mg and 7mg respectively and added to a 3mL sterile EP tube, and 1mg of 1-MT was also weighed and added to the tube, and then 3mL of DMSO was added to the tube and stirred to dissolve. The dissolved solution was slowly added to 7mL of PBS (pH 7.4, 10mM) to form micelles, and the addition process was stirred at room temperature. After the addition was completed, the formed micelles were dialyzed with ultrapure water for 12h, and the organic solvent DMSO was removed to obtain a clear and transparent composite micelle solution, that is, the composite micelle 1-MT@CTP / RPP co-loaded with immune checkpoint blockers.

[0052] Example 4

[0053] (1) Verification of the carrier material and drug-loaded micelles synthesized in the example by H NMR, TEM, DLS and DSC

[0054] First, the PCL prepared in Example 1 was 3K -TK-CVRARTR and PCL 3K -PEG 5K-RGD was analyzed by nuclear magnetic hydrogen spectrum, and the analysis results were as follows Figure 2 and Figure 3 As shown, it shows that the carrier material was successfully synthesized. The composite micelles obtained in Example 1 were characterized by TEM and DLS. Figure 5 and 6 As shown in Figure 2, the composite micelles are spherical in shape and about 100 nm in size. DSC was used to verify whether the drug-loaded composite micelles were successfully loaded with drugs. Figure 4 As shown, the CTP / RPP is prepared by the method of Example 1, but 1-MT is not added in (3). Figure 4 It can be seen that the crystallization peak of the drug disappears, indicating that the drug is successfully loaded into the micelles.

[0055] (2) Observation of cytotoxicity caused by composite micelles

[0056] The CCK reagent was mixed with RAPI-1640 or DMEM at a volume ratio of 1 / 9 (freshly prepared) to obtain a CCK working solution. 4T1 cells or 3T3 cells were inoculated at 5×10 cells per well using the composite micelles prepared in Example 1. 3 The cells were seeded at a density of 100 μL in a 96-well plate and incubated in 0.1 mL of RAPI-1640 (v / v) containing 10% fetal bovine serum for 12 h. 100 μL of free 1-MT and 1-MT@CTP / RPP (complex micelles) with different 1-MT concentrations were added to the cells (the concentration of 1-MT in the system ranged from 1 to 100 μg / ml). After incubation for 24 h, the cells were rinsed with PBS (pH 7.4, 10 mM), and then 100 μL of CCK working solution was added (the working solution formed by CCK reagent and RAPI-1640 was used for 4T1 cells, and the working solution formed by CCK reagent and DMEM was used for 3T3 cells), and then incubated for another 2 h. The absorbance (λ=450 nm) was measured by an ELISA instrument to quantitatively determine the cell viability. The cell viability was calculated based on the cell viability without any treatment.

[0057] The experimental results are as follows Figure 7 and 11 As shown by Figure 7 and 11 It can be seen that the composite micelles co-loaded with immune checkpoint blockers have no obvious toxicity to cells, indicating that the composite micelles have good safety and little side effects on cells.

[0058] (3) Observe the application of composite micelles in tumor site enrichment

[0059] In order to study the tumor accumulation ability of 1-MT@CTP / RPP (composite micelles) prepared in Example 1, 4T1 cells were subcutaneously injected into the right limb of Balb / c mice (the dose for each mouse was 1×106 cells). The mice were randomly divided into two groups (n=3). When the tumor volume was about 500 mm 3 The mice were injected with CTP / PP and CTP / RPP containing Cy7 via the tail vein. At different times after injection, the mice were imaged using the IVIS Lumina imaging system. The mice were then killed, and the main organs and tumors were collected for in vitro imaging. The fluorescent images were analyzed. The CTP / RPP containing Cy7 was prepared by replacing 1-MT with the fluorescent dye Cy7 according to the method in Example 1, and the CTP / PP containing Cy7 was prepared by replacing NHS-PEG according to the method in Example 1. 5K -RGD replaced by NHS-PEG 5K , in which 1-MT is replaced with the fluorescent dye Cy7.

[0060] The experimental results are as follows Figure 8 As shown in the figure, from left to right, the results of 2h, 4h, 8h, 12h, 24h after treatment and the results of major organs and tumors are shown in the figure. Figure 8 It can be seen that the composite micelles containing RGD polypeptide can be enriched in the tumor site faster and more efficiently than the control group.

[0061] (4) Observation of the inhibition experiment of composite micelle 4T1 subcutaneous tumor

[0062] To investigate the antitumor effects of 1-MT@CTP / RPP treatment, 4T1 tumor-bearing mice were established. Female Balb / c mice were subcutaneously inoculated with mouse tumor cells 4T1 at a cell number of 1×10 6 After 7 days, mice were injected with PBS, free 1-MT, CTP / RPP (prepared by the method of Example 1 but without adding 1-MT in (3), followed by ultrafiltration and concentration), 1-MT@RPP (prepared by the method of Example 1 but without adding PCL in (3)) and the like. 3K -TK-CVRARTR was prepared by ultrafiltration and concentration), 1-MT@CTP / PP (according to the method of Example 1, but PCL was replaced in (3) 3K -PEG 5K -RGD replaced with PCL 3K -PEG 5K Preparation and ultrafiltration, concentration) and 1-MT@CTP / RPP (prepared by the method of Example 1 and then ultrafiltration and concentration) were injected 5 times, once every 3 days, 100 μL per mouse each time, and the dosage of 1-MT was 2 mg / kg -1The growth of tumors and body weight were monitored every two days. The formula for calculating tumor volume was: tumor volume = 0.5 × length × width. On the 34th day after tumor implantation, all mice were sacrificed and the tumors were analyzed using H&E staining. Fig.10 As shown, from left to right, the results of PBS, 1-MT, CTP / RPP, 1-MT / @RPP, 1-MT@CTP / PP, and 1-MT@CTP / RPP are shown. Fig.10 It can be seen that the prepared drug-loaded micelles caused a large number of tumor apoptosis and effectively inhibited tumor growth.

[0063] Tumor growth and body weight experimental results are as follows Fig. 9 As shown, Fig. 9 It was shown that the composite micelles prepared according to Example 1 could effectively inhibit the growth of 4T1 subcutaneous tumor and had no significant effect on the body weight of mice.

[0064] In the present invention, a composite micelle with a "core-shell" structure is constructed. Through reasonable modification, the polypeptide antagonizing PD-L1 is encapsulated in the "shell", and the "core" is loaded with a small molecule chemical drug of a water-insoluble IDO inhibitor, and then PEG-RGD is connected to another chain, so that the "drug / carrier" complex after drug encapsulation remains inert in the blood circulation and normal tissues, preventing the polypeptide from being rapidly degraded in the body, and actively targeting the tumor to form an effective accumulation at the tumor site, achieving the purpose of tumor treatment. The preparation schematic diagram, delivery in mice, and specific efficacy are shown in the following figure. Figure 1 shown.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing composite micelles co-loaded with immune checkpoint blockers, characterized in that: The following steps are involved: S1. PCL 3K -TK-NHS, CVRARTR and solvent are mixed, stirred, dialyzed and dried to obtain PCL 3K -TK-CVRARTR; S2, PCL 3K -NH2 and NHS-PEG 5K -RGD is amidated to give PCL 3K -PEG 5K -RGD; S3, the obtained PCL 3k -TK-CVRARTR, PCL 3K -PEG 5K -RGD, 1-MT and a solvent are mixed, added to a PBS buffer to form micelles, dialyzed, and the solvent is removed to obtain the composite micelles co-loaded with the immune checkpoint blocker.

2. The method for preparing composite micelles co-loaded with immune checkpoint blockers according to claim 1, characterized in that: In S1, the solvent is DMF, and the stirring is stirring at room temperature for 3-5 h; in S3, the solvent is DMSO.

3. The method for preparing composite micelles co-loaded with immune checkpoint blockers according to claim 1, characterized in that: In S1, the PCL 3K -The mass ratio of TK-NHS and CVRARTR is 20-50:10-18.

4. The method for preparing composite micelles co-loaded with immune checkpoint blockers according to claim 1, characterized in that: In S1, the PCL 3K -The dosage ratio of TK-NHS to solvent is 10 mg: 1-2 ml.

5. The method for preparing composite micelles co-loaded with immune checkpoint blockers according to claim 1, characterized in that: In S2, the solvent used in the reaction process is DMF, and TEA is used as a catalyst.

6. The method for preparing composite micelles co-loaded with immune checkpoint blockers according to claim 5, characterized in that: In S2, the PCL 3K The dosage ratio of -NH2 to solvent is 4-7mg:1ml.

7. The method for preparing composite micelles co-loaded with immune checkpoint blockers according to claim 1, characterized in that: In S2, the PCL 3K -NH2 and NHS-PEG 5K -RGD mass ratio is 20-40:15-30.

8. The method for preparing composite micelles co-loaded with immune checkpoint blockers according to any one of claims 1 to 7, characterized in that: In S3, the PCL 3k -TK-CVRARTR, PCL 3K -PEG 5K The mass ratio of -RGD and 1-MT is 3-5:5-7:

1.

9. A composite micelle co-loaded with an immune checkpoint blocker, characterized in that: The composite micelle is prepared by the method for preparing a composite micelle co-loaded with an immune checkpoint blocker as described in any one of claims 1 to 8.

10. Use of the composite micelle co-loaded with an immune checkpoint blocker as claimed in claim 9 in the preparation of anti-tumor drugs.

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