Monocytic myeloid-derived suppressor cell-targeted long-circulating drug delivery system and method of making same

Abstract

The application discloses a monocyte-like myeloid-derived suppressor cell-targeting long-circulating drug delivery system and a preparation method thereof, and belongs to the field of pharmaceutical preparations. The drug delivery system comprises a long-circulating material and a monocyte-like myeloid-derived suppressor cell-targeting drug delivery system. The long-circulating material is an erythrocyte membrane. In the monocyte-like myeloid-derived suppressor cell-targeting drug delivery system, a first complex formed by a monocyte-like myeloid-derived suppressor cell-targeting functional molecule and a maleimide-based cationic liposome serves as an outer shell to coat a drug core. The monocyte-like myeloid-derived suppressor cell-targeting functional molecule is a thiolated Ly-6C monoclonal antibody. The drug core is a second complex formed by protamine and a gene drug. The monocyte-like myeloid-derived suppressor cell-targeting long-circulating drug delivery system has M-MDSCs targeting property, has a longer in-vivo circulation time, and can realize long-acting drug delivery to M-MDSCs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulations, specifically relating to a long-circulating drug delivery system targeting mononuclear myeloid-derived inhibitory cells and its preparation method. Background Technology

[0002] Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population composed of myeloid progenitor cells and precursor cells at different differentiation stages, playing a crucial role in tumor immunosuppression. Numerous studies have reported the presence of MDSCs in mouse tumors, and recent research has also demonstrated their presence in human glioblastoma, breast cancer, urothelial carcinoma, and pancreatic cancer. High MDSC levels in cancer patients nearly double the mortality rate; circulating MDSCs can predict higher cancer stage and poorer prognosis, increasing the risk of resistance to checkpoint inhibitors. Therefore, MDSCs are considered the most important protective cells in the tumor microenvironment, forming the cornerstone of the immunosuppressive shield and helping tumors evade the immune system and immunotherapy.

[0003] Based on their monocyte and granulocyte origin, MDSCs can be mainly divided into monocytic myeloid-derived suppressor cells (M-MDSCs) and multinucleated myeloid-derived suppressor cells (PMN-MDSCs). PMN-MDSCs are typically short-lived cells, while M-MDSCs recruited to tumors can release various T cell suppressor factors, such as arginase and nitrous oxide synthase, hindering effector T cell function. Furthermore, M-MDSCs restrict the proliferation of effector CD4 and CD8 T lymphocytes, inhibit their cytokine release, and induce apoptosis in these cells. M-MDSCs can also induce the production of regulatory T and regulatory B cells, limiting conventional B cell proliferation and antibody release. Moreover, M-MDSCs can inhibit NK cell and antigen-presenting cell activity, induce macrophage polarization towards a regulatory phenotype, and M-MDSCs migrating to tumor sites usually eventually differentiate into tumor-associated macrophages, further inducing a shift in anti-tumor immune responses towards tolerance.

[0004] In the prior art, some studies have reported the existence of inhibitors of myeloid-derived suppressor cells. For example, Chinese patent document CN108853129A discloses the application of cimicifugain in the preparation of drugs that inhibit myeloid-derived suppressor cells. The research results show that cimicifugain can inhibit the production and recruitment activation of MDSCs in the body and increase the content of CD3+CD8+ T cells, thereby improving the body's immune regulation capacity. Chinese patent document CN113975253A discloses that glycyrrhizin has a good killing effect on myeloid-derived suppressor cells MDSCs.

[0005] Targeted delivery is one of the main advantages of drug delivery systems over free drugs in cancer treatment. Through meticulous design and modification, drug delivery systems can maintain better specificity, bioavailability, larger drug loading capacity, longer half-life, and less cytotoxicity to normal tissues, overcoming the shortcomings of traditional chemotherapy. Over the past two decades, with the tremendous development of interdisciplinary approaches in polymer science, pharmaceutics, bioconjugated chemistry, and molecular biology, a large number of drug delivery materials for cancer treatment and diagnosis have emerged. For example, Chinese patent document CN111437399A discloses a gene and chemical small molecule co-delivery system. This co-delivery system, for the first time, co-encapsulates small interfering RNA targeting indoleamine 2,3-dioxygenase-1 with gemcitabine in a 2-methylimidazolium zinc metal-organic framework nanocage to alleviate immunosuppression associated with regulatory T cells and myeloid-derived suppressor cells. However, targeted drug delivery systems designed to target the M-MDSCs-induced anti-tumor immunosuppression process are still rarely reported. Furthermore, since tumors are a form of chronic inflammation, they can lead to the continuous abnormal generation of M-MDSCs, meaning that short-term treatments targeting M-MDSCs may be ineffective. Summary of the Invention

[0006] This invention provides a long-circulating drug delivery system targeting mononuclear myeloid-derived suppressor cells (M-MDSCs), which has M-MDSC targeting and a long circulation time in the body, enabling long-term drug delivery to M-MDSCs, and has good application prospects in the treatment of chronic inflammation such as cancer.

[0007] The specific technical solution adopted is as follows:

[0008] A mononuclear myeloid-derived suppressor cell targeted long-circulating drug delivery system includes a long-circulating material and a mononuclear myeloid-derived suppressor cell targeted drug delivery system, wherein the long-circulating material is coated on the outside of the mononuclear myeloid-derived suppressor cell targeted drug delivery system;

[0009] The long-circulating material mentioned above is a red blood cell membrane;

[0010] In the mononuclear myeloid-derived inhibitory cell targeted drug delivery system, a first complex formed by a mononuclear myeloid-derived inhibitory cell targeting functional molecule and a maleimide-modified cationic liposome serves as a shell to coat the drug core; the mononuclear myeloid-derived inhibitory cell targeting functional molecule is a thiolized Ly-6C monoclonal antibody; and the drug core is a second complex formed by protamine and a gene drug.

[0011] This invention utilizes the cationic properties of protamine to compress gene-based drugs into a negatively charged drug core. Since M-MDSCs express high levels of Ly6C-labeled protein, a Ly-6C monoclonal antibody is used as the targeting molecule for mononuclear myeloid-derived inhibitory cells (MDSCs). A first complex modified with the Ly-6C monoclonal antibody is formed by the reaction of maleimide and sulfhydryl groups, and its positive charge coats the negatively charged drug core. The erythrocyte membrane contains the special functional protein CD47. By modifying the erythrocyte membrane on the surface of the MDSC-targeted drug delivery system, a long-circulating MDSC-targeted drug delivery system is constructed, which reduces the phagocytosis of the drug delivery system by macrophages and enables long-circulating action of the drug delivery system in vivo.

[0012] Preferably, the preparation method of the maleimide-based cationic liposomes is as follows: cationic lipids, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL are dissolved in an organic solvent, the organic solvent is removed by rotary evaporation to obtain a lipid film, and after hydration with deionized water and ultrasonic dispersion, the maleimide-based cationic liposomes are obtained.

[0013] More preferably, the cationic lipid is selected from DOTAP, DOTMA, DODMA or DLin-MC3-DMA; the organic solvent is chloroform; and the mass ratio of cationic lipid, cholesterol, DSPE-PEG2000 to DSPE-PEG2000-MAL is 10:5-20:0.5-3:0.5-3.

[0014] Preferably, the gene drug is a PD-L1 trap plasmid.

[0015] The method for preparing the PD-L1 trap plasmid is as follows: the extracellular domain of PD-1 and the C-terminal trimer domain of cartilage matrix protein are fused with the flexible hinge region and the secretion signal peptide sequence, and cloned into the Nhe I and Xho I sites of pcDNA3.1 to obtain the PD-L1 trap plasmid.

[0016] Preferably, the preparation method of the drug core is as follows: protamine and gene drug are dissolved in glucose solution at a mass ratio of 0.5-2:1, stirred and incubated to obtain the drug core.

[0017] The thiolized Ly-6C monoclonal antibody is obtained by thiolization modification of Ly-6C monoclonal antibody. Preferably, the thiolization modification method is as follows: Ly-6C monoclonal antibody and 2-iminothione hydrochloride are dissolved in buffer solution, stirred and reacted, and then purified and concentrated to obtain thiolized Ly-6C monoclonal antibody.

[0018] Further preferred, the molar ratio of Ly-6C monoclonal antibody to 2-iminothionine hydrochloride is 1:5-20.

[0019] The present invention also provides a method for preparing the mononuclear-like myeloid-derived suppressor cell-targeted long-circulating drug delivery system, comprising the following steps:

[0020] (1) The mononuclear myeloid-derived suppressor cell targeting functional molecule and maleimide-modified cationic liposome were mixed, stirred and incubated to obtain the first complex;

[0021] (2) The drug core is mixed with the first complex, and after stirring and incubation, the mononuclear myeloid-derived inhibitory cell targeted drug delivery system is obtained.

[0022] (3) Collect blood cells, obtain red blood cell membranes, mix the red blood cell membranes with the mononuclear myeloid-derived inhibitory cell targeted drug delivery system, stir and incubate to obtain the mononuclear myeloid-derived inhibitory cell targeted long-circulation drug delivery system.

[0023] Preferably, in step (2), the drug core and the first complex are mixed at a mass ratio of 20-200:1000.

[0024] Preferably, in step (3), the red blood cell membrane is mixed with the mononuclear myeloid-derived inhibitory cell targeted drug delivery system at a mass ratio of 0.5-2:1.

[0025] The present invention also provides the application of the mononuclear myeloid-derived inhibitory cell-targeted long-circulating drug delivery system in the preparation of antitumor drugs.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The mononuclear myeloid-derived suppressor cell-targeting long-circulating drug delivery system prepared in this invention improves the targeting of the drug delivery system to M-MDSCs through targeted design and increases the circulation time of the drug delivery system in vivo through long-circulation modification. It can achieve long-acting drug delivery to immunosuppressive M-MDSCs during tumor progression during chronic inflammation such as cancer, and provide a therapeutic means to solve the problem of tumor immunosuppression related to M-MDSCs. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the preparation process of the M-MDSCs targeted long-circulation drug delivery system.

[0029] Figure 2 In vitro targeting characterization of M-MDSCs for long-circulating drug delivery systems.

[0030] Figure 3Characterization of the in vivo long-circulation properties of the M-MDSCs-targeted long-circulation drug delivery system. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0032] Example 1: Preparation of M-MDSCs Targeted Long-Circulation Drug Delivery System

[0033] A schematic diagram of the preparation process of the mononuclear-like myeloid-derived suppressor cell-targeted long-circulating drug delivery system is shown below. Figure 1 As shown;

[0034] (1) Preparation of maleimide-modified cationic liposomes: DOTAP, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL were dissolved in chloroform at a mass ratio of 10:10:1.5:1.5. The chloroform was removed by rotary evaporation to obtain a lipid film, which was then hydrated with deionized water and ultrasonically dispersed using an ultrasonic cell disruptor to obtain maleimide-modified cationic liposomes.

[0035] (2) Preparation of thiolated Ly-6C monoclonal antibody: Ly-6C monoclonal antibody (Bioxcell; catalog number BE0203) and 2-iminothione hydrochloride were dissolved in DPBS (Dupois phosphate buffer solution) at a molar ratio of 1:10. The mixture was stirred at room temperature for 1 h and then purified and concentrated by ultrafiltration centrifuge tube to obtain thiolated Ly-6C monoclonal antibody.

[0036] (3) Preparation of the first complex: The maleimide-modified cationic liposomes obtained in step (1) and the thiolized Ly-6C monoclonal antibody obtained in step (2) were mixed at a mass ratio of 66:1000 and incubated overnight at 4°C with stirring to obtain the first complex of M-MDSCs-targeting cationic liposomes.

[0037] (4) Preparation of PD-L1 trap plasmid: The extracellular domain of PD-1 (mouse PD-1 residues 21-150) and the C-terminal trimer domain of cartilage matrix protein (mouse CMP1 residues 458-500) were fused with the flexible hinge region and secretory signal peptide sequence (the gene sequence encoding the fusion protein is shown in SEQ ID NO.1), and cloned between the Nhe I and Xho I sites of pcDNA3.1 to obtain the PD-L1 trap plasmid (for detailed preparation method, see the literature Synergistic and low adverseeffect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap; doi: 10.1038 / s41467-018-04605-x);

[0038] (5) Preparation of drug core: Protamine (Aladdin; catalog number P123670) and PD-L1 trap plasmid were dissolved in 5% glucose solution at a mass ratio of 36:50 and incubated at room temperature for 10 min with stirring. The resulting second complex was used as the drug core.

[0039] (6) Preparation of M-MDSCs targeted drug delivery system: The drug core obtained in step (4) and the first M-MDSCs targeted cationic liposome complex obtained in step (3) are mixed at a mass ratio of 86:1000 and stirred and incubated at room temperature for 10 min to obtain the M-MDSCs targeted drug delivery system αLy-6C-LPD.

[0040] (7) Preparation of M-MDSCs-targeted long-circulation drug delivery system: Blood cells were collected to obtain red blood cell membranes. The red blood cell membranes were mixed with the M-MDSCs-targeted drug delivery system αLy-6C-LPD at a mass ratio of 1:1 and stirred and incubated at 37°C for 10 min to obtain the M-MDSCs-targeted long-circulation drug delivery system αLy-6C-LAMP. Specifically, the process of preparing the red blood cell membrane was as follows: C57BL / 6 mouse whole blood was centrifuged at 2000 rpm for 5 min to remove serum and leukocyte layer. The red blood cells were resuspended in 0.25×PBS, placed on ice for 20 min, centrifuged at 10000 rpm for 10 min to remove hemoglobin, and the light pink particles at the bottom were collected. The cells were resuspended in 1×PBS and dispersed by ultrasonic cell disruptor in an ice bath environment to obtain the red blood cell membrane.

[0041] (8) Preparation of DiI-labeled αLy-6C-LAMP: DOTAP, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL were added to a chloroform solution in a mass ratio of 10:10:1.5:1.5:1 / 100, and the cell membrane red fluorescent probe DiI was added in addition. DiI-labeled αLy-6C-LAMP was prepared in the same way as above.

[0042] Example 2: Preparation of a M-MDSCs-Targeted Long-Circulation Drug Delivery System

[0043] In this embodiment, the only difference between the preparation method of the M-MDSCs targeted long-circulation drug delivery system and the preparation method in Example 1 is that the cationic lipid is replaced with DOTMA.

[0044] Comparative Example 1: Preparation of a Drug Delivery System

[0045] Preparation of Iso-LAMP: The Ly-6C monoclonal antibody was replaced with the isotype control recombinant monoclonal antibody (Bioxcell; catalog number BE0089), and Iso-LAMP was prepared according to the same method as in Example 1, that is, the recombinant monoclonal antibody was modified by thiolization, and then it was combined with maleimide-modified cationic liposomes to prepare a complex. Then, the drug delivery system was prepared using the drug core and red blood cell membrane in Example 1.

[0046] (2) Preparation of DiI-labeled Iso-LAMP: DOTAP, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL were added to a chloroform solution in a mass ratio of 10:10:1.5:1.5:1 / 100, and the cell membrane red fluorescent probe DiI was added in addition to prepare DiI-labeled Iso-LAMP in the same way.

[0047] Example 3: In vitro targeting of M-MDSCs in a long-circulation drug delivery system

[0048] Bone marrow-derived M-MDSCs were 5 × 10 4 Cells were seeded at a density of 1 cell / well in 96-well plates and co-incubated with DiI-labeled αLy-6C-LAMP at a final concentration of 0.1 mg / mL at 4 °C. Cells were collected at 15, 30, 45, and 60 min, washed twice with cold PBS, stained with Hoechst 33342, and then the mean DiI fluorescence intensity of the cells was detected by flow cytometry.

[0049] M-MDSCs derived from bone marrow were extracted from C57BL / 6 mice. DiI-labeled αLy-6C-LAMP was co-incubated with the bone marrow-derived M-MDSCs, with DiI-labeled Iso-LAMP used as a non-targeted control. The uptake of αLy-6C-LAMP by M-MDSCs was investigated. Results are as follows: Figure 2 As shown, at 15 min, the uptake of αLy-6C-LAMP by M-MDSCs was about twice that of Iso-LAMP, suggesting that αLy-6C targeting molecule modification significantly enhanced the interaction between αLy-6C-LAMP and M-MDSCs, promoting the uptake of αLy-6C-LAMP by M-MDSCs.

[0050] Example 4: In vivo long-circulation characteristics of the M-MDSCs targeted long-circulation drug delivery system

[0051] C57BL / 6 mice were injected via tail vein with 150 μL of DiI-labeled αLy-6C-LAMP containing 50 μg of PD-L1 trap plasmid. Blood samples of 20 μL were collected from the tail at 1, 5, 15, and 30 mins and 1, 2, 4, 8, 12, 18, and 24 h post-injection. The collected blood samples were transferred to 96-well plates containing 30 μL of PBS, and fluorescence detection was performed using a microplate reader to estimate the remaining αLy-6C-LAMP levels in the mice at different time points.

[0052] To investigate the long-term circulation characteristics of αLy-6C-LAMP in vivo, the hydrophobic fluorescent dye DiI was encapsulated in αLy-6C-LAMP, and DiI-labeled αLy-6C-LAMP was injected into the tail vein of C57BL / 6 mice. Blood was collected from the tail vein at different time points after injection for fluorescence detection to estimate the remaining amount of αLy-6C-LAMP in the mice at different time points. αLy-6C-LPD was used as a control group with non-erythrocyte membrane coating. Figure 3 As shown, αLy-6C-LAMP has a longer blood retention time than αLy-6C-LPD. At 12 and 18 hours, the total retention rates of αLy-6C-LAMP were 38% and 30%, respectively, while those of αLy-6C-LPD were 12% and 5%. The circulating half-life can be derived from the slope of the semi-logarithmic signal. Using a two-compartment model to fit the circulation results of the drug delivery system, the clearance half-life of αLy-6C-LAMP was calculated to be 15 hours, while the clearance half-life of αLy-6C-LPD was only 5 hours.

[0053] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a mononuclear-like myeloid-derived suppressor cell-targeted long-circulating drug delivery system in the preparation of antitumor drugs, characterized in that, The mononuclear myeloid-derived suppressor cell targeted long-circulating drug delivery system includes a erythrocyte membrane and a mononuclear myeloid-derived suppressor cell targeted drug delivery system, wherein the erythrocyte membrane coats the outside of the mononuclear myeloid-derived suppressor cell targeted drug delivery system; the erythrocyte membrane and the mononuclear myeloid-derived suppressor cell targeted drug delivery system are mixed at a mass ratio of 0.5-2:

1. In the mononuclear myeloid-derived inhibitory cell targeted drug delivery system, a first complex formed by the mononuclear myeloid-derived inhibitory cell targeting functional molecule and maleimide-modified cationic liposomes serves as the outer shell, encapsulating the drug core; the mononuclear myeloid-derived inhibitory cell targeting functional molecule is a thiolized Ly-6C monoclonal antibody; the Ly-6C monoclonal antibody is dissolved in buffer with 2-iminothione hydrochloride, stirred, reacted, purified, and concentrated to obtain the thiolized Ly-6C monoclonal antibody; The drug core is a second complex formed by protamine and PD-L1 trap plasmid; the drug core and the first complex are mixed at a mass ratio of 20-200:1000. The preparation method of the maleimide-modified cationic liposomes is as follows: cationic lipids, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL are dissolved in an organic solvent, the organic solvent is removed by rotary evaporation to obtain a lipid film, and after hydration with deionized water and ultrasonic dispersion, the maleimide-modified cationic liposomes are obtained; the mass ratio of cationic lipids, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL is 10:5-20:0.5-3:0.5-3.

2. The application according to claim 1, characterized in that, The cationic lipid is selected from DOTAP, DOTMA, DODMA or DLin-MC3-DMA; the organic solvent is chloroform.

3. The application according to claim 1, characterized in that, The preparation method of the drug core is as follows: protamine and PD-L1 trap plasmid are dissolved in glucose solution at a mass ratio of 0.5-2:1, stirred and incubated to obtain the drug core.

4. The application according to claim 1, characterized in that, The preparation method of the mononuclear-like myeloid-derived suppressor cell-targeted long-circulating drug delivery system includes the following steps: (1) The mononuclear myeloid-derived suppressor cell targeting functional molecule and maleimide-modified cationic liposomes were mixed and incubated to obtain the first complex; (2) The drug core is mixed with the first complex, and after stirring and incubation, the mononuclear myeloid-derived inhibitory cell targeted drug delivery system is obtained; (3) Collect blood cells, obtain red blood cell membranes, mix the red blood cell membranes with the mononuclear myeloid-derived inhibitory cell targeted drug delivery system, stir and incubate to obtain the mononuclear myeloid-derived inhibitory cell targeted long-circulation drug delivery system.

Images