Liposome preparation based on myeloid cell regulation and chemotherapy synergistic effect, preparation method and anti-tumor application thereof

Abstract

This invention relates to a liposomal formulation based on myeloid cell regulation and chemotherapy enhancement, along with its preparation method and antitumor applications, belonging to the technical field of pharmaceutical formulations. The liposomal formulation uses liposomes as carriers to co-load the CD11b agonist leukadherin-1 (LA1) prodrug (DSPE-PEG2000-Azo-LA1) and the gemcitabine prodrug Gem-SS-Chol. DSPE-PEG2000-Azo-LA1 contains a hypoxia-responsive azobenzene linker, and Gem-SS-Chol contains a glutathione-responsive disulfide bond, enabling precise drug release within the tumor microenvironment. This formulation exhibits uniform particle size and good stability, synergistically exerting chemotherapeutic and immunomodulatory effects. It effectively inhibits the proliferation and migration of pancreatic cancer tumor cells, enhances tumor cell immunogenic cell death, regulates the phenotype of myeloid immune cells in the tumor immunosuppressive microenvironment, and improves the therapeutic effect of pancreatic cancer. This invention provides a novel and highly efficient liposomal formulation for pancreatic cancer treatment, focusing on myeloid cell regulation and chemotherapy enhancement.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and relates to liposomal formulations that co-load CD11b agonist prodrug and gemcitabine prodrug for myeloid cell regulation and chemotherapy enhancement, their preparation methods, and anti-tumor applications. Specifically, it relates to the construction of hypoxia-responsive CD11b agonist prodrug, glutathione-responsive gemcitabine prodrug, and liposomal formulations containing the two prodrugs, as well as their application in the preparation of drugs for treating pancreatic ductal adenocarcinoma. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is the most common pathological type of pancreatic cancer, characterized by high malignancy, poor prognosis, and significant clinical treatment challenges. Gemcitabine (Gem), a first-line chemotherapy drug for advanced pancreatic cancer, is widely used clinically, but it still suffers from problems such as a short plasma half-life, poor in vivo stability, easy development of drug resistance, and significant dose-dependent toxicity. Furthermore, gemcitabine is a nucleoside analog, and its transmembrane transport primarily relies on balanced nucleoside transporter (hENT1)-mediated endocytosis. However, pancreatic ductal adenocarcinoma often presents with low hENT1 expression or limited hENT1 function, resulting in insufficient intracellular delivery efficiency of gemcitabine and thus limiting its therapeutic efficacy. On the other hand, the tumor microenvironment of PDAC further exacerbates its resistance to gemcitabine through multiple mechanisms, specifically: dense matrix and hyaluronic acid hinder drug delivery, the formation of a hypoxic microenvironment induces epithelial-mesenchymal transition, matrix upregulation of cytidine deaminase inactivates gemcitabine, and the large number of immunosuppressive cells in the tumor microenvironment, such as myeloid-derived suppressor cells (MDSCs) and M2 tumor-associated macrophages (M2-TAMs), compete with drugs by regulating related pathways and releasing deoxycytidine, which together lead to the high resistance of PDAC to gemcitabine.

[0003] Leukadherin-1, a novel small-molecule allosteric agonist, specifically binds to the allosteric site of CD11b on the surface of M2-TAMs and myeloid-derived suppressor cells (MDSCs) and induces conformational changes. It precisely regulates the CD11b / FAK / ROS / STING / IFN signaling pathway, thereby reducing the number of MDSCs infiltrating and lowering immunosuppressive factors by modulating the polarization of immunosuppressive M2-TAMs towards a pro-inflammatory, anti-tumor phenotype (M1-TAMs), thus modulating the tumor immune microenvironment and potentially alleviating PDAC resistance to Gem. However, its oral administration is significantly affected by the acidic environment of the gastrointestinal tract, digestive enzyme degradation, and the intestinal mucosal barrier, severely limiting its drug stability and bioavailability. Furthermore, to achieve effective drug concentrations, the oral dosage is as high as 100 mg / kg, increasing the burden on the gastrointestinal tract and the metabolic stress on the liver.

[0004] Liposomes, as a commonly used nanomedicine delivery system, possess good biocompatibility, drug loading capacity, and in vivo safety. They can be used to improve the pharmacokinetic behavior of drugs and, to some extent, enhance drug accumulation in tumor tissues. After modification with polyethylene glycol, liposomes can further prolong circulation time and improve formulation stability. However, traditional liposomes typically lack the ability to respond to the tumor microenvironment, making it difficult to achieve precise drug release at the tumor site and potentially causing exposure to non-target tissues and systemic toxicity.

[0005] Therefore, gemcitabine in the current treatment of pancreatic cancer has many problems, such as short plasma half-life, insufficient in vivo stability, uptake restricted by nucleoside transporters, easy development of drug resistance and high dose-dependent toxicity, and multidrug resistance induced by the immunosuppressive microenvironment. At the same time, CD11b agonists have poor water solubility, insufficient in vivo stability, poor delivery efficiency, and difficulty in exerting efficient immunomodulatory effects. Summary of the Invention

[0006] Therefore, in response to the above key issues, this study designed and constructed a tumor microenvironment-responsive liposome that can co-load gemcitabine prodrug and CD11b agonist prodrug. This liposome can achieve myeloid immune cell regulation and chemotherapy enhancement. By modifying the prodrug to enhance drug stability, liposomes are used to achieve co-delivery of two drugs in vivo and tumor-targeted accumulation. Microenvironment-responsive release enables precise lesion drug delivery. The gemcitabine prodrug structure incorporates glutathione (GSH)-responsive disulfide bonds, which endow it with specific release capabilities. Furthermore, as a reducing substrate of glutathione peroxidase 4 (GPX4), GSH consumption leads to GPX4 inactivation, thereby triggering lipid peroxidation and ultimately inducing ferroptosis, synergistically enhancing gemcitabine-mediated immunogenic death (ICD) of tumor cells. Simultaneously, relying on CD11b agonists to regulate the immunosuppressive tumor microenvironment reverses M2-TAMs and MDSCs-mediated immunosuppression and chemotherapy resistance, further enhancing the antitumor effect of gemcitabine and providing a novel strategy for highly effective and low-toxicity combination therapy for PDAC.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a liposome-based formulation co-loaded with a CD11b agonist prodrug (DSPE-PEG2000-Azo-LA1) and a gemcitabine prodrug, Gem-SS-Chol. DSPE-PEG2000-Azo-LA1 conjugates the CD11b agonist leukadherin-1 (LA1) to the lipid material via a hypoxia-responsive azobenzene linker, enabling responsive cleavage and release of LA1 in the hypoxic tumor microenvironment. Gem-SS-Chol is a gemcitabine prodrug molecule formed by conjugation of gemcitabine to cholesterol via disulfide bonds, enabling responsive cleavage and release of gemcitabine under high glutathione conditions. This design endows the liposome formulation with dual hypoxia / glutathione microenvironment responsiveness.

[0009] This invention also provides a method for preparing the above-mentioned liposome formulation for regulating myeloid cells and enhancing chemotherapy efficacy, comprising the following steps: preparing DSPE-PEG2000-Azo-LA1 and Gem-SS-Chol respectively; dissolving the liposome carrier raw material, DSPE-PEG2000-Azo-LA1 and Gem-SS-Chol in an organic solvent, mixing them evenly and then removing the organic solvent to form a lipid film; hydrating the lipid film with an aqueous medium, and after dispersion treatment and membrane filtration, obtaining the liposome formulation.

[0010] The present invention also provides the use of the above-mentioned liposome formulation in the preparation of a medicament for treating pancreatic ductal adenocarcinoma.

[0011] The CD11b agonist prodrug DSPE-PEG2000-Azo-LA1 is composed of the CD11b agonist leukadherin-1 and DSPE-PEG2000-NHS linked by a hypoxia azoreductase-responsive linker.

[0012] The structure of the hypoxia azoreductase-sensitive linker is as follows:

[0013]

[0014] The specific CD11b agonist leukadherin-1 (LA1) molecule has the following structural formula:

[0015]

[0016] The specific structural formula of DSPE-PEG2000-NHS is as follows:

[0017]

[0018] Where m is an integer between 44 and 45.

[0019] The CD11b agonist prodrug DSPE-PEG2000-Azo-LA1 has the following structural formula:

[0020]

[0021] Where n is an integer between 44 and 45.

[0022] The specific synthetic steps of the CD11b agonist prodrug DSPE-PEG2000-Azo-LA1 include the following:

[0023] A hypoxia azo reductase-sensitive linker was synthesized using 4-nitrobenzyl alcohol and 4-aminobenzoic acid as raw materials.

[0024]

[0025] Starting with compound 2, its carboxyl terminus was first protected with Boc to obtain intermediate 3; then the benzyl alcohol group at the other end of the intermediate was esterified and coupled with the carboxyl group of leukadherin-1 to obtain intermediate 4; subsequently, the Boc protecting group was removed to obtain intermediate 5, which was further coupled with DSPE-PEG2000-NHS by amidation to obtain target compound 6 (DSPE-PEG2000-Azo-LA1).

[0026]

[0027] Where n is an integer between 44 and 45.

[0028] The gemcitabine prodrug Gem-SS-Chol is composed of gemcitabine (Gem) and cholesterol chloroformate linked by a glutathione-sensitive linker.

[0029] The structure of the glutathione-sensitive linker is as follows:

[0030]

[0031] Specifically, the structure of gemcitabine (Gem) is as follows:

[0032]

[0033] Specifically, the cholesterol chloroformate has the following structure:

[0034]

[0035] The structural formula of the gemcitabine prodrug Gem-SS-Chol is as follows:

[0036]

[0037] Specifically, the synthesis steps of the gemcitabine prodrug Gem-SS-Chol include the following:

[0038] Using 2-hydroxyethyl disulfide, methyl cholesterol chloroformate, and N,N'-disuccinimidyl carbonate as raw materials, methyl cholesterol chloroformate was coupled with 2-hydroxyethyl disulfide to obtain intermediate 1. The other end hydroxyl group of intermediate 1 was activated to form N-hydroxysuccinimidyl (NHS) active ester, which was then subjected to an amidation reaction with the amino group of gemcitabine to obtain the target compound Gem-SS-Chol.

[0039]

[0040] The DSPE-PEG2000-Azo-LA1 molecule is formed by coupling leukadherin-1 with DSPE-PEG2000-NHS through an azobenzene structure. In the hypoxic environment of the tumor microenvironment, the azo bond breaks and leukadherin-1 is released.

[0041] Gem-SS-Chol is a prodrug molecule formed by the coupling of the amino site of gemcitabine with cholesterol via a disulfide bond. Under the high glutathione environment of the tumor microenvironment, the disulfide bond breaks, specifically releasing free gemcitabine.

[0042] Based on the above, this invention provides a liposome formulation based on myeloid cell regulation and chemotherapy enhancement. The liposome formulation comprises soybean lecithin (SPC), distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), DSPE-PEG2000-Azo-LA1, and Gem-SS-Chol.

[0043] Preferably, the weight ratio of soybean lecithin (SPC), distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), DSPE-PEG2000-Azo-LA1, and Gem-SS-Chol is (5~12):(1~4):(1~4):(1~5).

[0044] More preferably, the weight ratio of soybean lecithin (SPC), distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), DSPE-PEG2000-Azo-LA1, and Gem-SS-Chol is (7~10):(2~4):(2~4):(2~4).

[0045] More preferably, the weight ratio of soybean lecithin (SPC), distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), DSPE-PEG2000-Azo-LA1, and Gem-SS-Chol is 9:2.2:2.5:4.

[0046] Preferably, the liposome formulation has a particle size of 50-400 nm.

[0047] More preferably, the liposome formulation has a particle size of 90~180 nm.

[0048] More preferably, the liposome formulation has a particle size of 120-145 nm.

[0049] This invention provides a method for preparing the above-mentioned liposome formulation, comprising the following steps:

[0050] (1) Soybean lecithin (SPC), DSPE-mPEG2000, DSPE-PEG2000-Azo-LA1, and Gem-SS-Chol were mixed evenly in a mass ratio of 9:2.2:2.5:4, dissolved in chloroform and mixed evenly. The mixture was transferred to a round-bottom flask, and the organic solvent chloroform was removed by rotary evaporation under reduced pressure. The mixture was then dried under vacuum to obtain a lipid film loaded with the drug.

[0051] (2) Add preheated PBS buffer to the eggplant-shaped flask, vortex to fully disperse the lipid film, and perform water bath hydration to obtain a multivesicular liposome suspension.

[0052] (3) After hydration, the multivesicular liposome suspension is ultrasonically treated using a water bath ultrasonic instrument (Ningbo SCIENTZ-IID), and then extruded through 0.45 μm and 0.22 μm aqueous polycarbonate membranes to obtain the liposome formulation; or the obtained formulation is further freeze-dried for preservation.

[0053] Preferably, in step (2), the hydration temperature is 55~65°C. o C, hydration time is 40~80 min.

[0054] More preferably, in step (3), after hydration, the lipid film basically disappears, and the lipids and drugs are evenly distributed in the system; after ultrasonic treatment, the liposome dispersion is in a uniformly dispersed state.

[0055] A method for preparing dual-drug-loaded liposomes, the method comprising the following steps:

[0056] ① Prepare a solution of lecithin, DSPE-mPEG2000, DSPE-PEG2000-Azo-LA1, and Gem-SS-Chol, and mix them evenly in a mass ratio of 9:2.2:2.5:4; the solvent of the solution is a low-boiling-point solvent that is easy to remove by rotary evaporation, specifically chloroform.

[0057] ② After removing the solvent from the solution obtained in step ① by rotary evaporation, the drug-loaded lipid film is dispersed in PBS solution; then hydration incubation is performed to obtain a co-loaded liposome suspension.

[0058] ③Use a water bath sonicator (Ningbo SCIENTZ-IID) to sonicate the liposomes from step ②, and then filter them using 0.45 μm and 0.22 μm aqueous polycarbonate membranes respectively to obtain dual-drug-loaded nanoliposomes.

[0059] The liposomes had a particle size of 120–145 nm, a polydispersity index (PDI) of (0.24±0.08–0.27±0.01), and a zeta potential of (-2.51±0.39–-1.71±0.21). The encapsulation efficiency of gemcitabine was (78.33±2.87%–86.31±3.52%), and the drug loading was (5.44±0.19%–6.00±0.24%). The encapsulation efficiency of the CD11b agonist (leukadherin-1) was (63.33±2.87%–71.66±2.87%), and the drug loading was (1.06±0.05%–1.21±0.05%).

[0060] The application of the liposome formulation for regulating myeloid cells and enhancing chemotherapy efficacy in the preparation of a drug for treating pancreatic ductal adenocarcinoma.

[0061] The nanomedicine is an injectable preparation, preferably an intravenous injection formulation.

[0062] The liposome formulation achieves specific drug release under hypoxia and glutathione conditions; gemcitabine prodrug is used to inhibit the proliferation, migration, and colony formation of pancreatic cancer cells and induce immunogenic cell death in tumor cells, enhance antigen presentation, and promote dendritic cell maturation; CD11b agonist prodrug is used to induce M2-type tumor-associated macrophages to polarize towards M1 type; the formulation can reduce the proportion of myeloid-derived suppressor cells (MDSCs) in pancreatic tumor tissue and increase CD8+. + The proportion of T cells reverses the tumor immunosuppressive microenvironment.

[0063] Compared with existing technologies, the present invention has the following beneficial effects: By co-constructing a CD11b agonist prodrug and a gemcitabine prodrug in the same liposome delivery system, the present invention facilitates the joint delivery of the two active ingredients. By introducing a hypoxia-responsive azobenzene linker and a glutathione-responsive disulfide bond, the formulation can achieve responsive drug release under tumor microenvironment conditions. The liposome formulation can improve the insufficient LA1 delivery efficiency and the problems of poor stability and limited uptake associated with direct gemcitabine administration, and helps enhance the synergistic effect of chemotherapy and immunomodulation. Experimental results show that the liposome formulation prepared by the present invention has good particle size uniformity and stability, can inhibit the progression of pancreatic cancer in situ, induce immunogenic cell death in tumor cells, and improve the tumor immunosuppressive microenvironment. Therefore, the present invention provides a liposome formulation for the treatment of pancreatic ductal adenocarcinoma that has tumor microenvironment responsive characteristics and combines myeloid cell regulation and chemotherapy enhancement, achieving the co-delivery of two drugs and exerting a synergistic effect of 1+1>2. Attached Figure Description

[0064] Figure 1 A schematic diagram of the structure of a liposome formulation based on myeloid cell regulation and chemotherapy enhancement.

[0065] Figure 2 Basic characterization diagram of liposome formulations;

[0066] (a) Macroscopic diagram of free drug and lipid formulation;

[0067] (b) Particle size distribution of liposome formulation;

[0068] (c), (d) Stability test graphs of liposome formulations over 7 days;

[0069] (e) Zeta potential diagram of liposome formulation;

[0070] (f) Transmission electron micrograph of the liposome formulation LipLA1@GSCH, scale bar 200 nm.

[0071] Figure 3 Figure showing the results of the MTT cytotoxicity assay.

[0072] Figure 4 Figure showing the validation results of the in vitro induced ICD effect of liposome formulation;

[0073] (a) Graph showing the results of ATP level detection in cell culture supernatant after drug treatment;

[0074] (b) Graph showing the results of CRT expression level detection on cell surface after drug treatment;

[0075] (c) Graph showing the results of HMGB1 expression level detection in cells after drug treatment.

[0076] Figure 5 Figure showing the results of in vitro regulation of macrophage repolarization;

[0077] (a) Flow cytometry results of M1 macrophage marker expression after treatment with different concentrations;

[0078] (b) Statistical chart of the proportion of M1 macrophages in F4 / 80⁺ cells;

[0079] (c) Flow cytometry results of M2 macrophage marker expression after different concentration treatments;

[0080] (d) Statistical chart of the proportion of M2 macrophages in F4 / 80⁺ cells.

[0081] Figure 6 In vivo antitumor therapeutic effect of liposomal formulations that regulate myeloid cells and enhance chemotherapy efficacy;

[0082] (a) Schematic diagram of in vivo antitumor experimental procedure and drug administration time;

[0083] (b) Curves showing the changes in body weight of mice in different treatment groups during the treatment period;

[0084] (c) Physical images of mouse pancreatic tumor tissues isolated at the experimental endpoint from different treatment groups;

[0085] (d) Statistical analysis of pancreatic tumor weight in mice of different treatment groups;

[0086] (e) Peritoneal tumor metastasis in mice in different treatment groups.

[0087] Figure 7 A diagram illustrating the regulatory effect of liposome formulations on immune cell subsets in the tumor microenvironment of mice with orthotopic pancreatic cancer.

[0088] (a) Statistical graph of the proportion of MDSCs (Gr-1⁺ CD11b⁺) in CD45⁺ cells in mouse pancreatic cancer tumor tissue;

[0089] (b) Statistical graph of the ratio of M2 / M1 macrophages in mouse pancreatic cancer tumor tissue;

[0090] (c) Statistical chart of the proportion of CD8⁺ CD3⁺ T cells to CD45⁺ cells in mouse pancreatic cancer tumor tissue. Detailed Implementation

[0091] The invention will be further described below with reference to the accompanying drawings and embodiments:

[0092] Example 1

[0093] (1) The synthesis of the CD11b agonist prodrug DSPE-PEG2000-Azo-LA1, the specific reaction steps are as follows:

[0094]

[0095] 4-Nitrobenzyl alcohol (3.5 g, 21.0 mmol), ammonium chloride aqueous solution (1.6 g, 29.4 mmol), and zinc powder (4.1 g, 63.0 mmol) were added sequentially to 40 mL of ethanol, and the mixture was stirred at room temperature for 30 min. After the reaction was complete, the insoluble matter was removed by filtration. The resulting filtrate was slowly added dropwise to ferric chloride hexahydrate solution (6.8 g, 25.2 mmol), and 20 mL of deionized water and 8 mL of ethanol were added to make up the difference. o Continue stirring at below °C for 30 min. Then dilute the reaction solution with saturated brine and extract with dichloromethane. Combine the organic phases, wash with water, and dry over anhydrous magnesium sulfate. Remove the solvent by vacuum distillation. The resulting crude product, 4-nitrosobenzyl alcohol, is used directly in the next reaction step.

[0096] 4-Nitrobenzyl alcohol (1.6 g) and 4-aminobenzoic acid (1.6 g, 21.0 mmol) were dissolved in 40 mL of acetic acid and stirred overnight at room temperature. After the reaction was complete, the precipitate was collected by centrifugation, washed with dichloromethane, and purified by silica gel column chromatography to give an orange-yellow solid compound 2 (1.6 g, yield 53%).

[0097] The NMR and mass spectrometry data of compound 2 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ8.15 (d, J =8.6 Hz, 2H), 7.96 (d, J = 8.6 Hz, 2H), 7.92 (d, J = 8.3 Hz, 2H), 7.56 (d, J =8.5 Hz, 2H), 4.62 (s, 2H). HRMS (ESI) m / z: C 14 H 12 Calculated N₂O₃: 256.0848; Measured: 255.0755 [M - H] - .

[0098]

[0099] Compound 2 (2.5 g, 9.76 mmol) was dissolved in 40 mL of N,N-dimethylformamide (DMF), followed by the addition of (2-aminoethyl)-tert-butyl carbamate (2.03 g, 12.67 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 2.43 g, 12.68 mmol), and 1-hydroxybenzotriazole (HOBt, 1.72 g, 12.68 mmol). The mixture was stirred overnight at room temperature. After the reaction was complete, the DMF was removed by concentration under reduced pressure. The residue was dispersed in dichloromethane (DCM) and filtered to remove insoluble matter. The filtrate was concentrated under reduced pressure. The resulting solid was washed with petroleum ether / ethyl acetate (PE / EA, 1:1) to give a brownish-yellow solid, compound 3 (2.7 g, 69% yield).

[0100] The NMR and mass spectrometry data of compound 3 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.05 (d, J =8.2 Hz, 2H), 7.93 (dd, J = 13.2, 8.2 Hz, 4H), 7.56 (d, J = 8.1 Hz, 2H), 4.62(d, J = 5.3 Hz, 2H), 3.31 (d, J = 7.9 Hz, 2H), 3.14 (d, J = 6.3 Hz, 2H), 1.38(s, 9H).. HRMS (ESI) m / z: C 21 H 26 Calculated N4O4: 398.1954 g; Measured: 421.1846 g [M + Na] + .

[0101]

[0102] Leukadherin-1 (421 mg, 1.0 mmol), 4-dimethylaminopyridine (DMAP, 183 mg, 1.5 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 288 mg, 1.5 mmol) were dissolved in 20 mL of N,N-dimethylformamide (DMF) and stirred at room temperature for 30 min to activate the carboxyl group of leukadherin-1. Compound 3 was then added, and the reaction was continued overnight at room temperature with stirring. After the reaction was complete, the mixture was purified by silica gel column chromatography to give a yellow solid compound 4 (304 mg, yield 38%).

[0103] The NMR and mass spectrometry data of compound 4 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J =8.2 Hz, 2H), 8.08-7.93 (m, 8H), 7.75 (s, 3H), 7.53 (d, J = 3.8 Hz, 1H), 7.43(d, J = 3.9 Hz, 1H), 7.33 (s, 5H), 5.50 (s, 2H), 5.25 (s, 2H), 3.32 (s, 2H), 3.14 (d, J = 6.4 Hz, 2H), 1.38 (s, 9H).

[0104] LRMS (ESI) m / z: C 43 H 39 Calculated value of N5O7S2: 801.2291; Measured value: 824.21 [M + Na] + .

[0105]

[0106] Compound 4 (100 mg, 0.12 mmol) was added to 5 mL of 1,4-dioxane hydrochloride solution and stirred at room temperature for 4 h. The solvent was removed by vacuum distillation, and the residue was dispersed in n-hexane by ultrasonication. The mixture was filtered to obtain a solid, which was dried to give an orange-red solid, compound 5 (92 mg, 85% yield). The NMR and mass spectrometry data of compound 5 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ8.20 (d, J = 8.1 Hz, 1H), 8.13 (d, J = 8.2 Hz, 1H), 8.00 (t, J =8.8 Hz, 8H), 7.75 (t, J = 4.3 Hz, 3H), 7.53 (d, J = 3.8 Hz, 1H), 7.43 (d, J =3.8 Hz, 1H), 7.32 (q, J = 12.2, 9.4 Hz, 5H), 5.51 (s, 2H), 5.26 (s, 2H), 3.57(d, J = 5.7 Hz, 2H), 3.03 (s, 2H). LRMS (ESI) m / z: C 38 H 31 Calculated value of N5O5S2: 701.1767; Measured value: 702.21 [M + H] + .

[0107]

[0108] Compound 5 (10 mg, 0.014 mmol) was dissolved in 3 mL of dimethyl sulfoxide (DMSO), and N,N-diisopropylethylenediamine (DIPEA, 20 μL, 0.115 mmol) and DSPE-PEG2000-NHS (42 mg, 0.014 mmol) were added sequentially. The mixture was sonicated until completely dissolved, yielding an orange-yellow solution. The reaction was stirred overnight at room temperature. After the reaction was complete, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed. Deionized water was changed every 2 hours for the first 4 hours, followed by dialysis overnight. After dialysis, the dialysate was lyophilized for 24 hours to obtain a yellow solid product, DSPE-PEG2000-Azo-LA1 (40 mg, 80% yield).

[0109] (2) The synthesis of the gemcitabine prodrug Gem-SS-Chol, the specific reaction steps are as follows:

[0110] 2-Hydroxyethyl disulfide (652 μL, 5.3 mmol) and pyridine (800 μL, 9.93 mmol) were added to 25 mL of dichloromethane (DCM) and stirred until homogeneous. Separately, methyl cholesterol chloroformate (2 g, 4.4 mmol) was dissolved in 10 mL of DCM and slowly added dropwise to the above system, stirred overnight at room temperature. After the reaction was complete, deionized water was added to terminate the reaction, and the mixture was extracted with DCM. The organic phases were combined, and the crude product was obtained by vacuum distillation. The crude product was purified by silica gel column chromatography to give colorless compound 11 (1.2 g, 47% yield).

[0111] The NMR and mass spectrometry data of compound 11 are as follows: 1H NMR (400 MHz, CDCl3) δ 5.43-5.37 (m,1H), 4.54-4.44 (m, 1H), 4.39 (t, J = 6.8 Hz, 2H), 3.89 (t, J = 5.8 Hz, 2H), 2.96 (t, J = 6.7 Hz, 2H), 2.90 (t, J = 5.8 Hz, 2H), 2.40 (dd, J = 5.4, 2.0Hz, 2H), 2.04-1.94 (m, 3H), 1.86 (d, J = 3.8 Hz, 2H), 1.50 (d, J = 4.6 Hz,6H), 1.34 (d, J = 7.9 Hz, 3H), 1.14 (dd, J = HRMS(ESI) m / z: C 32 H 54 Calculated O4S2: 566.3464; Measured: 589.3356 [M + Na] + .

[0112]

[0113] Compound 11 (1.7 g, 3.0 mmol) was added to 10 mL of DCM and stirred until completely dissolved. N,N'-disuccinimidyl carbonate (1.2 g, 4.5 mmol) and triethylamine (631 μL, 4.5 mmol) were added sequentially, and the mixture was stirred overnight at room temperature. After the reaction was complete, the solvent was removed by vacuum distillation to obtain the crude product, which was purified by silica gel column chromatography to give colorless compound 12 (0.9 g, 43% yield).

[0114] The NMR and mass spectrometry data of compound 12 are as follows: 1H NMR (400 MHz, CDCl3) δ 5.40 (d, J =5.0 Hz, 1H), 4.57 (t, J = 6.8 Hz, 2H), 4.48 (tdd, J = 10.6, 6.0, 4.4 Hz, 1H), 4.38 (t, J = 6.5 Hz, 2H), 3.00 (dt, J = 18.0, 6.7 Hz, 4H), 2.84 (s, 4H), 2.40(d, J = 3.1 Hz, 2H), 2.04-1.94 (m, 3H), 1.86 (d, J = 3.7 Hz, 2H), 1.60-1.45(m, 6H), 1.26 (t, J = 7.1 Hz, HRMS (ESI) m / z: C 37 H 57 Calculated NO₈S₂: 707.3526; Measured: 730.3415 [M + Na] + .

[0115]

[0116] Compound 12 (0.9 g, 1.3 mmol) was added to 10 mL of tetrahydrofuran (THF) and stirred until homogeneous. Separately, gemcitabine (513 mg, 1.95 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF) and mixed thoroughly with the above THF solution. Then, 250 μL of N,N-diisopropylethylamine was added, and the mixture was stirred at room temperature for 24 h. After the reaction was completed, the solvent was removed by vacuum distillation to obtain the crude product, which was purified by silica gel column chromatography to obtain colorless compound 13 (333 mg, yield 30%).

[0117] The NMR and mass spectrometry data of compound 13 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s,1H), 8.23 ​​(d, J = 7.7 Hz, 1H), 7.09 (d, J = 7.7 Hz, 1H), 6.31 (d, J = 6.5 Hz,1H), 6.16 (s, 1H) , 4.35 (s, 2H), 4.30 (s, 2H), 4.19 (s, 1H), 3.88 (d, J =8.6 Hz, 1H), 3.79 (s, 1H), 3.70-3.61 (m, 1H), 3.02 (d, J = 2.1 Hz, 4H), 2.31(t, J = 9.5 Hz, 2H), 1.99-1.77 (m, 5H), 1.51 (d, J = 6.5 Hz, 6H), 1.35-1.27(m, 3H), 1.18-1.03 (m, 7H), 0.96 (s, 5H), 0.89 (d, J = 6.3 Hz, 4H), 0.84 (dd,J = 6.6, 2.0 Hz, 7H), 0.65 (s, 4H). LRMS (ESI) m / z: C 42 H 63 Calculated value of F2N3O9S2: 855.3974; Measured value: 878.21 [M + Na] + .

[0118]

[0119] Example 2

[0120] Control group: Preparation of liposome formulation I (single-loaded LA1 liposome, LipLA1)

[0121] (1) Accurately weigh 9 mg of soybean lecithin, 2.2 mg of DSPE-mPEG2000, 2.5 mg of DSPE-PEG2000-Azo-LA1 and 3.9 mg of cholesterol into a 250 mL round-bottom flask, dissolve them in 5 mL of chloroform, distill under reduced pressure on a rotary evaporator to form a film, and dry it overnight in a vacuum drying oven to ensure it is fully dried.

[0122] (2) Add 3 mL of PBS to the above lipid film for hydration, and stir at 400 rpm at 65°C under nitrogen protection. o The solution was stirred in a water bath for 1 hour, and then sonicated until the solution became clear.

[0123] (3) The obtained solution was passed through a 0.45 μm and 0.22 μm polycarbonate membranes in sequence by an extruder to obtain a leukadherin-1 liposome formulation, denoted as LipLA1.

[0124] Control group: Preparation of liposome formulation II (single-loaded Gem-SS-Chol liposomes, Lip@GSCH)

[0125] (1) Accurately weigh 9 mg of soybean lecithin, 4.7 mg of DSPE-mPEG2000 and 3.9 mg of Gem-SS-Chol into a 250 mL round-bottom flask, dissolve them in 5 mL of chloroform, distill under reduced pressure on a rotary evaporator to form a film, and dry in a vacuum drying oven overnight to allow it to dry thoroughly.

[0126] (2) Add 3 mL of PBS to the above lipid membrane for hydration. Under nitrogen protection, stir with a magnetic stirrer at 400 rpm at 65°C. o The solution was stirred in a water bath for 1 hour, and then sonicated until the solution became clear.

[0127] (3) The obtained solution was passed through a 0.45 μm and 0.22 μm polycarbonate membranes in sequence by an extruder to obtain a single Gem-SS-Chol liposome formulation, denoted as Lip@GSCH.

[0128] Experimental group: Preparation of liposome formulation III (co-loaded liposome formulation, LipLA1@GSCH)

[0129] (1) Accurately weigh 9 mg of soybean lecithin, 2.2 mg of DSPE-mPEG2000, 4 mg of Gem-SS-Chol, and 2.5 mg of DSPE-PEG2000-Azo-LA1 into a 250 mL round-bottom flask, dissolve them in 5 mL of chloroform, distill under reduced pressure on a rotary evaporator to form a film, and dry it overnight in a vacuum drying oven to ensure it is fully dried.

[0130] (2) Add 3 mL of PBS to the above lipid membrane for hydration. Under nitrogen protection, stir with a magnetic stirrer at 400 rpm at 65°C. o The solution was stirred in a water bath for 1 hour, and then sonicated until the solution became clear.

[0131] (3) The obtained solution was passed through a 0.45 μm and 0.22 μm polycarbonate membranes in sequence by an extruder to obtain liposome formulation III, denoted as LipLA1@GSCH.

[0132] Example 3

[0133] Basic characterization was performed on the liposome formulation prepared in Example 1.

[0134] (1) Characterization Experiment 1: Morphological analysis of the co-loaded liposome formulation LipLA1@GSCH

[0135] like Figure 2 As shown in (f), the co-loaded liposome formulation LipLA1@GSCH has a regular spherical structure with a uniform particle size distribution, mainly distributed in the range of 120~160 nm.

[0136] (2) Characterization Experiment 2: Particle size, zeta potential and stability analysis of liposome formulation

[0137] The prepared co-loaded liposome formulation LipLA1@GSCH, the mono-loaded liposome formulation Lip@GSCH, and LipLA1 were diluted with PBS to appropriate concentrations at a dilution ratio of approximately 1:10. One mL of each sample was placed in a sample cell, and the system was kept in water at a temperature of 25°C. o Under C conditions, the particle size and zeta potential of each liposome formulation were determined using a zeta potential and particle size analyzer (Malvern Zetasizer, Nanozs90). For example... Figure 2 As shown in Figure (b), all three liposome formulations exhibit a uniform particle size distribution. The average particle size of LipLA1@GSCH is approximately 135 nm, the average particle size of Lip@GSCH is approximately 120 nm, and the average particle size of LipLA1 is approximately 145 nm, all of which meet the design requirements for nanoliposome formulations.

[0138] Stability test results are as follows Figure 2 As shown in (c) and (d), LipLA1, Lip@GSCH, and LipLA1@GSCH, dispersed in PBS buffer, all exhibited good storage stability during the 7-day observation period, with polydispersity index (PDI) less than 0.3, indicating that the three liposome formulations all possess excellent dispersion uniformity and no significant aggregation. Furthermore, the Zeta potentials of LipLA1, Lip@GSCH, and LipLA1@GSCH all exhibited slightly negative potential characteristics, effectively preventing rapid metabolic clearance of the liposome formulations in vivo and prolonging their circulation time. In summary, the construction schemes for LipLA1, Lip@GSCH, and LipLA1@GSCH are all reasonable and feasible, and the resulting formulations possess good physicochemical properties.

[0139] Example 4

[0140] (1) MTT assay for the in vitro cytotoxicity and in vitro antitumor activity of liposome preparations

[0141] To evaluate the in vitro cytotoxicity of each formulation, pancreatic cancer cells (KPC) were seeded in 96-well plates, with 3000 cells in logarithmic growth phase per well. Cells were incubated at 37°C. o Incubate overnight in a C20 incubator. Then, according to the experimental groups, different concentration gradients of drug formulations were added to each well. The LipLA1@GSCH group had a gradient based on the final Gem-SS-Chol concentration, with concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 μM. The drug concentration gradients for the other groups were consistent with those for the LipLA1@GSCH group. After drug addition, incubation was performed for 48 h. After incubation, the drug incubation solution in each well was discarded, and 100 μL of 1 mg / mL 3(4,5-dimethylthiazolium-2)2,5-diphenyltetrazolium bromide (thiazolium blue, MTT) solution was added to each well and incubated at 37°C. o C. Incubate in a 5% CO2 incubator in the dark for 4 h to allow succinate dehydrogenase in the mitochondria of live cells to reduce MTT to blue-purple formazan crystals. After incubation, discard the MTT solution in each well, avoiding contact with the formazan crystals at the bottom of the well. Add 200 μL of dimethyl sulfoxide (DMSO) to each well and shake on a multi-plate reader for 2 min to fully dissolve the formazan crystals. Measure the absorbance (OD) at 490 nm using a multi-plate reader and calculate the cell viability using the following formula:

[0142] Cell viability (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%.

[0143] The results are as follows Figure 3 As shown, under low drug concentration conditions, free Gem-SS-Chol was significantly less toxic to KPC cells than free gemcitabine (Gem), indicating that prodrugation modification of gemcitabine helps reduce its non-specific toxicity. Compared with Lip@GSCH, LipLA1@GSCH showed significantly enhanced killing ability against KPC cells, indicating that LipLA1@GSCH can enhance antitumor activity through the synergistic effect of LA1.

[0144] (2) In vitro ICD effect measurement

[0145] KPC cells were loaded at 2×10 4 Cells were seeded at a density of cells / well in 48-well cell culture plates and incubated at 37°C. oC. KPC cells were cultured in a 5% CO2 incubator for 12 hours. The blank control group (Con) was supplemented with culture medium, while the other groups were supplemented with complete culture medium containing the corresponding drugs. Six groups were set up: Con, Gem, Gem-SS-Chol, Lip@GSCH, LipLA1, and LipLA1@GSCH. After 48 hours of incubation, flow cytometry was used to detect the expression level of CRT protein on the cell surface of each group, quantifying the regulatory effect of different drug formulations on CRT protein expression in KPC cells. Simultaneously, the culture supernatant was collected, and ATP release was measured using an enhanced ATP detection kit (Beyotime Biotechnology). The results showed that a significant CRT positive signal was detected on the surface of KPC cells treated with LipLA1@GSCH, and the ATP release in the culture supernatant was high.

[0146] Place a sterile glass slide flat at the bottom of a 12-well cell culture plate, and add KPC cells at a density of 2 × 10⁶ cells / well. 4 Seeds were applied to the surface of a glass slide at a density of 1 / 2 well to 37°C. o C. After culturing in a 5% CO2 constant temperature incubator for 12 h, the same grouping treatment as above was performed. After incubation for 48 h, 4% paraformaldehyde was added for fixation for 15 min, and then anti-HMGB1 antibody was added at 4... o Incubated overnight at C, then incubated for 2 h at room temperature with AF594-labeled secondary antibody (1:200). After nuclear staining with DAPI, the expression level of HMGB1 was detected using a fluorescence imaging system. Results are as follows: Figure 4 As shown in (c), the expression level of HMGB1 in KPC cells treated with LipLA1@GSCH was significantly reduced. These results indicate that LipLA1@GSCH enhances immunogenic cell death in tumor cells.

[0147] (3) In vitro M2 macrophage repolarization experiment

[0148] C57BL / 6J mice were euthanized, and the femur and tibia were aseptically separated. Muscle tissue was removed, and the bone marrow was disinfected with 70% ethanol and washed with PBS. The epiphysis was removed to expose the bone marrow cavity. The bone marrow suspension was obtained by rinsing with PBS, filtered through a 200-mesh filter, and centrifuged at 1800 rpm for 5 min to collect cells. Cells were treated with erythrocyte lysis buffer for 4 min, centrifuged at 1800 rpm for 5 min to collect cells, resuspended in M-CSF medium, and seeded in 24-well plates at 37°C. oC. Cells were cultured under 5% CO2 conditions, designated as day 0. On day 3, medium containing M-CSF was added, and on day 7, the medium was replaced with IL-4 to induce polarization for 24 h, establishing an M2 macrophage polarization model. LipLA1 groups were set up with drug concentrations of 5, 2.5, 1.25, and 0.625 μM, with three replicates per group. Cells were collected after drug treatment, and the signals of anti-F4 / 80 FITC, anti-CD206 APC, and anti-CD86 PE were detected by flow cytometry to analyze the macrophage repolarization level. Results are as follows: Figure 5 As shown, this indicates that LA1 still has the function of regulating macrophage polarization after nano-processing.

[0149] Example 5

[0150] Evaluation of the in vivo antitumor efficacy of liposomal formulations based on myeloid cell regulation and chemotherapy enhancement

[0151] KPC cells in the logarithmic growth phase and in good growth condition were collected, digested with 0.25% trypsin to prepare a single-cell suspension, and the cell density was adjusted to 1×10⁻⁶. 7 Cells / mL were collected and kept in an ice bath for later use. Mice were anesthetized by a slow intraperitoneal injection of 200 μL of avorin. The hair on the right abdomen was removed, and the skin of the surgical area was disinfected with 75% alcohol. Under aseptic conditions, the abdominal skin and subcutaneous tissue were incised layer by layer to expose the pancreas. 25 μL of cell suspension was slowly injected into the pancreatic parenchyma, and the needle was left in place for a short time before being slowly withdrawn. After confirming the absence of intraperitoneal bleeding and abnormalities, the peritoneum and skin were sutured sequentially with aseptic sutures. The wound was disinfected again, completing the model construction.

[0152] Seven days after tumor implantation, 30 female C57BL / 6J tumor-bearing mice (6-8 weeks old) with uniform tumor size and good health were randomly divided into 5 groups of 6 mice each: PBS control group, Gem group, LipLA1 group, Lip@GSCH group, and LipLA1@GSCH group. All groups received intravenous administration via tail vein injection. The control group received an equal volume of PBS; the LipLA1 group and LipLA1@GSCH group received DSPE-PEG at the following dosages. 2000 The Azo-LA1 dosage, calculated as LA1, was 30 μg / mouse. In the Gem, Lip@GSCH, and LipLA1@GSCH groups, the dosage, calculated as Gem, was 3 mg / kg. The dosing regimen consisted of six injections, each 24 hours apart. In vivo experimental data were collected on day 12 (two days after the last administration). Results are as follows: Figure 6 As shown in (b), no significant abnormal fluctuations were observed in the overall body weight of mice in each group. Figure 6As shown in (c) and (d), the tumor weight in the LipLA1@GSCH group was significantly lower than that in other groups, indicating that it has a significant inhibitory effect on tumor growth. Figure 6 As shown in (e), compared with the PBS group, the LipLA1@GSCH group had fewer peritoneal tumor metastases, indicating that LipLA1@GSCH effectively inhibits the distant invasion of tumor cells.

[0153] After treatment, pancreatic tumor tissue from mice was collected, placed in DMEM medium, minced and ground, and filtered through a 70 μm filter to obtain a single-cell suspension. Immune cells were enriched by 30% Percoll density gradient centrifugation. The obtained immune cells were immediately stained and analyzed by flow cytometry. Results are as follows: Figure 7 As shown in (a) and (b), compared with the PBS group, the proportion of MDSCs (myeloid-derived suppressor cells) in the LipLA1@GSCH group was significantly reduced, and the ratio of M2 / M1 macrophages decreased; Figure 7 As shown in the results in (c), CD8+ was present in the tumor tissue of the LipLA1@GSCH group. + The proportion of T cells was significantly increased. These results indicate that the immunomodulatory nanoliposome formulation LipLA1@GSCH of this invention helps improve the tumor immunosuppressive microenvironment and enhance the anti-tumor immune response.

[0154] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any equivalent changes, modifications, or substitutions made by those skilled in the art based on the technical solutions disclosed in this invention should be covered within the scope of protection of this invention. The scope of protection of this invention should be determined by the claims.

Claims

1. A liposomal formulation based on myeloid cell regulation and chemotherapy enhancement, characterized in that, The product comprises a liposome carrier, a CD11b agonist prodrug, and a gemcitabine prodrug; the CD11b agonist prodrug is coupled to the surface of the phospholipid bilayer of the liposome carrier via a hypoxia-responsive azobenzene linker, and the gemcitabine prodrug is encapsulated within the phospholipid bilayer of the liposome carrier; the CD11b agonist prodrug is DSPE-PEG2000-Azo-LA1, and its structural formula is shown in Formula I. ； Where n is an integer between 44 and 45; The gemcitabine prodrug is Gem-SS-Chol, which is obtained by coupling the amino site of gemcitabine to cholesterol via a glutathione-responsive disulfide bond, and its structural formula is shown in Formula II: 。 2. The liposome formulation according to claim 1, characterized in that, The DSPE-PEG2000-Azo-LA1 molecule is formed by coupling leukadherin-1 with DSPE-PEG2000-NHS through an azobenzene structure. In the hypoxic environment of the tumor microenvironment, the azo bond breaks and leukadherin-1 is released. The structural formula of leukadherin-1 is: ; The structural formula of azobenzene is: ; The structural formula of DSPE-PEG2000-NHS is: 。 3. The liposome formulation according to claim 1, characterized in that, Gem-SS-Chol is a prodrug molecule formed by the coupling of the amino site of gemcitabine with cholesterol via a disulfide bond. Under the high glutathione environment of the tumor microenvironment, the disulfide bond breaks, specifically releasing free gemcitabine.

4. The liposomal formulation for regulating myeloid cells and enhancing chemotherapy efficacy according to any one of claims 1 to 3, characterized in that, The raw materials of the liposome formulation include lecithin, DSPE-mPEG2000, DSPE-PEG2000-Azo-LA1 and Gem-SS-Chol, with a mass ratio of (5~12):(1~4):(1~4):(1~5); preferably, the mass ratio is (7~10):(2~4):(2~4):(2~4); the optimal mass ratio is 9:2.2:2.5:

4.

5. The liposome formulation according to claim 4, characterized in that, The lecithin is at least one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, and PEGylated phospholipids, preferably soybean lecithin.

6. The liposome formulation according to claim 1, characterized in that, The liposome formulation has a particle size of 50-400 nm; preferably, the liposome formulation has a particle size of 90-180 nm; most preferably, The liposome formulation has a particle size of 120~145nm.

7. The liposome formulation according to any one of claims 1-6, characterized in that, The polydispersity index (PDI) of the liposome formulation in PBS buffer was 0.24±0.08 ~ 0.27±0.01, and the zeta potential was -2.51±0.39 ~ -1.71±0.

21. The encapsulation efficiency of gemcitabine in the liposome formulation was 78.33±2.87% ~ 86.31±3.52%, and the drug loading was 5.44±0.19% ~ 6.00±0.24%; the encapsulation efficiency of the CD11b agonist leukadherin-1 was 63.33±2.87% ~ 71.66±2.87%, and the drug loading was 1.06±0.05% ~ 1.21±0.05%.

8. A method for preparing a liposomal formulation for regulating myeloid cells and enhancing chemotherapy efficacy as described in any one of claims 1 to 7, characterized in that, Includes the following steps: (1) Synthesize DSPE-PEG2000-Azo-LA1 and Gem-SS-Chol functional molecules respectively; (2) Dissolve the liposome carrier raw material, DSPE-PEG2000-Azo-LA1 and Gem-SS-Chol in chloroform in proportion, mix evenly, remove chloroform by rotary evaporation under reduced pressure, and vacuum dry to form a drug-loaded lipid film, and then vacuum dry. (3) The lipid film is hydrated with PBS buffer, sonicated, and then passed through a polycarbonate membrane to obtain the rice liposome preparation.

9. The use of the liposomal formulation for myeloid cell regulation and chemotherapy enhancement according to any one of claims 1 to 8 in the preparation of a medicament for treating pancreatic ductal adenocarcinoma.

10. The application according to claim 9, characterized in that, The liposome formulation is an injectable preparation, preferably an intravenous injection preparation.

Images