Cascade response type drug delivery system for targeting matrix-rich solid tumor as well as construction method and application of cascade response type drug delivery system

Abstract

The invention discloses a cascade response type drug delivery system targeting matrix-rich solid tumors as well as a construction method and application of the cascade response type drug delivery system, and belongs to the technical field of biological medicines. The cascade response type drug delivery system is composed of a polymer core and a lipid shell layer, the polymer core has the function of responding to high-level glutathione / esterase in tumor cells to release anti-tumor drugs, and the lipid shell layer can target the tumor cells and make FAP-alpha + CAFs static. According to the drug delivery system provided by the invention, the defects of poor responsiveness, insufficient intra-tumor infiltration and relatively low immunogenicity of the existing immunotherapy are overcome, and the combined chemotherapy immunotherapy curative effect of the matrix-rich solid tumor is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a cascade-responsive drug delivery system targeting stroma-rich solid tumors, a construction method thereof, and an application thereof. Background Art

[0002] Immunotherapy represented by immune checkpoint inhibitors has completely changed the treatment mode of solid tumors and shows great potential in improving the prognosis of patients. However, most patients have immune resistance, especially those stroma-rich solid tumors such as breast cancer, pancreatic cancer, and prostate cancer.

[0003] Mechanomedicine is an emerging field that focuses on targeted tissue mechanics treatment, either by directly regulating the mechanical cues acting on cells or by impairing the cell's response to mechanics. Abnormal physical factors can not only affect the biological behavior of tumors but also change the immune phenotype of tumors by interfering with the tumor immune cycle. Cancer-associated fibroblasts (CAFs), as the main source of the extracellular matrix (ECM), change the tumor microenvironment (TMME) by secreting enzymes and cytokines required for matrix deposition and cross-linking. In addition, CAFs can make the immune ecosystem tend to a tolerant and immunosuppressive microenvironment through multiple mechanisms, thereby mediating the recruitment and functional differentiation of innate immune cells and adaptive immune cells. Therefore, targeting immune-CAFs communication may be a promising strategy to regulate immune dysregulation to limit cancer progression.

[0004] Regrettably, treatments that massively inhibit or deplete CAFs have produced contrary treatment results, which may be attributed to the loss of all myofibroblast-like CAFs. On the contrary, specifically knocking out FAP-α+ CAFs at the genetic level can enhance the anti-tumor immune response and limit tumor growth. This suggests that anti-tumor immunity can be enhanced by depleting specific subsets of CAFs.

[0005] Drugs such as angiotensin receptor blockers (ARBs) can block the angiotensin II signaling pathway and inactivate the myofibroblast state of CAFs, but direct injection of drugs may have an impact on normal physiological signaling pathways. For example, the AngII / AT1 signal can regulate blood pressure, and ARBs that inhibit this signal can induce hypotension and CAF reprogramming. Therefore, these therapies can only be applied to cancer in limited doses.

[0006] An early clinical trial for the treatment of advanced pancreatic cancer showed that ARBs have a moderate anti-cancer effect only at doses that cause hypotension. Therefore, how to increase the enrichment of ARBs in tumors and maximize the anti-tumor immune effect is a problem to be solved in the present invention. In addition, another obstacle to tumor immunotherapy is the poor intrinsic tumor immunogenicity. To improve tumor immunogenicity, current methods mostly utilize certain chemotherapeutic drugs, such as camptothecin derivatives (e.g., irinotecan), which can induce immunogenic cell death effects to promote the recruitment and activation of T cells. However, the extremely low conversion rate of active cytotoxic compounds, insufficient bioavailability, and potential off-target side effects seriously affect their clinical efficacy. These obstacles prevent patients from achieving a stable and complete immune response, thus driving the development of drug delivery systems that can synchronously and selectively regulate CAFs and enhance tumor immunogenicity. Summary of the Invention

[0007] Based on the above background analysis, the technical personnel of the present invention believe that the abnormal tumor mechanical microenvironment caused by the FAP-α positive cancer-associated fibroblast (CAF) subset and the low tumor immunogenicity caused by the low conversion efficiency of active chemotherapeutic drugs are two key obstacles that prevent patients with stroma-rich tumors from achieving a stable and lasting immune response. To solve this problem, the present invention ingeniously designs a cascade reaction nano-lipopeptide prodrug, which can render FAP-α+ CAFs quiescent and amplify tumor immunogenicity to enhance the mechanical immunotherapy efficacy of stroma-rich solid tumors.

[0008] The present invention includes the following technical solutions:

[0009] In the first aspect of the present invention, the present invention provides a cascade-responsive drug delivery system, characterized in that the drug delivery system has a core-shell structure, the core is a polymer core loaded with a drug and having glutathione-esterase cascade responsiveness, and the shell is a lipid shell layer loaded with an angiotensin II receptor blocker and an FAP-α cleavable peptide.

[0010] Specifically, the polymer core is prepared by a glutathione-responsive amphiphilic block polymer loading an esterase-responsive drug, and the glutathione-responsive amphiphilic block polymer is selected from the commonly used block polymers in the art that have amphiphilicity and contain glutathione-sensitive groups in their structures.

[0011] The glutathione-sensitive group can be reduced by glutathione, causing the chemical bond to break. In some embodiments of the present invention, the glutathione-sensitive group is selected from at least one of a monothiol bond, a disulfide bond, a trithiol bond, a dithioacetal bond, a monoselenide bond, and a diselenide bond.

[0012] In a specific embodiment of the present invention, the glutathione-sensitive group is selected from a disulfide bond.

[0013] In a preferred embodiment of the present invention, the glutathione-responsive amphiphilic block polymer is a poly(lactic-co-glycolic acid)-disulfide-methoxypolyethylene glycol copolymer (PLGA-SS-mPEG). The PLGA-SS-mPEG described in the present invention can be prepared by itself or obtained through commercial channels.

[0014] The esterase-responsive drug described in the present invention is selected from any anti-tumor active drug modified with an ester bond. The "ester bond modification" means constructing an ester bond, i.e., -COO-, in the molecular structure of the anti-tumor active drug using a modifying group. Those skilled in the art can select common modifying groups containing -COOH or -OH and couple them with -OH or -COOH in the anti-tumor active drug molecule through conventional technical means to form an ester bond. The anti-tumor active drug modified with an ester bond can be cleaved under the action of esterase to release the free anti-tumor drug, thereby playing a role in killing tumor cells.

[0015] In some embodiments of the present invention, the anti-tumor active drugs include but are not limited to SN-38, paclitaxel, paclitaxel derivatives, doxorubicin, epirubicin, camptothecin, camptothecin derivatives, cisplatin drugs, vinblastine, vincristine, docetaxel, gemcitabine, curcumin or salvianolic acid.

[0016] In a specific embodiment of the present invention, the anti-tumor active drug is SN-38, the modifying group is selected from vitamin E succinate, SN-38 contributes -OH, vitamin E succinate contributes -COOH, and the ester bond-modified SN-38 obtained by coupling is SN-38-vitamin E succinate (denoted as SV in the present invention), and its structural formula is as follows:

[0017]

[0018] Those skilled in the art of the present invention select vitamin E succinate as the modifying group to modify SN-38 with an ester bond, and use -COOH in the structure of vitamin E succinate and -OH in the structure of SN-38 to form an ester bond, thereby completing the ester bond modification of SN-38. Those skilled in the art should understand that vitamin E succinate is only used to illustrate the specific embodiment, rather than a limitation on the technical solution of the present invention. Those skilled in the art can select the modifying group capable of forming an ester bond according to the specific anti-tumor active drug loaded, and optimize and screen the modifying group according to parameters such as coupling stability and drug release rate. The selection, screening and optimization processes are all within the scope of the technical solution of the present invention.

[0019] In a specific embodiment of the present invention, the glutathione-responsive amphiphilic block polymer constituting the polymer core is PLGA-SS-mPEG, the esterase-responsive drug is SN-38-vitamin E succinate, and the mass ratio of PLGA-SS-mPEG to SN-38-vitamin E succinate is 10:(0.5-1.5), such as 10:0.5, 10:1, 10:1.5. Preferably, the mass ratio of PLGA-SS-mPEG to SN-38-vitamin E succinate is 10:1.

[0020] The shell of the cascade-responsive drug delivery system described in the present invention is a lipid layer formed by thin film hydration of phospholipids containing an angiotensin II receptor blocker and an FAP-α cleavable peptide.

[0021] The phospholipids are selected from one or a combination of two or more of dipalmitoyl phosphatidylcholine (DPPC), lecithin, cholesterol, and distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-mPEG).

[0022] In a specific embodiment of the present invention, the phospholipids are a combination of dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-mPEG).

[0023] In the present invention, the angiotensin II receptor blocker is selected from the sartan drugs, including but not limited to losartan, valsartan, irbesartan, telmisartan, candesartan, olmesartan medoxomil, and alisartan esters.

[0024] In a specific embodiment of the present invention, the angiotensin II receptor blocker is losartan.

[0025] The amino acid sequence of the FAP-α cleavable peptide described in the present invention is: Ac-Asp-Ala-Thr-Gly-Pro-Ala-Cys.

[0026] In a specific embodiment of the present invention, the lipid shell layer of the drug delivery system is formed by dispersing losartan and the FAP-α cleavable peptide in dipalmitoyl phosphatidylcholine, cholesterol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000, and then forming a lipid layer by thin film hydration.

[0027] Among them, the mass ratio of dipalmitoyl phosphatidylcholine, cholesterol, losartan, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, and the FAP-α cleavable peptide is 10:2:(0.5-1.5):1:(0.5-1). In a specific embodiment of the present invention, the mass ratio of dipalmitoyl phosphatidylcholine, cholesterol, losartan, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, and the FAP-α cleavable peptide is 10:2:1.5:1:0.5.

[0028] In the lipid shell of the drug delivery system provided by the present invention, since losartan is a lipophilic drug, it is uniformly dispersed inside the lipid layer; since the FAP-α cleavable peptide is an amphiphilic molecule, the FAP-α cleavable peptide is inserted on the outer surface of the lipid layer, which is more conducive to the FAP-α cleavable peptide exerting its targeting effect.

[0029] The principle of action and the cascade response process of the cascade-responsive drug delivery system provided by the present invention are as follows:

[0030] First-level response: The FAP-α cleavable amphiphilic peptide in the lipid layer can actively target the tumor site (FAP-α is a membrane-bound serine protease that is specifically expressed in CAFs and malignant tumor cells in more than 90% of human cancers), and cleavage occurs in the high FAP-α environment of the tumor tissue (the Gly-Pro-Ala site of the targeting peptide can be cleaved by FAP-α, and an acetyl group is designed at the N-terminus of the targeting peptide to prevent cleavage by dipeptidyl peptidase IV), which causes the lipid membrane to lose stability;

[0031] Second-level response: The unstable lipid membrane releases the angiotensin II receptor blocker losartan to regulate the quiescence of FAP-α+CAFs (losartan can reduce the expression of type I collagen by binding to AGTR and inhibiting the RhoA-YAP signaling axis of CAFs);

[0032] Third-level response: After the quiescence of FAP-α+CAFs, the secreted extracellular matrix components decrease, making the tumor mechanical microenvironment softer. Matrix softening can improve blood vessel perfusion and oxygenation, and then promote the infiltration of T lymphocytes, enabling more polymer cores to penetrate deep into the tumor and be phagocytosed; through programmed dissociation, the polymer core (PLGA-SS-mPEG-SV) responds to the high level of glutathione (GSH) in tumor cells and releases SV;

[0033] Fourth-level response: SV responds to the high level of esterase in tumor cells and cleaves to release the cytotoxic drug SN-38 to produce a strong tumor-killing effect and induce immunogenic death of tumor cells.

[0034] The cascade-responsive drug delivery system designed by the present invention solves the problems of poor responsiveness of existing immunotherapy, insufficient intratumoral infiltration, and low immunogenicity, and significantly improves the efficacy of combined chemotherapy immunotherapy for stroma-rich solid tumors.

[0035] In the second aspect of the present invention, the present invention provides a method for constructing the cascade-responsive drug delivery system described in the first aspect of the present invention, characterized in that the method comprises the following steps:

[0036] a1) Select a modifying group containing -COOH or -OH and couple it with -OH or -COOH in the anti-tumor active drug molecule to prepare an anti-tumor active drug modified with an ester bond;

[0037] a2) Mix the anti-tumor active drug modified with an ester bond with a glutathione-responsive amphiphilic block polymer to prepare a polymer core;

[0038] a3) Dissolve an angiotensin II receptor blocker, an FAP-α cleavable peptide, and phospholipids in an organic solvent, rotate and evaporate to form a liposome film, and add an aqueous solution containing the polymer core to the liposome film for hydration treatment to obtain a cascade-responsive drug delivery system.

[0039] In a specific embodiment of the present invention, the method for constructing the cascade-responsive drug delivery system includes the following steps:

[0040] b1) Couple vitamin E succinate with SN-38 through an ester bond to prepare SN-38 modified with an ester bond, namely SN-38-vitamin E succinate;

[0041] b2) Mix SN-38-vitamin E succinate with poly(lactic-co-glycolic acid)-disulfide-methoxypolyethylene glycol copolymer (PLGA-SS-mPEG) according to a mass ratio of (0.5 - 1.5):10, and obtain a polymer core by ultrasonic oscillation;

[0042] b3) Dissolve dipalmitoyl phosphatidylcholine, cholesterol, losartan, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, and FAP-α cleavable peptide in an organic solvent according to a mass ratio of 10:2:(0.5 - 1.5):1:(0.5 - 1), rotate and evaporate to form a liposome film, add an aqueous solution containing the polymer core to the liposome film for hydration treatment, and extrude the solution through a 220 - 420 nm filter membrane to obtain a cascade-responsive drug delivery system.

[0043] Preferably, in step b3), the mass ratio of the polymer core to the liposome film is (1 - 3):(1 - 2), specifically such as 1:1, 2:1, 3:2, and preferably 3:2.

[0044] In the third aspect of the present invention, the present invention provides an application of the cascade-responsive drug delivery system described in the first aspect of the present invention in at least one of the following:

[0045] c1) Application in the preparation of an anti-tumor product for treating matrix-rich solid tumors;

[0046] c2) Application in the preparation of an adjuvant for enhancing tumor immunotherapy.

[0047] The anti-tumor product has the efficacy of inhibiting tumor growth, tumor metastasis and / or tumor recurrence.

[0048] The matrix-rich solid tumor described in the present invention refers to a solid tumor with abundant cell matrix and a dense tumor surface. In the tumor microenvironment of this type of solid tumor, the fibrotic matrix will produce a hard extracellular matrix protein (ECM) to transmit strong signals, encouraging tumor cells to evade any immune therapy attack. Some solid tumor types, especially those associated with extensive fibrotic tumor stroma, respond poorly to immunotherapy, such as pancreatic ductal adenocarcinoma (PDAC). In addition, some cancers with inflammatory-derived chronic fibrosis as their susceptibility factors, such as hepatocellular carcinoma and breast cancer, also respond weakly to immunotherapy. Tumors with the above properties are all within the scope of the matrix-rich solid tumors described in the present invention.

[0049] In some embodiments of the present invention, the matrix-rich solid tumors include, but are not limited to, breast cancer, pancreatic cancer, prostate cancer, and liver cancer.

[0050] The tumor immunotherapy described in the present invention refers to enhancing the attack of immune cells on tumors by inhibiting inhibitory signaling pathways in the immune system through immune checkpoint inhibitors. Commonly used immune checkpoint inhibitors include anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA-4 antibodies.

[0051] The drug delivery system provided by the present invention improves the disadvantages of the conventional liposome delivery system, such as lack of targeting in intravenous administration, easy leakage and fusion of loaded drugs, by loading a FAP-α cleavable amphiphilic peptide on the surface of the lipid shell layer, and improves the tumor targeting of the drug delivery system; secondly, aiming at the disadvantage of the strong toxicity of the conventional chemotherapy drug SN-38, an SN-38-vitamin E succinate prodrug modified with an ester bond is designed, which can improve the bioavailability of SN-38 and reduce off-target toxicity, and make SN-38 have esterase responsiveness. By using a polylactic-co-glycolic acid copolymer modified with a disulfide bond to load an esterase-responsive drug as the polymer core, the disadvantage of slow drug release of the conventional polylactic-co-glycolic acid copolymer is improved.

[0052] In summary, the cascade-responsive drug delivery system provided by the present invention has the following three major innovative advantages:

[0053] Innovation in treatment strategy: The drug delivery system is used for combined chemotherapy and immunotherapy of matrix-rich solid tumors. It can not only specifically target the FAP-α+CAFs subpopulation in the extracellular matrix of tumor cells to restore them to a quiescent state, but also can be site-specifically decomposed into SN-38 cytotoxic drugs under the environment of high esterase and high glutathione in tumor cells. Benefiting from the precise elimination of external obstacles (rigid mechanical microenvironment) and the removal of internal obstacles (enhanced tumor immunogenicity), the body's immune response is effectively activated, achieving excellent tumor killing effects, and providing new ideas for the treatment of matrix-rich solid tumors.

[0054] Innovation of drug delivery system: The drug delivery system consists of a polymer core (disulfide-modified polylactic-co-glycolic acid copolymer loaded with ester-modified SN-38-vitamin E succinate), a lipid shell (loaded with angiotensin II receptor blocker losartan) and a targeting peptide (FAP-α cleavable peptide), which can sequentially target FAP-α+CAFs to make them static, and then the polymer core penetrates deep into the tumor, responding to high esterase and high glutathione in tumor cells to release the cytotoxic drug SN-38. This prodrug can improve the conversion efficiency of active ingredients in chemotherapeutic drugs, improve bioavailability, and significantly reduce off-target toxicity.

[0055] Has great transformation potential: The drug delivery system has tolerable biocompatibility for mechanical immunotherapy of matrix-rich solid tumors, overcoming the shortcomings of existing immunotherapy such as poor responsiveness, insufficient intratumoral penetration and low immunogenicity. It is expected to be expanded to other fibroproliferative solid tumors with similar properties. The ingredients used are all FDA-certified formulas, and have great prospects for clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The schematic diagram of the synthesis of the cascade response drug delivery system in the implementation of the present invention.

[0057] Figure 2 It is a transmission electron microscopy image of the cascade response drug delivery system in the implementation of the present invention.

[0058] Figure 3 Graph showing particle size distribution and dispersion stability of the cascade response drug delivery system in the implementation of the present invention.

[0059] Figure 4 UV-visible spectrum of the cascade response drug delivery system in the implementation of the present invention

[0060] Figure 5 The drug release kinetics curve of the cascade response drug delivery system in the implementation of the present invention is

[0061] Figure 6The cascade-responsive drug delivery system in the implementation of the present invention improves the performance of the extracellular matrix

[0062] Figure 7 The cascade-responsive drug delivery system in the implementation of the present invention inhibits the invasion and migration of tumor cells

[0063] Figure 8 The cascade-responsive drug delivery system in the implementation of the present invention has an effect of killing tumor cells in vitro

[0064] Figure 9 The cascade-responsive drug delivery system in the implementation of the present invention induces the effect of tumor immunogenic death

[0065] Figure 10 The effect diagram of the cascade-responsive drug delivery system in the implementation of the present invention inhibiting the growth and metastasis of matrix-rich triple-negative breast cancer tumors in mice

[0066] Figure 11 The effect diagram of the cascade-responsive drug delivery system in the implementation of the present invention improving the tumor mechanical microenvironment

[0067] Figure 12 The effect diagram of the cascade-responsive drug delivery system in the implementation of the present invention activating the tumor immune response

[0068] Figure 13 The effect diagram of the cascade-responsive drug delivery system in the implementation of the present invention resisting tumor recurrence

[0069] Figure 14 The effect diagram of the cascade-responsive drug delivery system in the implementation of the present invention inducing the immune memory response

[0070] Figure 15 The biosafety evaluation effect of the cascade-responsive drug delivery system in the implementation of the present invention Detailed implementation manners

[0071] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0072] Preparation and characterization of the cascade-responsive drug delivery system

[0073] The structural schematic diagram and synthesis process of the cascade-responsive drug delivery system described in the present invention are as Figure 1 shown. In this embodiment, the specific preparation process of the cascade-responsive drug delivery system is as follows:

[0074] S1: Preparation of SN-38-Vitamin E Succinate (SV)

[0075] Dissolve SN-38 (1 g), Vitamin E Succinate (2 g) and anhydrous pyridine (10 mL) in N,N-dimethylformamide (DMF, 100 mL) and cool in an ice bath. Subsequently, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.95 g) dissolved in DMF (20 mL) to the mixture and continue stirring at room temperature for 24 h. Remove DMF by rotary evaporation and add dichloromethane. Wash the organic phase successively with water and saturated sodium chloride, and dry with anhydrous sodium sulfate. Purify the SV sample by silica gel column chromatography using petroleum ether / ethyl acetate (3:2) as the eluent.

[0076] S2: Preparation of Polymer Core (PLGA-SS-mPEG-SV)

[0077] Dissolve PLGA-SS-mPEG and SV separately in DCM and mix them at different mass ratios (5%, 10% and 15%), preferably 10%. Place the mixture in an ice-water bath and ultrasonically oscillate for 15 s, then add 5% polyvinyl alcohol (PVA) solution and continue ultrasonication for 15 s. Finally, remove the solvent in the solution by vacuum distillation and wash and centrifuge (10000 rpm, 10 min) 3 times to obtain the PLGA-SS-mPEG-SV sample.

[0078] When SV forms a polymer core with PLGA-SS-mPEG at different ratios, the drug loading rate and encapsulation rate of the polymer core are as follows:

[0079] SV: PLGA-SS-mPEG 5％ 10％ 15％ Drug loading rate 3.93±0.25 6.86±0.40 8.36±0.65 Entrapment efficiency 71.53±5.62 68.96±2.07 59.30±4.27

[0080] S3: Preparation of Cascade Response-Type Drug Delivery System (PLGA-SS-mPEG-SV@LOS-Lip-FAP-α)

[0081] To achieve tumor targeting, dipalmitoylphosphatidylcholine (DPPC), cholesterol, losartan, distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG), and FAP-α cleavable peptide (sequence: Ac-Asp-Ala-Thr-Gly-Pro-Ala-Cys) were dissolved in a chloroform / methanol mixed solution at a mass ratio of 10:2:1.5:1:0.5, and a liposome film was obtained by rotary evaporation. Subsequently, PLGA-SS-mPEG-SV and the liposome film were added to the film at different mass ratios (1:1, 2:1, and 3:2) for hydration treatment. Subsequently, the solution was extruded through filters with pore sizes of 420 nm and 220 nm for 20 cycles to obtain the cascade-responsive drug delivery system PLGA-SS-mPEG-SV@LOS-Lip-FAP-α. After comparison, when the mass ratio of PLGA-SS-mPEG-SV to the liposome film was 3:2, the prepared drug delivery system had a more uniform particle size distribution and high nanoparticle consistency.

[0082] The prepared cascade-responsive drug delivery system (PLGA-SS-mPEG-SV@LOS-Lip-FAP-α) was characterized, and the results are as Figures 2 - 4 shown. According to the characterization results, it can be seen that the cascade-responsive drug delivery system prepared in the present invention has a uniform particle size distribution and good dispersion stability.

[0083] In vitro drug release experiment

[0084] Furthermore, to verify the drug release performance of the cascade-responsive drug delivery system prepared in the present invention, first, solutions with pH = 7.4, pH = 6.5, and pH = 6.5 + FAP-α protein were respectively prepared to detect the release rate of losartan (LOS) in the cascade-responsive drug delivery system (PLGA-SS-mPEG-SV@LOS-Lip-FAP-α). The results are as Figure 5 shown in A. When at pH 6.5 and in the presence of recombinant FAP-α protein, accelerated release of losartan was detected using liquid chromatography, indicating that under acidic conditions, hydrolysis and protonation of the FAP-α cleavable peptide disrupted the stability of the lipid layer, accelerating the release of losartan in the lipid layer. In addition, solutions with pH = 7.4, pH = 6.5, pH = 6.5 + FAP-α protein, and pH = 6.5 + FAP-α protein + GSH were respectively prepared to detect the release rate of SV in the cascade-responsive drug delivery system (PLGA-SS-mPEG-SV@LOS-Lip-FAP-α). The results are as Figure 5As shown in B, under the protection of the polymer core, the release kinetics of the SV prodrug was slightly slower than that of LOS under the same conditions. However, after exposure to 10 mM GSH, the release kinetics of SV was significantly accelerated, and approximately 49% was released within 48 hours, which was attributed to the disruption of the GSH-responsive polymer core structure and rapid payload release.

[0085] The biological activity of the cascade-responsive drug delivery system prepared in the present invention was detected, and the specific scheme was as follows:

[0086] 1. Effect on the extracellular matrix

[0087] To clarify the effect of the drug delivery system provided by the present invention on the extracellular matrix, technicians constructed nanoparticles with different formulations for comparison, and the specific grouping was as follows: ① control, ② PLGA-SS-mPEG@Lip-LOS (PL), ③ PLGA-SS-mPEG-SV (PS), ④ PS@Lip-LOS (PSL), and ⑤ PS@Lip-LOS-FAP-α (PSLF). Unless otherwise explained, the meanings represented by PL, PS, PSL, and PSLF in the following are the same as those in this experiment.

[0088] Subsequently, the present invention constructed 3D cell spheroids composed of 4T1 tumor cells and cancer-associated fibroblasts (CAFs) to evaluate the expression of extracellular matrix components. Cellular immunofluorescence showed that nanoparticles containing LOS significantly inhibited the expression of collagen-I and fibronectin in 3D cell spheroids, especially in the PSLF group, where this inhibitory effect was the strongest, due to the targeting effect of the FAP-α peptide further inhibiting the activity of CAFs.

[0089] 2. Effect on the migratory and invasive phenotypes of tumor cells

[0090] Considering that inactivated CAFs can weaken the crosstalk between them and tumor cells, the present invention further studied the effect of different treatment regimens on the migratory and invasive phenotypes of 4T1 tumor cells. The supernatant of CAFs co-cultured with different nanoformulations was collected and then cultured with tumor cells. The groupings G1-G5 in the figure represent control, PL, PS, PSL, and PSLF, respectively. The wound healing assay showed that the conditioned medium of CAFs pretreated with LOS-containing formulations could significantly reduce tumor migratory behavior, especially in the PSLF group ( Figure 7A - B). In contrast, the conditioned medium treated with PS had little effect on the migration of 4T1 tumor cells, indicating that the SV prodrug does not interfere with the function of CAFs. Meanwhile, similar experimental results were also observed in the transwell assay (with or without Matrigel coating). The conditioned medium of untreated CAFs promoted the downward migration of 4T1 cells, while in the PSLF pretreatment group, tumor invasion was significantly reduced ( Figure 7 C - E). These results indicate that the PSLF prepared in the present invention can reprogram CAFs, affect the interaction between CAFs and tumor cells by downregulating the expression of ECM proteins, thereby inhibiting the invasion and migration phenotypes of tumors.

[0091] 3. Analysis of tumor cell apoptosis staining

[0092] Flow cytometry apoptosis staining analysis showed that a large number of 4T1 tumor cell apoptosis was observed after co - incubation with nanoparticles containing SV, indicating that the SV prodrug can be converted into SN - 38 in tumor cells and exert a strong cytotoxic effect. Notably, the addition of LOS did not improve the cell - killing efficacy of the SV prodrug, which may be due to the lack of ECM components in in vitro cell culture ( Figure 8 ).

[0093] 4. Analysis of CRT and HMGB1 staining during tumor cell apoptosis

[0094] During tumor cell apoptosis, damage - associated molecular patterns, such as calreticulin (CRT), high - mobility group protein 1 (HMGB1), and adenosine triphosphate (ATP), can bind to pattern recognition receptors on the surface of dendritic cells (DCs), ultimately activating innate and adaptive immune responses. Consistent with expectations, the group containing the SV prodrug showed more CRT exposure and HMGB1 release ( Figure 9 ), indicating that PSLF can effectively kill tumor cells and induce stress - related immunogenic cell death, thereby activating the immune response.

[0095] 5. Evaluation of anti - tumor efficacy

[0096] Taking triple - negative breast cancer as an example, 1×10 6 4T1 cells were mixed with 5×10 5 CAFs cells in 100 μL cell suspension and injected in situ into the third pair of mammary pads of 6 - week - old female Balb / c mice to construct a mouse stroma - rich triple - negative breast cancer (TNBC) model. When the tumor volume increased to 50 mm 3 (about 7 days after tumor inoculation), the tumor - bearing mice were injected via the tail vein with PBS, PL, PS, PSL, or PSLF (equivalent to 20 mg kg -1 LOS and 10 mg kg-1 of SN-38), once every other day for a total of 5 times ( Figure 10 A). Subsequently, the tumor volume change and the survival time of the mice were monitored. When the tumor volume exceeded 1000 mm 3 or cachexia symptoms appeared, the mice were euthanized. Meanwhile, on the 35th day after tumor inoculation, the lung tissues of the mice were collected to evaluate the efficacy of inhibiting lung metastasis.

[0097] The control group and the PL group had little effect on tumor growth. In contrast, after treatment with the formulations containing the SV prodrug (including the PS, PSL, and PSLF groups), tumor growth was significantly inhibited ( Figure 10 B). Importantly, compared with PSL, PSLF could inhibit tumor growth to a greater extent and significantly prolong the survival time of the mice, which was due to the enhanced drug enrichment induced by the FAP-α peptide ( Figure 10 C). In addition, during the observation period, all treatment formulations had little effect on the body weight change of the mice, indicating the tolerance of the treatment ( Figure 10 D). Considering that triple-negative breast cancer is highly prone to spontaneous metastasis, we further evaluated the therapeutic efficacy of PSLF NPs on lung metastasis on the 35th day after tumor inoculation. Gross examination and histological observation of the lung tissues both showed that PSLF treatment significantly reduced the volume and number of metastatic tumor nodules ( Figure 10 E and Figure 10 F). The above results indicate that PSLF is a tolerable and safe treatment regimen that can effectively inhibit the growth and metastasis of tumors in the orthotopic stroma-rich mouse triple-negative breast cancer model.

[0098] 6. Effects on the tumor mechanical microenvironment

[0099] Considering that CAFs are the main components of the extracellular matrix, the present invention first analyzed the changes in the TMME after treatment. Immunofluorescence and immunohistochemical staining showed that after treatment with PL, PSL, and PSLF, the α-SMA + Ki67 + cell density in the tumor was significantly reduced, indicating that the activity and proliferation ability of CAFs were inhibited ( Figure 11 A). The groups containing LOS led to a significant decrease in the expression of collagen fibers and fibronectin ( Figure 11 B and Figure 11 C). And after treatment with PL, PSL, and PSLF, lysyl oxidase (LOX), the key enzyme catalyzing ECM protein cross-linking, was also significantly reduced ( Figure 11 D). The above data indicate that PSLF treatment can improve the tumor mechanical microenvironment by reprogramming CAFs.

[0100] 7. Activating the adaptive immune response

[0101] Since a rigid stroma restricts the distribution of T lymphocytes within tumors, we wondered whether normalization of the tumor mechanical microenvironment would alter the tumor immune phenotype. The enrichment of immune cells within tumors after treatment with different formulations was analyzed by flow cytometry. PSLF treatment induced the most infiltration of DCs, most of which were mature DCs( Figure 12 A). Mature DCs can effectively activate naive T cells and initiate an immune response. The proportion of CD8 + T cells in the PSLF group was 4.5-fold higher than that in the control group( Figure 12 B). More importantly, the infiltration of tumor-associated macrophages (TAMs) in the PSLF group was significantly reduced, with the greatest reduction in the immunosuppressive M2-TAMs subtype( Figure 12 C). Correspondingly, key indicators of immune positive responses, such as the CD8 + T / Tregs and M1-TAMs / M2-TAMs ratios were significantly increased in the PSLF group, which was attributed to the disruption of communication between CAFs and immune cells( Figure 12 D and Figure 12 E). To verify this conjecture, we further evaluated the expression of key serum cytokines. By ELISA, interleukin 6 (IL-6) that stimulates the proliferation of immune cells and tumor necrosis factor α (TNF-α) that promotes cytotoxic T cells were significantly upregulated after PSLF treatment, indicating that PSLF treatment induced a strong adaptive immune response( Figure 12 F and Figure 12 G).

[0102] 8. Induce long-term immune memory effect

[0103] Considering that TNBC is basically unresponsive to T lymphocyte-mediated immunotherapy, we were curious whether the immune-stimulating effect of PSLF NPs could potentially overcome the resistance of TNBC to immune checkpoint blockade therapy. The previously constructed orthotopic stroma-rich murine triple-negative breast cancer (TNBC) model was randomly divided into 4 groups: (1) control group; (2) aPD-L1 group; (3) PSLF group; (4) PSLF + aPD-L1 group. According to the treatment plan, PSLF NPs were intravenously injected once every other day for a total of 5 times, and at the same time, aPD-L1 antibody (3.75 mg kg -1 ) was intraperitoneally injected on days 8, 11, and 14 after tumor inoculation. On the 5th day after treatment, the primary tumor was resected. And on the 20th day after treatment, 1 × 10 6 4T1 cells and 5 × 10 5The growth of tumors in each group was then observed, and the spleen cells of the mice were collected to analyze the proportion of central memory T cells and effector memory T cells. Age- and sex-matched Naive mice were used as controls. Compared with Naive mice with rapid tumor progression, the tumor growth kinetics of mice treated with PSLF+aPD-L1 were significantly delayed ( Figure 13 A and Figure 13 B).

[0104] Correspondingly, effector memory T cells (TEM, CD44 + CD62L - ) subgroup was also significantly higher than that of naive mice ( Figure 14 A and Figure 14 B). These results provide compelling evidence that PSLF combined with aPD-L1 therapy can establish effective long-term memory and prevent tumor recurrence.

[0105] Given that combination therapy may also bring additional side effects, extensive toxicology testing was performed after PSLF+aPD-L1 treatment. H&E staining of the main organs of mice did not show obvious morphological damage ( Figure 15 A), and blood biochemical indices were all maintained within the normal range, indicating that the combined treatment was tolerable and biosafe ( Figure 15 B).

[0106] Benefiting from the continuous removal of external barriers and the unwinding of internal barriers, the drug delivery system-assisted mechanical immunotherapy provided by the present invention initiated a powerful anti-tumor immune response and established a long-lasting immune memory, providing a new perspective for the treatment of stroma-rich solid tumors.

[0107] 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 above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all 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 scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cascade response drug delivery system, characterized in that: The drug delivery system has a core-shell structure, wherein the core is a polymer core loaded with drugs and having glutathione-esterase cascade responsiveness, and the shell is a lipid shell layer loaded with angiotensin II receptor blocker and FAP-α cleavable peptide.

2. The drug delivery system according to claim 1, characterized in that The polymer core is prepared by loading an esterase-responsive drug on a glutathione-responsive amphiphilic block polymer; the glutathione-responsive amphiphilic block polymer is selected from commonly used block polymers in the art that are amphiphilic and contain a glutathione-sensitive group in the structure; the esterase-responsive drug is selected from any ester-modified drug with anti-tumor activity, and the ester-modification refers to constructing an ester bond in the molecular structure of the anti-tumor active drug using a modifying group, that is, selecting a common modifying group containing -COOH or -OH, and coupling it with -OH or -COOH in the anti-tumor active drug molecule through conventional technical means to form an ester bond.

3. The drug delivery system according to claim 2, characterized in that The glutathione sensitive group can be reduced by glutathione to break the chemical bond, and the glutathione sensitive group is selected from at least one of a monosulfide bond, a disulfide bond, a trisulfide bond, a dithioketal bond, a monoselenide bond, and a diselenide bond.

4. The drug delivery system according to claim 3, characterized in that The glutathione sensitive group is selected from disulfide bonds, and the glutathione responsive amphiphilic block polymer is polylactic acid glycolic acid-disulfide bond-methoxy polyethylene glycol copolymer, namely PLGA-SS-mPEG.

5. The drug delivery system according to claim 2, characterized in that The drugs with anti-tumor activity include SN-38, paclitaxel, paclitaxel derivatives, doxorubicin, epirubicin, camptothecin, camptothecin biologics, cisplatin drugs, vinblastine, vincristine, docetaxel, gemcitabine, curcumin or salvianolic acid.

6. The drug delivery system according to claim 5, characterized in that The drug with anti-tumor activity is SN-38, the modification group is selected from vitamin E succinic acid, and the prepared ester-bond modified SN-38 is SN-38-vitamin E succinic acid.

7. The drug delivery system according to any one of claims 2 to 6, characterized in that The glutathione-responsive amphiphilic block polymer constituting the polymer core is PLGA-SS-mPEG, the esterase-responsive drug is SN-38-vitamin E succinic acid, and the mass ratio of PLGA-SS-mPEG to SN-38-vitamin E succinic acid is 10:(0.5-1.5).

8. The drug delivery system according to claim 1, characterized in that The shell of the cascade responsive drug delivery system is a lipid layer formed by film hydration of phospholipids containing angiotensin II receptor blockers and FAP-α cleavable peptides, wherein the phospholipids are selected from one or a combination of two or more of dipalmitoylphosphatidylcholine, lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol; the angiotensin II receptor blocker is selected from sartan drugs, including losartan, valsartan, irbesartan, telmisartan, candesartan, olmesartan medoxomil, and olisartan medoxomil.

9. The drug delivery system according to claim 8, characterized in that The phospholipid is a combination of dipalmitoylphosphatidylcholine, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol; the angiotensin II receptor blocker is losartan; and the amino acid sequence of the FAP-α cleavable peptide is: Ac-Asp-Ala-Thr-Gly-Pro-Ala-Cys.

10. The drug delivery system according to claim 9, characterized in that The lipid shell layer of the drug delivery system is prepared by dispersing losartan and FAP-α cleavable peptide in dipalmitoylphosphatidylcholine, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000, and forming a lipid layer by film hydration; wherein the dipalmitoylphosphatidylcholine, cholesterol, losartan, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and FAP-α cleavable peptide are in a mass ratio of 10:2:(0.5-1.5):1:(0.5-1).

11. The method for constructing a cascade response drug delivery system according to any one of claims 1 to 10, characterized in that: The method comprises the following steps: a1) selecting a modifying group containing -COOH or -OH to couple with -OH or -COOH in the anti-tumor drug molecule to prepare an ester-bond-modified anti-tumor drug; a2) mixing the ester-modified anti-tumor active drug with the glutathione-responsive amphiphilic block polymer to prepare a polymer core; a3) dissolving angiotensin II receptor blocker, FAP-α cleavable peptide and phospholipid in an organic solvent, rotary evaporating to form a liposome film, adding an aqueous solution containing a polymer core into the liposome film for hydration treatment, and obtaining a cascade response drug delivery system.

12. The construction method according to claim 11, characterized in that: The method for constructing the cascade response type drug delivery system comprises the following steps: b1) coupling vitamin E succinic acid and SN-38 via an ester bond to prepare ester-modified SN-38, i.e., SN-38-vitamin E succinic acid; b2) mixing SN-38-vitamin E succinic acid and poly(lactic acid glycolic acid)-disulfide bond-methoxy polyethylene glycol copolymer (PLGA-SS-mPEG) at a mass ratio of (0.5-1.5):10, and ultrasonically vibrating to obtain a polymer core; b3) dissolving dipalmitoylphosphatidylcholine, cholesterol, losartan, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and FAP-α cleavable peptide in an organic solvent at a mass ratio of 10:2:(0.5-1.5):1:(0.5-1), rotary evaporating to form a liposome film, adding an aqueous solution containing a polymer core to the liposome film for hydration, and extruding the solution through a 220-420 nm filter membrane to obtain a cascade response drug delivery system.

13. Use of the cascade response drug delivery system according to any one of claims 1 to 10 in at least one of the following: c1) Use in the preparation of anti-tumor products for treating matrix-rich solid tumors; c2) Application in the preparation of tumor immunotherapy enhancing adjuvant; The anti-tumor product has the efficacy of inhibiting tumor growth, tumor metastasis and / or tumor recurrence.

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