An acoustophoretic nanocatalyst, a preparation method and application thereof

Abstract

The application discloses a sonodynamic nanocatalyst and a preparation method and application thereof, the preparation method constructs a copper-centered organometallic molecule, which adopts an acceptor-donor-acceptor (A-D-A) structure design and can generate active oxygen under ultrasonic stimulation after being prepared into nanoparticles. In order to enhance tumor targeting and immune regulation functions, the sonodynamic nanocatalyst is wrapped with M1 macrophage membranes overexpressing PD-1, and is jointly loaded into a 3D printed bioadhesive GelMA-based hydrogel together with lipid nanoparticles loaded with a targeted CD300ld small interfering RNA, so that precise implantation of the cervical canal is realized. After ultrasonic activation, the system realizes tumor-killing ROS generation and selective silencing of PMN-MDSC-derived immunosuppression.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an acoustic-dynamic nanocatalyst, its preparation method, and its application. Background Technology

[0002] Cervical cancer remains one of the leading causes of cancer-related morbidity and mortality among women worldwide, especially in advanced or recurrent cases, where traditional interventions such as surgery, radiotherapy, and chemotherapy have limited efficacy. Although the advent of immune checkpoint inhibitors (ICIs) has brought new hope to cervical cancer treatment, their clinical benefit is primarily limited to a small number of patients, which is related to the immunosuppressive nature of the tumor microenvironment (TME). This immunosuppressive microenvironment not only endows tumor cells with persistent immune evasion capabilities but also severely impairs the cytotoxic function of key effector cells (including CD8+ T lymphocytes and natural killer (NK) cells), thereby limiting the efficacy of existing immunotherapy strategies.

[0003] Recent research indicates that the tumor microenvironment (TME) is regulated by a complex network of intercellular signaling pathways and metabolic interactions, which collectively influence tumor progression, immunosuppression, and treatment resistance. Among the major cellular components of this network, myeloid-derived suppressor cells (MDSCs), particularly polymorphonuclear subtypes (PMN-MDSCs), play a crucial role in shaping the "immune-cold tumor" phenotype. PMN-MDSCs, by secreting immunosuppressive factors such as IL-17 and TGF-β, not only directly inhibit T cell and NK cell activity but also promote the recruitment of regulatory T cells (Tregs), matrix remodeling, and tumor angiogenesis. These combined effects construct a robust anti-tumor immune barrier, contributing to tumor recurrence and metastasis. Therefore, targeting and regulating the recruitment, activation, and functional mechanisms of PMN-MDSCs has become an important strategy for reprogramming the TME immune environment and overcoming treatment resistance in cervical cancer.

[0004] Furthermore, the immunosuppressive properties of PMN-MDSCs are closely related to the high expression of their cell surface receptor CD300ld. Activation of CD300ld triggers the STAT3 signaling cascade, thereby upregulating key immunosuppressive genes and driving metabolic reprogramming, enhancing the function and persistence of MDSCs in the TME. Small interfering RNAs (siRNAs) targeting CD300ld can achieve precise molecular targeting, restoring effector immune cell function by reducing MDSC-mediated immunosuppression, and transforming "immunely cold" tumors into "hot" tumors with better immune responses, thus improving the efficacy of immunotherapy.

[0005] However, successfully eradicating solid tumors requires not only immune modulation but also overcoming the physical and biochemical barriers of the tumor itself. In this regard, sonodynamic therapy (SDT), as an emerging treatment, activates sonosensitive agents through low-intensity ultrasound, generating reactive oxygen species (ROS), which then exert anti-tumor effects through organelle destruction and immunogenic cell death (ICD). Damage-associated molecular patterns (DAMPs) released during ICD, including calreticulin (CRT) and high-mobility group box 1 (HMGB1), can enhance antigen presentation and promote systemic anti-tumor immune responses. However, traditional inorganic sonosensitive agents (such as titanium dioxide (TiO2) and zinc oxide (ZnO) suffer from low ultrasound responsiveness and poor biodegradability, severely limiting their clinical translational potential.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a sonodynamic nanocatalyst, its preparation method, and its application. The sonodynamic nanocatalyst of this invention is based on a copper-organometallic hybrid molecule of porphyrin (CH-Cu), which combines the redox activity of copper ions with the extended π-conjugated system of the porphyrin skeleton. This structure can promote efficient intramolecular charge transfer and optimal frontier orbital energy level arrangement, thereby enhancing the ability to generate ROS under ultrasound irradiation. The highly conjugated and planar molecular structure of CH-Cu ensures its excellent chemical stability and dispersibility, which can support the continuous oxidative stress required for efficient sonodynamic therapy.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] The first aspect of this invention provides a method for preparing acoustic-dynamic nanocatalysts, the method comprising the following steps:

[0010] (a) Compound 1 and Ni-4 were added to an organic solvent to react. After the reaction was completed, the solvent was removed and the mixture was purified to obtain Ni-5. The structural formula of compound 1 is shown in Formula 1, and the structural formula of Ni-4 is shown in Formula II.

[0011]

[0012] In Formula I, R1 is n-undecyl; R2 is 2-hexyldecyl; in Formula II, R 3 It is phenyl;

[0013] (b) Under argon protection, LiAlH4 was added to Ni-5 tetrahydrofuran solution and stirred. Then, glacial acetic acid was added and extracted with dichloromethane. The organic phase was collected, dried, solvent removed and purified to obtain H-1.

[0014] (c) Under argon protection, phosphorus oxychloride was added to a 1,2-dichloroethane solution containing H-1 and dimethylformamide and refluxed. After the reaction was completed, the mixture was cooled, and then a saturated sodium acetate solution was added and stirred for a period of time. Subsequently, the mixture was extracted with dichloromethane, the organic phase was collected, and the organic phase was dried, the solvent was removed, and the mixture was purified to obtain H-CHO.

[0015] (d) Under argon protection, copper acetate was added to a tetrahydrofuran solution containing H-CHO and N,N-dimethylformamide and stirred. After the reaction was completed, the organic phase was extracted, collected, dried, solvent removed and purified to obtain Cu-CHO.

[0016] (e) Under argon protection, Cu-CHO, 2-(5,6-difluoro-3-oxo-2,3-dihydro-1H-inden-1-ylidene) malononitrile and chloroform were mixed, and pyridine was added dropwise to carry out the reaction under heating. After the reaction was completed, the mixture was cooled, and then methanol was added, filtered, and purified to obtain CH-Cu.

[0017] (f) Under ultrasonic and water bath conditions, CH-Cu tetrahydrofuran solution and DSPE-mPEG 5000 tetrahydrofuran emulsion were mixed, and after continuous ultrasonic treatment for a period of time, deionized water was added, the mixture was allowed to stand, filtered through a membrane, ultrafiltered, concentrated and dried to obtain the sonic dynamic nanocatalyst.

[0018] In this invention, compounds 1 and Ni-4 are existing products (Ni-4 is synthesized from Ni-1 according to the chemical synthesis steps shown in the diagram below), see existing literature KPBirin, IAAbdulaeva, AIPoddubnaya, YGGorbunova, AYTsivadze, Dyes Pigm. 2020, 181, 108550; and T.Duan, W.Feng, Y.Li, Z.Li, Z.Zhang, H.Liang, H.Chen, C.Zhong, S.Jeong, C.Yang, S.Chen, S.Lu, OARakitin, C.Li, X.Wan, B.Kan, Y.Chen, Angew.Chem.Int.Ed. 2023, 62, e202308832.

[0019] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0020] This invention relates to a sonodynamic nanocatalyst based on a porphyrin copper-organic metal hybrid molecule (CH-Cu), which combines the redox activity of copper ions with the extended π-conjugated system of the porphyrin skeleton. This structure promotes efficient intramolecular charge transfer and optimal leading orbital energy level alignment, thereby enhancing ROS generation capacity under ultrasound irradiation. The highly conjugated and planar molecular structure of CH-Cu ensures its excellent chemical stability and dispersibility, supporting the sustained oxidative stress required for efficient sonodynamic therapy. To achieve precise anatomical delivery, a customized 3D-printed smart hydrogel suppository was designed to precisely fit the cervix and posterior vaginal fornix structure of mice. This platform can simultaneously deliver the sonodynamic nanocatalyst and lipid nanoparticles encapsulating CD300ldsiRNA, achieving local enrichment and sustained release in the tumor region. After ultrasound activation, this dual-loaded hydrogel system, on the one hand, induces efficient tumor ablation mediated by ROS, and on the other hand, reverses PMN-MDSCs-driven immunosuppression by silencing CD300ld. To enhance immune regulation and tumor targeting, the sonodynamic nanocatalyst was biomimeticly coated onto the membrane of M1 macrophages overexpressing PD-1, giving it dual characteristics of immune mimicry and tumor homing. In vivo experiments showed that local cervical administration combined with ultrasound irradiation significantly inhibited the growth of primary and metastatic tumors, reduced malignant ascites, and potently activated anti-tumor immunity, while exhibiting good biocompatibility. In summary, this invention proposes a comprehensive therapeutic strategy integrating high-performance organometallic sonosensitive agents, biomimetic nanocarriers, and anatomically targeted local delivery. By simultaneously disrupting tumor structure, reshaping the immunosuppressive microenvironment, and reactivating immune effector function, it provides a novel and systematic local immunotherapy combined with sonodynamic therapy for immune tolerance issues not only in cervical cancer but also in other solid tumors, potentially changing existing treatment paradigms and concepts in this field. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0022] Figure 1 The results of characterization of CH-Cu, CH-CuNPs, LNPs, LNPs-siCD300ld and 3D printed hydrogel suppositories in Experimental Example 1 of this invention;

[0023] Figure 2 The results of ultrasound-activated CH-CuNPs generating reactive oxygen species (ROS) and the characterization of M1 macrophage membranes with high PD-1 expression in Experiment Example 2 of this invention;

[0024] Figure 3 The results are in vitro evaluations of material uptake, cytotoxicity, and ROS-induced apoptosis in U14 cells in Experiment Example 3 of this invention.

[0025] Figure 4 This is the experimental evaluation result of cell damage, immunogenic cell death, and PMN-MDSC-related mechanisms in Experiment Example 3 of the present invention;

[0026] Figure 5 The results of tumor enrichment, gene silencing and in vivo antitumor effects analysis of the 3D hydrogel suppository in Experimental Example 4 of this invention;

[0027] Figure 6 The results of H&E and immunohistochemical staining analysis and peritoneal metastasis inhibition assessment of mouse tumor tissue after treatment in Experiment Example 4 of this invention;

[0028] Figure 7 Flow cytometry analysis of immune cell populations in the spleen and tumor microenvironment after different treatments in Experiment Example 4 of this invention;

[0029] Figure 8 This is a comprehensive analysis of the differentially expressed genes and immune-related pathways in the orthotopic cervical cancer model after treatment with different preparations in Experimental Example 5 of this invention;

[0030] Figure 9 This is a flowchart illustrating the chemical synthesis from Ni-1 to Ni-4 and then to CH-Cu in an embodiment of the present invention. Detailed Implementation

[0031] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0032] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0033] This invention provides a method for preparing acoustic-dynamic nanocatalysts, the method comprising the following steps:

[0034] (a) Compound 1 and Ni-4 were added to an organic solvent to react. After the reaction was completed, the solvent was removed and the mixture was purified to obtain Ni-5. The structural formula of compound 1 is shown in Formula 1, and the structural formula of Ni-4 is shown in Formula II.

[0035]

[0036] In Formula I, R1 is n-undecyl; R2 is 2-hexyldecyl; in Formula II, R3 It is phenyl;

[0037] (b) Under argon protection, LiAlH4 was added to Ni-5 tetrahydrofuran solution and stirred. Then, glacial acetic acid was added and extracted with dichloromethane. The organic phase was collected, dried, solvent removed and purified to obtain H-1.

[0038] (c) Under argon protection, phosphorus oxychloride was added to a 1,2-dichloroethane solution containing H-1 and dimethylformamide and refluxed. After the reaction was completed, the mixture was cooled, and then a saturated sodium acetate solution was added and stirred for a period of time. Subsequently, the mixture was extracted with dichloromethane, the organic phase was collected, and the organic phase was dried, the solvent was removed, and the mixture was purified to obtain H-CHO.

[0039] (d) Under argon protection, copper acetate was added to a tetrahydrofuran solution containing H-CHO and N,N-dimethylformamide and stirred. After the reaction was completed, the mixture was extracted, the organic phase was collected, and the organic phase was dried, the solvent was removed, and the mixture was purified to obtain Cu-CHO.

[0040] (e) Under argon protection, Cu-CHO, 2-(5,6-difluoro-3-oxo-2,3-dihydro-1H-inden-1-ylidene) malononitrile and chloroform were mixed, and pyridine was added dropwise to carry out the reaction under heating. After the reaction was completed, the mixture was cooled, and then methanol was added, filtered, and purified to obtain CH-Cu.

[0041] (f) Under ultrasonic and water bath conditions, CH-Cu tetrahydrofuran solution and DSPE-mPEG 5000 tetrahydrofuran emulsion were mixed, and after continuous ultrasonic treatment for a period of time, deionized water was added, the mixture was allowed to stand, filtered through a membrane, ultrafiltered, concentrated and dried to obtain the sonic dynamic nanocatalyst.

[0042] In this invention, compounds 1 and Ni-4 are existing products (Ni-4 is synthesized from Ni-1 according to the chemical synthesis steps described below), see existing literature KPBirin, IAAbdulaeva, AIPoddubnaya, YGGorbunova, AYTsivadze, Dyes Pigm. 2020, 181, 108550; and T.Duan, W.Feng, Y.Li, Z.Li, Z.Zhang, H.Liang, H.Chen, C.Zhong, S.Jeong, C.Yang, S.Chen, S.Lu, OARakitin, C.Li, X.Wan, B.Kan, Y.Chen, Angew.Chem.Int.Ed. 2023, 62, e202308832. The synthetic reaction process from Ni-1 to Ni-4, and the preparation of the sonic nanocatalyst using the method of this invention, is as follows: Figure 9 As shown.

[0043] In some embodiments, in step (a), the reaction temperature is 100–120°C and the time is 20–28 h.

[0044] In some embodiments, in step (b), the stirring process is carried out at room temperature for 0.8 to 2 minutes.

[0045] In some embodiments, in step (c), the reflux reaction temperature is 85–90°C, the reaction time is 10–14 h, the temperature is cooled to 0°C, and the stirring treatment temperature is room temperature for 1.5–3 h.

[0046] In some embodiments, in step (d), the stirring reaction temperature is 60–80°C and the time is 1–3 hours;

[0047] In step (e), the heating reaction temperature is 60–80°C and the time is 10–14 h;

[0048] In step (f), the ultrasonic treatment is continued for 15 to 25 minutes, and the settling time is 20 to 28 hours.

[0049] Another embodiment of the present invention provides an acoustic dynamic nanocatalyst prepared by the above preparation method.

[0050] Another embodiment of the present invention provides the application of the acoustic dynamic nanocatalyst prepared by the above preparation method in combination with siRNA targeting CD300ld in the preparation of a drug for treating cervical cancer.

[0051] Another embodiment of the present invention provides a method for preparing a GelMA-based hydrogel for local treatment of cervical cancer, the preparation method comprising the following steps:

[0052] (1) The acoustic-dynamic nanocatalyst prepared by any of the preparation methods described in claims 1 to 5 is encapsulated in the membrane of M1 macrophages overexpressing PD-1 to obtain cell membrane-coated nanoparticles.

[0053] (2) Cell membrane-coated nanoparticles and lipid nanoparticles loaded with siRNA targeting CD300ld were added to a mixture of GelMA-based hydrogel prepolymers to obtain a precursor solution.

[0054] (3) The precursor solution was 3D printed to obtain GelMA-based hydrogel for the treatment of cervical cancer.

[0055] Preferably, the cell membrane-coated nanoparticles are prepared by the following method:

[0056] RAW264.7 macrophages were cultured to the logarithmic growth phase, and then IL-1β was added to a final concentration of 40-60 ng / mL. Palmitic acid DMSO solution was then added to a palmitic acid concentration of 20-30 μM and the cells were cultured for another 20-28 h. The cells were then collected and the cell membranes were extracted.

[0057] The cell membrane and the acoustic-dynamic nanocatalyst prepared by any of the preparation methods described in claims 1 to 5 are mixed and repeatedly co-extruded to obtain the cell membrane-coated nanoparticles.

[0058] The lipid nanoparticles carrying siRNA targeting CD300ld were prepared by the following method:

[0059] DSPE-PEG, DOTAP, DSPE-PEG-SA, DSPC and cholesterol were co-dissolved in chloroform, and the solvent was removed by rotary evaporation to form a uniform lipid film and then hydrated to obtain a lipid vesicle suspension.

[0060] The siRNA targeting CD300ld was added to a lipid vesicle suspension for incubation, repeated extrusion, and then ultrafiltration to obtain lipid nanoparticles loaded with the siRNA targeting CD300ld.

[0061] Another embodiment of the present invention provides a GelMA-based hydrogel for treating cervical cancer prepared by the above preparation method.

[0062] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0063] Example 1

[0064] This embodiment describes a method for preparing a sonic dynamic nanocatalyst, which includes the following steps:

[0065] (a) Compound 1 (117 mg, 0.1 mmol) and Ni-4 (84 mg, 0.12 mmol) were dissolved in glacial acetic acid (5 mL) and toluene (15 mL) in a round-bottom flask. The reaction mixture was stirred at 110 °C for 24 h. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The crude product was separated by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 5:1, volume ratio) to give a deep yellow solid Ni-5 (103 mg, yield 56%).

[0066] The above Ni-5 was characterized as follows:

[0067] ¹H NMR (400 MHz, chloroform-d): δ 8.74–8.70 (multiplet, 4H), 8.51 (doublet, J = 3.7 Hz, 2H), 8.23–8.19 (multiplet, 4H), 8.13–8.05 (multiplet, 4H), 7.92 (quartet, J = 5.0, 4.6 Hz, 6H), 7.72 (quintet, J = 2.8, 2.4 Hz, 6H), 7.10 (Single peak, 2H), 4.77 (double peak, J = 5.9 Hz, 4H), 3.07 (triple peak, J = 5.8 Hz, 4H), 2.23 (multiple peak, 2H), 2.12 (triple peak, J = 5.7 Hz, 4H), 1.72 (multiple peak, 5H), 1.64 (multiple peak, 5H), 1.48–1.35 (multiple peak, 31H), 1.12–0.82 (multiple peak, 57H).

[0068] 13C NMR (101 MHz, chloroform-d): δ 147.3, 144.3, 142.7, 140.8, 140.5, 139.6, 139.3, 135.9, 135.6, 134.0, 133.0, 132.5, 132.5, 131.4, 131.0, 130.3 (×2), 129.8, 128.6, 127.5, 127.1, 126.7, 125.9, 122 .4,122.0,119.3,117.6,117.2,114.6,53.8,37.4,30.9,30.7,30.6,29.3(×2),29.0,28.8,28.7(×2),28.6(×2),28.3(×2),28.2,28.1,24.5,24.3(×2),21.6,21.5,21.4,13.1,13.0(×2).

[0069] (b) Under argon protection, LiAlH4 (106 mg, 2.64 mmol) was added to a Ni-5 (970 mg, 0.53 mmol) tetrahydrofuran (THF, 50 mL) solution and stirred at room temperature for 1 minute. Glacial acetic acid (3 mL) was slowly added, followed by extraction with dichloromethane. The organic phase was collected and dried over anhydrous sodium sulfate for 1 hour. After removing the solvent, the crude product was separated by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 1:2, v / v) to give a deep yellow solid H-1 (536 mg, yield 57%).

[0070] The above H-1 is characterized as follows:

[0071] ¹H NMR (400 MHz, chloroform-d): δ 8.90 (doublet, J = 5.0 Hz, 2H), 8.76 (doublet, J = 11.3 Hz, 4H), 8.55 (double quartet, J = 6.5, 2.9 Hz, 4H), 8.31 (double quartet, J = 6.6, 2.8 Hz, 4H), 8.03–7.98 (multiplet, 6H), 7.81 (double quartet, J = 4.8, 1.8 Hz, 6H). H), 7.08 (single peak, 2H), 4.71 (double peak, J = 7.9 Hz, 4H), 3.11 (triple peak, J = 7.7 Hz, 4H), 2.15 (triple peak, J = 7.6 Hz, 6H), 1.75 (single peak, 4H), 1.64 (single peak, 4H), 1.51–1.30 (multiple peak, 33H), 1.13–0.75 (multiple peak, 65H), -2.35 (single peak, 2H);

[0072] 13C NMR (101 MHz, chloroform-d): δ 153.6, 148.6, 145.4, 142.6, 142.1, 140.9, 139.7, 137.0, 136.0, 135.6, 134.2, 133.8, 133.7, 132.7, 130.3, 127.4, 127.2, 126.8, 126.7, 126.6, 125.8, 122.2, 122.1 ,119.9,117.6,117.5,116.7,53.8,37.5,30.9(×2),30.7,30.6,29.3,29.2,29.1,28.9,28.8(×2),28.7(×2),28.6,28.4,28.3(×2),28.0,24.5,24.3,21.7,21.5,21.4,13.1,13.0,12.9.

[0073] (c) Under argon protection, phosphorus oxychloride (0.3 mL) was added to a solution of 1,2-dichloroethane (DCE, 15 mL) containing H-1 (355 mg, 0.20 mmol) and dimethylformamide (DMF, 0.6 mL). The mixture was heated under reflux for 12 h, cooled to 0 °C, and then saturated sodium acetate solution (20 mL) was slowly added. The mixture was stirred at room temperature for 2 h, extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate for 1 h to remove the solvent. The crude product was separated by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 1:1, volume ratio) to give H-CHO as a red solid (289 mg, yield 79%).

[0074] The above H-CHO was characterized as follows:

[0075] ¹H NMR (400 MHz, chloroform-d): δ 10.28 (single peak, 2H), 8.98 (double peak, J = 5.0 Hz, 2H), 8.82 (double peak, J = 10.7 Hz, 4H), 8.61 (double quartet, J = 6.5, 2.8 Hz, 4H), 8.35 (double quartet, J = 6.4, 2.9 Hz, 4H), 8.04 (quartet, J = 3.0 Hz, 6H), 7.83 (double quartet). , J = 4.4, 1.9 Hz, 6H), 4.80 (doublet, J = 7.8 Hz, 4H), 3.47 (triplet, J = 7.8 Hz, 4H), 2.23 (multiplet, 6H), 1.82 (singlet, 4H), 1.67 (triplet, J = 7.7 Hz, 4H), 1.55–1.32 (multiplet, 30H), 1.15–0.78 (multiplet, 60H), -2.32 (singlet, 2H).

[0076] 13C NMR (101 MHz, chloroform-d): δ 181.8, 155.1, 150.2, 146.9, 145.6, 145.0, 143.4, 141.9, 140.8, 138.4, 136.9, 136.6, 135.6, 135.1, 134.9, 134.2, 132.7, 129.4, 128.7, 128.5, 128.3, 128.0, 127 .9,127.0,121.4,119.5,117.9,55.2,38.9,32.1,31.9,31.7,31.1,30.5,30.4,30.2,29.9(×2),29.8,29.5(×2),29.4,29.2,28.8,25.7,25.6,25.4,22.8,22.7,22.6,14.2(×2),14.1.

[0077] (d) Under argon protection, copper acetate (Cu(CH3COO)2·H2O, 22 mg) was added to H-CHO (92 mg, 0.05 mmol) in a mixed solution of N,N-dimethylformamide (DMF, 4 mL) and tetrahydrofuran (THF, 15 mL). The reaction mixture was stirred at 70 °C for 2 hours. After the reaction was complete, the mixture was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate for 1 hour to remove the solvent. The crude product was separated by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 1:1, v / v) to give a red solid Cu-CHO (90 mg, 95% yield).

[0078] The above Cu-CHO was characterized as follows:

[0079] High-resolution mass spectrometry (HR-MS, MALDI): Calculated value C118H140CuN8O2S4[M+]: 1893.9312, Measured value: 1893.9324.

[0080] (e) Under argon protection, Cu-CHO (189 mg, 0.1 mmol), 2-(5,6-difluoro-3-oxo-2,3-dihydro-1H-inden-1-yl)malononitrile (115 mg, 0.5 mmol) and dry chloroform (10 mL) were added to a 100 mL double-necked flask; then pyridine (0.2 mL) was added dropwise; the reaction mixture was stirred at 70 °C for 12 h and cooled to room temperature; 70 mL of methanol was added to the reaction solution to precipitate the product, and after filtration, the crude product was separated by silica gel column chromatography (eluent: petroleum ether / chloroform = 4:5, volume ratio) to give a black solid CH-Cu (197 mg, yield 85%).

[0081] The above CH-Cu was characterized as follows:

[0082] High-resolution mass spectrometry (HR-MS, MALDI): Calculated value C142H144CuF4N12O2S4[M+]: 2317.9684, Measured value: 2318.9594.

[0083] (f) CH-Cu (0.5 mg) and DSPE-mPEG 5000 (20 mg) were dissolved in 1 mL of tetrahydrofuran (THF). The two solutions were then mixed in an ultrasonic water bath and subjected to continuous sonication for 20 minutes. 10 mL of deionized water (THF:water volume ratio of 1:9) was then rapidly injected. The resulting suspension was placed in a fume hood and allowed to stand for at least 24 hours to allow the THF to evaporate completely. The suspension was then filtered through a 0.45 μm microporous membrane and subjected to three rounds of centrifugation ultrafiltration using a Millipore centrifugal ultrafiltration system to remove residual THF and impurities. The final supernatant was dark green and homogeneous, which was the purified acoustic-dynamic nanocatalyst (denoted as CH-CuNPs).

[0084] Example 2

[0085] This embodiment describes a method for preparing cell membrane-coated nanoparticles, the method comprising:

[0086] RAW264.7 macrophages were cultured in complete culture medium (DMEM) to the logarithmic growth phase. To induce M1 polarization and PD-1 overexpression, IL-1β was added to the cells to a final concentration of 50 ng / mL. Palmitic acid (PA) was dissolved in sterile DMSO to prepare a 10 mM stock solution, which was then added to the culture system at a final concentration of 25 μM. After 24 hours of treatment, cell morphology was observed using an inverted microscope, and cells were collected. To extract the cell membrane, the collected cells were washed with pre-cooled PBS and subjected to five freeze-thaw cycles (alternating between a 37°C water bath and liquid nitrogen). Subsequently, the cell lysate was centrifuged at 1,000 × g for 5 minutes at 4°C to remove cell nuclei and impurities. The supernatant was collected and centrifuged again at 18,000 × g for 1 hour at 4°C. The supernatant was discarded, and the precipitate was the isolated cell membrane. Each 10 cm culture dish of cell membrane was mixed with 100 μl of PBS and stored at -80°C.

[0087] The extracted cell membrane was mixed with CH-CuNPs from Example 1 at a 1:1 ratio and repeatedly co-extruded using an Avanti PolarLipids (USA) micro extruder to coat the cell membrane onto the surface of the nanoparticles. The resulting cell membrane-coated nanoparticles (denoted as CH-CuNPs@MM) ​​were stored at 4°C for subsequent experiments.

[0088] Example 3

[0089] This embodiment describes a method for preparing lipid nanoparticles loaded with siRNA targeting CD300ld. The preparation method includes:

[0090] DSPE-PEG (10 mg), DOTAP (5 mg), DSPE-PEG-SA (10 mg), DSPC (25 mg), and cholesterol (2.5 mg) were co-dissolved in 10 mL of chloroform; the solvent was removed under reduced pressure using a rotary evaporator to form a uniform lipid film; the obtained lipid film was hydrated in 5 mL of water preheated to 65 °C with diethyl pyrophosphate (DEPC) under vigorous stirring for 10 minutes to obtain a lipid nanoparticle (LNPs) suspension.

[0091] A separate 1 OD-level siRNA targeting CD300ld (purchased from Suzhou Gemma Gene Co., Ltd., catalog number A10001) was diluted in 125 μL of DEPC-treated water and then slowly added dropwise to the above lipid nanoparticle suspension. The resulting mixture was incubated at 55°C with continuous stirring for 1 hour to achieve efficient siRNA encapsulation. The suspension was repeatedly extruded through a 200 nm polycarbonate membrane using an Avanti PolarLipids (USA) microextruder to obtain nanoparticles with uniform particle size. To remove unencapsulated siRNA and concentrate the nanoparticles, the suspension was centrifuged five times at 4,500 rpm using a centrifugal ultrafilter with a molecular weight cutoff of 50 kDa. The resulting lipid nanoparticles encapsulating CD300ld siRNA (denoted as LNPs-siCD300ld or LNPs-si) were resuspended in DEPC-treated water and adjusted to the required concentration for subsequent in vitro and in vivo experiments. The sample was stored at 4°C for later use.

[0092] Example 4

[0093] This embodiment describes a method for preparing a GelMA-based hydrogel suppository for treating cervical cancer, the preparation method comprising the following steps:

[0094] Prepare a prepolymer mixed solution by dissolving 5% (w / v) gelatin-methacrylamide (GelMA), 0.25% (w / v) photoinitiator LAP, and 0.05% (w / v) UV absorber in PBS; to protect from light, wrap the solution in aluminum foil and store at room temperature.

[0095] According to experimental requirements, CH-CuNPs@MM and LNPs-siCD300ld were added to the prepolymer mixture in a 1:1 ratio to obtain the precursor solution;

[0096] The obtained precursor solution was used for photopolymerization 3D printing. The printed structure included a lower base (3mm×3mm×2.5mm) and an upper cylinder (1.5mm in diameter and 1mm in height), arranged in a 5×5 matrix. The printing conditions were a light intensity of 16mW / cm². 2Each layer is exposed for 16 seconds, and the initial bottom layer is exposed for 18 seconds; the formed three-dimensional hydrogel suppository (denoted as 3D printed hydrogel suppository) is immediately placed in a -80℃ refrigerator for storage.

[0097] Experimental Example 1

[0098] This experiment characterizes CH-Cu, CH-CuNPs, LNPs, LNPs-siCD300ld, and 3D-printed hydrogel suppositories.

[0099] This application utilizes rational molecular engineering strategies to design a novel acceptor-donor-acceptor (A–D–A) porphyrin-based small-molecule sonosensitive agent, CH-Cu. The molecule uses an electron-rich copper porphyrin macrocycle as the central donor, linked to an S,N-heterofused aromatic donor unit, with strongly electron-withdrawing indanone (INCN) groups derived from Y-series acceptors introduced at both ends. The resulting A–D–A configuration enhances intramolecular charge separation and transfer efficiency, with the central Cu... 2+ Ions significantly promote intersystem crossing, further endowing CH-Cu with excellent reactive oxygen species (ROS) generation ability under ultrasonic (US) excitation. Figure 1 (a) The electrostatic potential surface distribution shows that the molecule has space charge polarization characteristics, with positive charges mainly concentrated on the porphyrin ring and the S,N-heterofused aromatic skeleton, which facilitates the directional migration of electrons. Figure 1 b. Calculation of molecular orbitals using density functional theory (DFT) Figure 1 As shown in c), HOMO and LUMO are mainly distributed on the porphyrin ring and conjugated framework, with energy levels of –5.24 eV and –3.42 eV, respectively. Furthermore, cyclic voltammetry tests ( Figure 1 Figure d) shows band gaps of -5.59 eV and -3.63 eV, indicating that the molecule has a reasonable energy level structure. These results demonstrate that CH-Cu possesses an electrical structure that facilitates efficient charge separation and ROS generation, providing a solid foundation for its excellent acoustic-dynamic performance under ultrasonic activation.

[0100] To improve water solubility, biocompatibility, and stability, CH-Cu was co-assembled with DSPE-mPEG and prepared into uniform nanoparticles (CH-CuNPs) using a nanoprecipitation method. UV-Vis spectroscopy analysis showed a significant red shift in the absorption peak from the molecular state of CH-Cu (763 nm) to CH-CuNPs (820 nm), attributed to the π–π stacking and molecular aggregation effects within the nanostructure. Figure 1 (e). These interactions reduce the HOMO–LUMO band gap, stabilize the excited states, and contribute to enhanced acoustic-dynamic reactivity. Transmission electron microscopy (TEM) imaging confirmed that CH-CuNPs are monodisperse spherical nanoparticles. Figure 1(f) Further encapsulation with M1 macrophage membranes revealed a clear continuous coating layer, and successful membrane encapsulation was confirmed by SDS-PAGE and Coomassie Brilliant Blue staining, showing typical membrane protein bands. Stability tests of CH-CuNPs demonstrated excellent colloidal and thermal stability; the particle size remained stable and turbidity-free within 10 days of suspension in different media (DMEM, PBS, and deionized water). Furthermore, its UV-Vis spectrum remained largely unchanged under thermal stress ranging from 30–90℃, exhibiting good environmental adaptability.

[0101] Meanwhile, lipid nanoparticles (LNPs) and their siRNA-loaded derivatives (LNPs-si) were prepared via thin-film hydration-extrusion and ultrafiltration. TEM observations showed that both LNPs and LNPs-si particles exhibited uniform spherical shapes, and siRNA loading had minimal impact on their particle size and structural integrity. Figure 1 (g). Zeta potential analysis showed that the CH-CuNPs surface was negatively charged (-20mV), tending to be neutral (-14mV) after encapsulating the macrophage membrane, consistent with the native membrane potential (approximately -8mV), indicating that the surface modification was effective. Figure 1 (j). Dynamic light scattering (DLS) analysis indicated that the coated particle size increased from 137 nm to 193 nm. Figure 1 (k) further confirmed successful integration into the cell membrane. For LNPs-si, siRNA encapsulation reversed its surface charge, increasing the particle size to 177 nm while maintaining a narrow distribution. Figure 1 The results (in lm) confirm that siRNA loading can regulate its physicochemical properties and colloidal behavior.

[0102] To achieve localized and sustained drug release, we developed a hydrogel suppository conforming to cervical morphology, formulated with 5% GelMA prepolymer, in combination with CH-CuNPs@MM and LNPs-siCD300ld. The precursor solution was then 3D printed using digital light processing (DLP) to obtain a personalized suppository conforming to the anatomical structure of the mouse cervix and vaginal fornix. Figure 1 (i). Scanning electron microscopy (SEM) revealed a uniform porous structure inside the hydrogel. Figure 1 The presence of a pH value (h) is beneficial for high drug loading and controlled release. To simulate the acidic conditions of the vaginal microenvironment, a FITC-PEG probe was loaded into the hydrogel and incubated in pH 4.5 PBS. The results showed that the fluorescence gradually decreased over time, suggesting that it has pH-responsive degradation characteristics. Figure 1 (n). Meanwhile, in vitro release studies showed that GelMA-encapsulated CH-CuNPs could achieve sustained drug release for over 72 hours. Figure 1 The study verified the ability of hydrogels to sustain drug release and local delivery.

[0103] In summary, the experimental results in this section confirm the successful construction and systematic characterization of this multifunctional nanoplatform—which integrates a copper-centered organic molecular sonosensitive agent, gene-silencing lipid nanoparticles, and a 3D-printed hydrogel suppository targeting the cervix. It exhibits excellent physicochemical properties, biocompatibility, and functional tunability, providing a solid foundation for subsequent clinical translation in cervical cancer treatment.

[0104] Figure 1 In the image, (a) the chemical structure of the organic molecule CH-Cu; (b) the electrostatic potential (ESP) distribution of the CH-Cu molecule, with color gradients representing changes in charge density; (c) the DFT-calculated structure of the CH-Cu molecule, showing the electron density distribution and energy levels (-5.24 eV and -3.42 eV) of the frontier molecular orbitals (HOMO and LUMO); (d) the energy levels of CH-Cu determined by electrochemical testing; (e) the UV-Vis absorption spectra of CH-Cu in CHCl3 solution and in nanoparticle form; (f) TEM images of CH-CuNPs and macrophage membrane-coated CH-CuNPs (CH-CuNPs@MM), scale bar = 100 nm; and (g) the synthesized LNPs and LNPs-siCD3. (h) TEM image of 00ld(LNPs-si); (i) SEM images of the surface and cross-section of the 3D hydrogel suppository loaded with two types of nanoparticles simultaneously, scale bar = 10 μm; (j) Photograph of the 3D printed hydrogel suppository; (k) Zeta potential test results of CH-CuNPs, CH-CuNPs@MM and MM; (l) DLS particle size distribution curves of CH-CuNPs and CH-CuNPs@MM; (m) Zeta potential test results of LNPs, si-CD300ld and LNPs-si; (m) DLS particle size distribution curves of LNPs-si and LNPs; (n) Fluorescence microscopy images showing the dissolution behavior of the 3D hydrogel suppository loaded with CH-CuNPs under PBS (pH 4.5) conditions at different time points (0, 6, 12, 24, 48, 72 h); (o) Release curve of CH-CuNPs from the 3D hydrogel suppository in PBS (pH 4.5).

[0105] Experimental Example 2

[0106] This experiment demonstrates the preparation of M1 macrophage membranes with high PD-1 expression and reactive oxygen species (ROS) generated by CH-CuNPs under ultrasonic stimulation.

[0107] Under ultrasound (US) stimulation, CH-Cu generates localized high temperature and high pressure through acoustic cavitation, effectively inducing water decomposition and generating hydroxyl radicals (·OH). This also promotes the excitation of electrons in the CH-Cu molecule from the HOMO to the LUMO, forming electron-hole pairs. The excited-state electrons further react with molecular oxygen to generate reactive oxygen anions (·O2).- The generation of these ROS was verified using Rhodamine B (Rh.B) and DPBF as selective probes. Figure 2 (a)

[0108] Quantitative analysis of ·OH generation ( Figure 2 As shown in b), ·OH formation is dependent on the US power density and the concentration of CH-CuNPs. Under the condition of a CH-CuNPs concentration of 25 μg / mL, the formation of ·OH increased with increasing ultrasonic power density (0, 0.5, 0.8, 1.0 W / cm²). 2 As the concentration of β-B gradually increases, the characteristic absorption peak of Rh.B decreases continuously within 25 minutes, with a maximum decrease of 60%. Furthermore, at a constant US power density of 1.0 W / cm², the absorption peak... 2 Under these conditions, as the CH-CuNPs concentration increased from 0 to 12.5, 25, and 50 μg / mL, the Rh.B absorption peak showed a concentration-dependent decrease, with a maximum decrease of nearly 80%. DPBF was used as a detector for O2. - A similar trend was observed when using indicators ( Figure 2 c). At 25 μg / mL CH-CuNPs, with increasing US power density (0, 0.3, 0.5, 0.8, 1.0 W / cm²), 2 As the concentration of CH-CuNPs increases, the absorbance of DPBF at 420 nm decreases rapidly within 5 minutes, with a maximum reduction of approximately 70%. Similarly, with the US parameter remaining constant, increasing the concentration of CH-CuNPs also leads to a more significant decrease in DPBF absorbance, further demonstrating its obvious concentration dependence, with a maximum decrease exceeding 70%.

[0109] To further verify the type of ROS generated, 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) was used as a spin trapping agent, and electron spin resonance (ESR) spectroscopy was used to simultaneously analyze hydroxyl radicals and superoxide anion radicals. Figure 2 (d). The obtained ESR spectrum exhibits a quartet characteristic corresponding to ·OH, accompanied by ·O2. - The diffuse, broad signal is consistent with previous reports. In summary, these results strongly demonstrate that CH-CuNPs can efficiently and synchronously generate ·OH and ·O2 under ultrasonic activation. - Free radicals. The generation of ROS by CH-CuNPs is closely related to the US power density, nanoparticle concentration, and exposure time. This controllable and potent ROS generation activity under ultrasound irradiation highlights the great potential of CH-CuNPs as a highly efficient sonication agent in therapeutic applications.

[0110] Previous studies have shown that IL-1β and / or palmitic acid (PA) can enhance PD-1 expression on the cell membrane surface and promote macrophage polarization towards the M1 type. To obtain M1 macrophage membranes with high PD-1 expression, we systematically evaluated the regulatory effects of IL-1β and / or PA on PD-1 expression and macrophage polarization in RAW264.7 cells. Western blot analysis ( Figure 2 (e) showed that both IL-1β and PA significantly upregulated PD-1 protein expression. Compared with the control group, the synergistic effect of the combination of the two was most pronounced in PD-1 overexpression. Based on this, subsequent experiments all used a combined stimulation protocol of IL-1β and PA. Flow cytometry analysis of macrophage polarization dynamics revealed that with prolonged stimulation time (0, 12, 24 h), the expression of the M2 marker CD206 gradually decreased, while the expression of the M1 marker CD86 continuously increased, leading to a continuous increase in the M1 / M2 ratio. Figure 2 (fg). These results indicate that macrophages are gradually transforming into the pro-inflammatory and anti-tumor M1 phenotype.

[0111] Cell morphological changes were systematically observed at different time points (0, 12, 24 h) using an inverted microscope. The results showed that after treatment with IL-1β and PA, RAW264.7 cells increased in size, had abundant cytoplasm, significantly increased pseudopodia, and exhibited a more rounded cell morphology, all reflecting M1 polarization characteristics. Figure 2 h). Correspondingly, confocal laser scanning microscope (CLSM, Figure 2 (i) It was also found that the expression of CD86 and PD-1 significantly increased in a time-dependent manner after combined stimulation, which was consistent with the results of flow cytometry and Western blot. After the above treatment, M1 macrophage membranes with high PD-1 expression were extracted by multiple freeze-thaw cycles and water baths and stored at -80°C. Subsequently, the above membranes were coated onto the surface of CH-CuNPs using a membrane extruder. Figure 2 The experimental procedures and mechanistic relationships of macrophage phenotypic transformation, PD-1 upregulation, and membrane separation are systematically described in the Chinese paper.

[0112] In summary, synergistic stimulation of IL-1β and PA can significantly induce macrophages to polarize to the anti-tumor M1 type and significantly upregulate PD-1 expression.

[0113] Figure 2(a) Schematic diagram of ultrasonic activation of CH-CuNPs to generate reactive oxygen species, detected by Rhodamine B (Rh.B) and DPBF methods; (b) Quantitative analysis of ·OH generated by CH-CuNPs under different ultrasonic power densities (left) and different CH-CuNPs concentrations (fixed ultrasonic power, right), all characterized by Rh.B degradation; (c) ·O2 generated by CH-CuNPs under different ultrasonic power densities (left) and different CH-CuNPs concentrations (right). - Quantitative analysis was performed using DPBF absorption attenuation as the characterization method; (d) ESR spectroscopy confirmed that CH-CuNPs produced ·OH (left) and ·O2 under ultrasonic treatment. - (Right) Free radicals; (e) Western blot analysis of PD-1 protein expression in RAW264.7 cells under different treatment conditions; (f) Flow cytometry analysis of macrophage polarization in RAW264.7 cells stimulated with PA and IL-1β for 0, 12, and 24 hours; (g) Quantitative analysis of the M1 / M2 macrophage ratio in RAW264.7 cells based on flow cytometry data; (h) Representative morphological images of RAW264.7 cells under an inverted microscope at different stimulation times (0, 12, and 24 hours), scale bar = 50 μm; (i) CLSM images of RAW264.7 cells stimulated with PA and IL-1β, showing CD86, PD-1, and DAPI nuclear staining expression, scale bar = 50 μm; (j) Schematic diagram of IL-1β and PA-induced M1 polarization in RAW macrophages and extraction of the membrane of M1 macrophages with high PD-1 expression.

[0114] Experimental Example 3

[0115] This experimental example is an evaluation of in vitro antitumor performance:

[0116] To systematically evaluate the biocompatibility of CH-CuNPs, the effects of different concentrations of CH-CuNPs on the viability of U14, HeLa, and NCM460 cells were first examined using a CCK-8 assay. The results showed that even at concentrations as high as 150 μg / mL, the viability of all three cell lines exceeded 90%, indicating that CH-CuNPs exhibited extremely low toxicity to both tumor and normal cells in the absence of ultrasound stimulation. Furthermore, primary PMN-MDSCs isolated from the spleens of cervical cancer-bearing mice were co-incubated with different concentrations of LNPs; even at the highest tested concentration (150 μg / mL), cell viability remained at a high level. These results demonstrate that both CH-CuNPs and LNPs possess excellent biocompatibility, highlighting their promising prospects for biomedical applications.

[0117] To further evaluate the effects of different surface modifications on cellular uptake of CH-CuNPs, three control groups were designed. First, CH-CuNPs were thoroughly mixed with FITC-PEG for 12 hours as a bare nanoparticle control group. Subsequently, fluorescently labeled CH-CuNPs were coated with ordinary macrophage membranes and PD-1-overexpressing M1 macrophage membranes, respectively, and CH-CuNPs with different surface modifications were obtained through multiple extrusion mixing. The obtained bare CH-CuNPs, CH-CuNPs coated with ordinary macrophage membranes, and CH-CuNPs coated with high PD-1-expressing M1 macrophage membranes were co-incubated with U14 cells and analyzed using a confocal laser scanning microscope (CLSM). Figure 3 (a) Cellular uptake of nanoparticles was observed and quantitatively analyzed. Results showed that the intracellular fluorescence intensity of the naked CH-CuNP group gradually increased with prolonged incubation time, indicating a time-dependent uptake. In contrast, the macrophage membrane-coated group, especially the CH-CuNPs group coated with high PD-1-expressing M1 macrophage membranes, showed a significant increase in intracellular green fluorescence within 1–6 hours, indicating a substantial improvement in cellular uptake efficiency. In conclusion, PD-1-highly-expressing M1 macrophage membrane coating significantly promotes the uptake of CH-CuNPs by U14 cells. Therefore, subsequent experiments used CH-CuNPs@MM modified with high PD-1-expressing M1 macrophage membranes for further research.

[0118] The effects of different treatments on U14 cell viability were further evaluated using a CCK-8 assay. Figure 3 (b) The results showed that cell viability did not change significantly in the PBS control group, ultrasound group, and CH-CuNPs treatment group. However, CH-CuNPs significantly reduced cell viability after ultrasound stimulation (CH-CuNPs+US), and the group treated with CH-CuNPs@MM combined with ultrasound (CH-CuNPs@MM+US) showed the largest decrease in cell viability, approximately 60%, highlighting a synergistic anti-tumor effect. This confirms that membrane coating of macrophages with high PD-1 expression combined with ultrasound stimulation can significantly enhance the therapeutic effect of CH-CuNPs on tumor cells.

[0119] To verify the ability of CH-CuNPs to promote ROS generation under ultrasound stimulation, the intracellular ROS content of U14 cells under different treatments was analyzed by fluorescence microscopy and flow cytometry using the DCFH-DA fluorescent probe. Figure 3(cd). The results showed that ROS production was low in the PBS control, ultrasound alone, and CH-CuNPs-only groups, while ROS levels were significantly increased in the CH-CuNPs+US group, with the highest level observed in the group using PD-1-overexpressing M1 macrophage membrane-coated CH-CuNPs combined with ultrasound (CH-CuNPs@MM+US). These results indicate that PD-1-overexpressing M1 macrophage membrane coating can enhance the tumor-targeted uptake effect of CH-CuNPs, thereby further enhancing ultrasound-induced ROS production, strongly supporting the development of efficient and precise cancer treatment strategies.

[0120] Under ultrasound, CH-CuNPs can be activated to produce reactive oxygen species, inducing oxidative stress in tumor cells, leading to apoptosis or necrosis. To further verify the promoting effect of US-activated CH-CuNPs on tumor cell death, cell viability and cell death in each treatment group were quantitatively analyzed. Calcein-AM / PI double staining (… Figure 3 (e) Combined with ImageJ quantification, the number of PI-positive (dead) cells was significantly increased in the CH-CuNPs+US group and the CH-CuNPs@MM+US group, while the PBS control, ultrasound alone, and CH-CuNPs alone groups were dominated by surviving (calcein-positive) cells. Annexin V-FITC apoptosis assay further confirmed that the proportion of late apoptotic cells in the CH-CuNPs+US group and the CH-CuNPs@MM+US group was as high as 42.3% and 48.6%, respectively, which was significantly higher than that in the control group (e). Figure 3 (f). The above results collectively demonstrate that US-stimulated CH-CuNPs can efficiently generate reactive oxygen species, induce oxidative stress-mediated and programmed tumor cell death, and exhibit significant sonodynamic antitumor potential.

[0121] Under ultrasound activation, CH-CuNPs generate potent ROS, directly leading to loss of mitochondrial membrane potential (ΔΨm) and mitochondrial dysfunction in tumor cells, and causing irreversible damage to nuclear DNA. Furthermore, excessive oxidative stress can promote the exposure or release of damage-related molecules such as CRT and HMGB1, thereby triggering immunogenic cell death (ICD), activating specific anti-tumor immune responses, and effectively enhancing the overall therapeutic effect in vivo. CLSM observation after JC-1 staining (…) Figure 4 As shown in (a), the PBS control, ultrasound alone, and CH-CuNPs group exhibited predominantly red fluorescence, indicating that the mitochondrial membrane potential remained normal. In the CH-CuNPs+US group, especially the CH-CuNPs@MM+US group, green fluorescence was significantly enhanced while red fluorescence decreased, with the latter being particularly pronounced, suggesting severely impaired mitochondrial function. γ-H2AX immunofluorescence analysis ( Figure 4(b) shows that the γ-H2AX nuclear green fluorescence signal was significantly enhanced in the CH-CuNPs+US and CH-CuNPs@MM+US groups, indicating DNA damage. CLSM was used for the detection of ICD markers ( Figure 4 The results (CCD) showed that the CH-CuNPs+US group, especially the CH-CuNPs@MM+US group, exhibited significantly increased CRT exposure and HMGB1 translocation from the nucleus to the cytoplasm, indicating a markedly enhanced ICD effect. In summary, these results strongly confirm that ultrasound-activated CH-CuNPs can synergistically induce oxidative stress, mitochondrial and nuclear damage, and induce immunogenic tumor cell death, thereby enhancing the host's anti-tumor immune response. This provides a solid mechanistic basis for highly efficient and immunosynergistic sonodynamic tumor therapy.

[0122] PMN-MDSCs are a class of myeloid cells with potent immunosuppressive functions that accumulate in large numbers in the tumor microenvironment (TME). Through metabolic reprogramming and the secretion of various cytokines, they significantly inhibit the activity of T cells and NK cells, thereby promoting immune escape and tumor progression. CD300ld, a transmembrane immune regulatory receptor, is highly expressed on PMN-MDSCs, further enhancing their migration ability and immunosuppressive function. In cervical cancer, CD300ld+ PMN-MDSCs are highly enriched at the tumor site, exacerbating immunosuppression in the local microenvironment and promoting tumor growth and metastasis. Therefore, these cells are a key factor driving cervical cancer progression. Based on these mechanisms, using siRNA to target and silence CD300ld is a promising strategy to attenuate PMN-MDSC-mediated immunosuppression. Downregulating CD300ld expression can effectively weaken the immunosuppressive function of PMN-MDSCs, thereby enhancing the anti-tumor immune response of effector T cells and NK cells.

[0123] To ensure a reliable cellular basis for subsequent functional experiments, PMN-MDSCs were efficiently isolated from the spleen of cervical cancer-bearing mice, and their purity was rigorously verified by flow cytometry. (Ly6G) + CD11b + The cell proportion was as high as 85.7% ( Figure 4 (e). Comprehensive characterization, including negative controls during sorting, Giemsa staining, and DAPI confocal imaging, showed that the isolated cells possessed typical polymorphonuclear cell characteristics, demonstrating high cell population purity and good enrichment. Subsequently, the expression level of CD300ld in different tumor-associated cell populations was detected. qRT-PCR results showed that CD300ld mRNA expression in PMN-MDSCs from the spleen of cervical cancer-bearing mice was significantly higher than that in neutrophils, RAW264.7 cells, and U14 tumor cells from the spleen of healthy mice, and U14 cells showed almost no CD300ld expression. Figure 4(f). Considering that CD300ld expression could be detected in RAW264.7 cells and that the in vitro activity of primary PMN-MDSCs was limited, the screening of CD300ld-targeting siRNAs was conducted in RAW264.7 cells. Mouse CD300ld siRNA (sense strand: 5′-CCACAGUUCUGAAAUCCAUTT-3′, antisense strand: 5′-AUGGAUUUCAGAACUGUGGTT-3′) used for cell experiments and siRNA (sense strand: 5′-CCACAGUUCUGAAAUCCAU-3′, antisense strand: 5′-AUGGAUUUCAGAACUGUGGUU-3′) used for animal experiments (ESCs) were purchased from Gemma Pharmaceuticals (Shanghai, China). Four different siCD300ld sequences were purchased for cell experiments. After transfection into cells, qRT-PCR analysis verified that si-CD300ld-01 had the highest silencing efficiency, and this sequence was selected for all subsequent functional experiments.

[0124] Efficient LNP delivery systems are crucial for the intracellular stability and functional realization of siRNA. In our LNP preparation system, DSPE-PEG-SA not only endows neutrophils with targeting capabilities but also significantly enhances the uptake efficiency of PMN-MDSCs, while DOTAP facilitates endosome escape after endocytosis. LNPs not only significantly improve cellular uptake efficiency but also promote endosome escape after endocytosis. To systematically evaluate the efficiency of LNP-mediated siRNA delivery and intracellular transport, LNPs loaded with FAM-labeled siRNA were co-incubated with PMN-MDSCs for 12 hours. Abundant green fluorescence signal was observed in the cells under a fluorescence microscope, indicating efficient siRNA transfection. Furthermore, the distribution of LNPs-FAM-siRNA in cells at different time points (1, 6, and 12 h) was dynamically tracked using CLSM. The results showed that PMN-MDSCs continuously and efficiently uptake LNPs-FAM-siRNA, and the intracellular green fluorescence signal increased in a time-dependent manner. Meanwhile, the colocalization of green fluorescence with LysoTracker Deep Red-labeled lysosomes gradually decreased, suggesting that some siRNAs had successfully escaped from the endosome. Figure 4 (g). The effect of LNPs-siCD300ld on the migration ability of PMN-MDSCs was systematically evaluated using transwell cell migration experiments. Figure 5 (H schematic diagram). Both fluorescence microscopy and flow cytometry analysis showed that the number of PMN-MDSCs migrating to the lower compartment in the LNPs-siCD300ld group was significantly lower than that in the negative control group. Figure 4 The results (i.j) indicate that siCD300ld effectively inhibits the migration ability of PMN-MDSCs.

[0125] To further investigate the effect of LNPs-siCD300ld on the regulation of T cell effector function by PMN-MDSCs, PMN-MDSCs isolated from the spleen of cervical cancer-bearing mice were transfected with LNPs-siNC or LNPs-siCD300ld, respectively. After 48 hours, they were co-incubated with mouse splenic T cells for 24 hours. Flow cytometry was then used to detect the expression of T cell activation markers and effector cytokines. The results showed that, compared with the LNPs-siNC group, PMN-MDSCs treated with LNPs-siCD300ld, after co-culture with T cells, showed significantly higher levels of CD4+IFN-γ. + and CD4+TNF-α + The proportion of T cells increased significantly. Figure 4 (k), CD8+IFN-γ + and CD8+TNF-α + The proportion of T cells also increased similarly. Figure 4 These results indicate that CD300ld silencing can significantly alleviate the inhibition of T cell effector function by PMN-MDSCs, providing a solid experimental basis for improving the tumor immune microenvironment.

[0126] Furthermore, to assess the effect of CD300ld knockdown on the inhibitory effect of PMN-MDSCs-mediated T cell proliferation, T cells were uniformly labeled with CFSE and co-incubated with siRNA-transfected PMN-MDSCs at a 1:2 ratio. The proliferation status was assessed by detecting the CFSE fluorescence decay of T cells at 24, 48, and 72 hours. Compared with the LNPs-siNC group, the LNPs-siCD300ld group showed a more significant decrease in T cell CFSE fluorescence intensity, suggesting that its proliferation capacity significantly increased over time (especially at 48 and 72 hours). Figure 4 (m). In summary, the results indicate that CD300ld is highly expressed in PMN-MDSCs. Its downregulation not only significantly weakens the migration ability of PMN-MDSCs, but also effectively alleviates their inhibitory effect on T cell effector function and proliferation, thereby significantly reducing the immunosuppressive effect mediated by PMN-MDSCs.

[0127] Figure 3In the figures, (a) CLSM images of U14 cells taking up different materials at different time points. I: CH-CuNPs; II: CH-CuNPs coated with macrophage membranes; III: CH-CuNPs coated with M1 macrophage membranes with high PD-1 expression. Imaging times were 1, 3, and 6 hours, scale bar = 25 μm; (b) Cell viability of U14 cells after different treatments; (c) Fluorescence microscopy and 3D reconstruction showing the generation of reactive oxygen species (ROS) in U14 cells under different treatment conditions, scale bar = 200 μm; (d) Flow cytometry detection of intracellular ROS levels in treated U14 cells; (e) Fluorescence microscopy staining of live / dead cells, green for live cells and red for dead cells, scale bar = 200 μm; (f) Flow cytometry analysis of apoptosis in U14 cells after different treatments. Data are expressed as mean ± standard deviation (SD).

[0128] Figure 4 In the images, (a) JC-1 stained CLSM images show the disruption of mitochondrial membrane potential in U14 cells after different treatments, scale bar = 50 μm; (b) γ-H2AX stained CLSM images assess DNA damage in U14 cells under different conditions; (c) CLSM images show the expression of calreticulin (CRT) on the surface of U14 cells after different treatments, scale bar = 25 μm; (d) CLSM images show the nuclear localization of HMGB1 in U14 cells after treatment; (e) Flow cytometry identification of mouse PMN-MDSCs; (f) Relative mRNA expression levels of CD300ld in different cell types; (g) LNPs-FAM-s i. CLSM images of lysosome escape at 1, 6, and 12 hours after treatment with PMN-MDSCs, scale bar = 25 μm; (h) Schematic diagram of the PMN-MDSC migration assay; (i) Fluorescence microscopy images showing the migration of PMN-MDSCs; (j) Flow cytometry quantitative analysis of PMN-MDSC migration, scale bar = 200 μm; (k) Flow cytometry quantitative analysis of IFN-γ and TNF-α production in CD4+ T cells under different treatment groups; (l) Flow cytometry quantitative analysis of IFN-γ and TNF-α production in CD8+ T cells under different treatment groups; (m) Flow cytometry analysis of T cell proliferation under different treatments.

[0129] Experiment Example 4

[0130] This experimental example is an in vivo evaluation of antitumor performance:

[0131] To further evaluate the local tumor accumulation and in vivo metabolism of 3D-printed nanomedicine hydrogel suppositories in an orthotopic cervical cancer mouse model, the study used a speculum to precisely implant the hydrogel suppositories into the cervix and vaginal fornix of mice. Figure 5(a) Anatomical dissection of the reproductive system combined with Coomassie brilliant blue staining can clearly show the suppository implantation process. Figure 5 (b) CH-CuNPs and CH-CuNPs@MM labeled with m-THPP were blended with GelMA, 3D printed, and implanted into the tumor site. In vivo fluorescence imaging showed that the CH-CuNPs@MM group showed a stronger and more persistent fluorescence signal in the tumor area, and significant liver accumulation was observed after 24 hours, indicating that it is mainly metabolized by the liver ( Figure 5 (middle CD). Ex vivo organ analysis (36 hours) further demonstrated that CH-CuNPs@MM accumulation in tumor tissue was slightly higher than in the control group. Figure 5 (f). Furthermore, qRT-PCR analysis showed that local administration of LNPs-siCD300ld significantly reduced CD300ld mRNA expression in both the spleen and tumor tissue of mice, demonstrating a gene silencing effect far superior to the LNPs-siNC control group (f). Figure 5 (gh). In summary, 3D-printed hydrogel suppositories not only achieve efficient local enrichment of nanomedicines at tumor sites, but also effectively inhibit CD300ld expression, providing strong support for precise local treatment of cervical cancer.

[0132] Based on previous findings: CH-CuNPs can efficiently generate reactive oxygen species and induce tumor cell death under ultrasound stimulation; coating M1 macrophages with high PD-1 expression significantly enhances tumor targeting and accumulation and reverses T cell suppression; LNPs-siCD300ld can inhibit PMN-MDSC migration and regulate T cell function and proliferation. Therefore, these functional components were integrated into a 3D-printed hydrogel suppository to achieve sustained local release within the cervical cancer tumor microenvironment. To systematically evaluate the antitumor effect of the hydrogel suppository, an orthotopic cervical cancer mouse model was established, and tumor progression was dynamically monitored using an in vivo imaging system (IVIS). Seven days after tumor inoculation, mice were randomly divided into six groups (n=5) based on imaging data: Group I (PBS control), Group II (3D hydrogel suppositories containing CH-CuNPs@MM), Group III (3D hydrogel suppositories containing LNPs-siCD300ld), Group IV (solution CH-CuNPs@MM + LNPs-siCD300ld + ultrasound), Group V (3D hydrogel suppositories containing CH-CuNPs@MM + ultrasound), and Group VI (3D hydrogel suppositories containing CH-CuNPs@MM + LNPs-siCD300ld + ultrasound). Each group received the medication on days 1 and 5, and underwent ultrasound stimulation 24 hours after administration, with tumor volume monitored. Animals were sacrificed on day 12 for systematic dissection and pathological analysis. Figure 5 e, Figure 5 (Middle K).

[0133] The results showed that cervical tumors in mice in groups I and II continued to grow without significant inhibition. In group III, tumors in two mice shrank significantly, with one achieving complete remission. In group IV, one mouse experienced complete tumor elimination after the first treatment, while the tumors in the other four continued to develop. Tumor growth was significantly inhibited in groups V and VI, with group VI showing the most significant therapeutic effect, resulting in near-complete tumor clearance in several animals. Figure 5 (i, 6k). Significant differences in the weight of cervical tumors after dissection were observed among different groups, highly consistent with IVIS imaging results. Throughout the treatment process, there were no significant differences in body weight, food and water intake, and general activity among the groups, indicating that the CH-CuNPs and LNPs-siCD300ld co-loaded 3D-printed hydrogel suppositories have excellent biocompatibility and safety for local administration. Survival analysis showed that the survival time of mice in group VI was significantly prolonged, with some animals surviving for more than 90 days (i, 6k). Figure 5 (j).

[0134] Pathological and immunofluorescence analyses of tumor tissues from different groups revealed significant differences in tumor structural destruction, apoptosis, and immune cell infiltration. H&E staining showed that group VI exhibited the most disordered tumor structure, with extensive necrosis, disordered cell arrangement, and a significantly decreased cell density. Figure 6 (a) TUNEL fluorescence labeling showed a significant increase in tumor apoptosis in groups V and VI. Figure 6 (b) CD8 + T-cell immunofluorescence results showed that CD8+ T cell infiltration was highest in these two groups. Ly6G immunostaining showed a significant decrease in pro-tumor myeloid cells (such as PMN-MDSCs) in groups V and VI, suggesting significant remodeling of the tumor immune microenvironment and activation of anti-tumor immunity. Furthermore, in group III, the number of Ly6G-positive cells in tumor tissue was significantly reduced after CD300ld siRNA targeted silencing. Figure 6 (c) In summary, the ultrasound-activated multifunctional 3D-printed hydrogel suppository exhibited a significant and safe antitumor effect in a mouse cervical cancer model, highlighting its promising clinical translation prospects.

[0135] To elucidate the potential molecular mechanisms of immune regulation following CD300ld silencing, Western blot analysis was performed. Figure 6(d) Compared with the control group, P-STAT3 expression decreased to varying degrees in groups III and V, with the most significant decrease in group VI; while the levels of STAT3 and GAPDH proteins were basically consistent across groups. This indicates that CH-CuNPs@MM+ ultrasound can not only directly induce tumor cell death through the generation of reactive oxygen species by potent ROS, but also inhibit the JAK / STAT3 pathway by reducing protein phosphorylation levels (including P-STAT3) through ROS. In addition, the high PD-1 expression of M1 macrophage membrane endows nanoparticles with stronger immunomodulatory capabilities, which helps regulate pro-inflammatory factors and immune signaling pathways, indirectly inhibiting STAT3 phosphorylation. More importantly, CD300ld silencing effectively weakens the recruitment and function of PMN-MDSCs, alleviates their inhibition of T cells and remodeling of the immunosuppressive microenvironment, and further promotes STAT3 pathway inhibition. Under the synergistic effect of multiple mechanisms, the P-STAT3 level is significantly reduced, effectively inhibiting tumor progression, remodeling the tumor microenvironment, and enhancing anti-tumor immunity, providing strong molecular evidence for efficient treatment.

[0136] Advanced cervical cancer often involves peritoneal metastasis. To further investigate the inhibitory effect of hydrogel suppositories co-loaded with two types of nanoparticles on peritoneal implantation metastasis, a mouse model of cervical cancer with in situ peritoneal implantation metastasis was established to highly simulate the clinical process. Patients were randomly divided into four groups: Group I was a PBS control; Group II received 3D hydrogel containing LNPs-siCD300ld; Group III received 3D hydrogel containing CH-CuNPs@MM + ultrasound; and Group IV received 3D hydrogel co-loaded with CH-CuNPs@MM and LNPs-siCD300ld + ultrasound. Drug administration was performed on days 1 and 5, and the endpoint anatomical analysis was conducted on day 14. Figure 6 (e).

[0137] During the experiment, some mice developed significant ascites. After removing the abdominal skin, the intact peritoneum was first exposed, and bloody ascites was recorded by peritoneal imaging. The abdominal cavity was then opened to evaluate the distribution and number of intra-abdominal metastases. In the PBS group, the primary cervical tumor enlarged and spread to the uterine body, with widespread metastatic nodules in the abdominal cavity, particularly dense in the mesentery and subhepatic region, accompanied by significant bloody ascites. In contrast, the IV group not only showed a significant reduction in peritoneal metastases and ascites, but also complete removal of the primary cervical tumor. Figure 6 Quantitative analysis showed that the ascites volume and number of metastatic nodules in group IV were significantly lower than those in group PBS. Figure 6 (zhonggh).

[0138] In summary, these results indicate that 3D hydrogel suppositories co-loaded with CH-CuNPs@MM or LNPs-siCD300ld, combined with ultrasound therapy, can significantly inhibit the growth of primary cervical tumors and the formation of peritoneal metastases, demonstrating strong synergistic antitumor activity and good clinical translational potential.

[0139] To further elucidate the immune activation mechanism mediated by the co-loaded two types of nanoparticles in the 3D hydrogel suppository, we systematically analyzed the dynamic changes of major immune cell populations in the spleen and tumor microenvironment of mice after different treatments. This hydrogel suppository exerts a potent immunomodulatory effect through a triple mechanism: First, ultrasound-activated CH-CuNPs efficiently generate reactive oxygen species (ROS), which not only directly induce tumor cell apoptosis but also promote the release of tumor-associated antigens and DAMPs, thereby activating innate and adaptive immune responses dominated by dendritic cells (DCs). Second, the encapsulation of M1 macrophage membranes with high PD-1 expression not only enhances the tumor-targeting ability of the nanosystem but also alleviates T cell exhaustion and enhances the killing activity of effector T cells and NK cells through PD-1 / PD-L1 axis blockade. Third, the delivery of LNPs-siCD300ld effectively inhibits the recruitment and immunosuppressive function of PMN-MDSCs, further alleviating the inhibitory effect on T cells and NK cells and significantly improving the tumor immunosuppressive microenvironment.

[0140] To verify the above mechanism, we performed flow cytometry analysis and quantitative statistics on the major immune subsets in the spleen and tumor tissues of six groups of mice. The results showed that CD8+ in the spleen of group VI mice... + The proportion of T cells increased significantly from 42.7% in the PBS group to 60.1%. Figure 7 (a) This suggests enhanced systemic immune activation. Simultaneously, the proportion of peripheral PMN-MDSCs (CD11b+Ly6G+Ly6C-) decreased significantly from 44.1% in the control group to 19.8%. Figure 7 (b) showed a significant reduction in systemic immunosuppression. In the tumor microenvironment, the expression of the dendritic cell co-stimulatory molecules CD80 / CD86 in group VI increased to 13.02%, more than twice that of the PBS group (5.97%). Figure 7 (c) indicates a significant enhancement in local antigen presentation and immune activation. The M1 / M2 macrophage ratio increased nearly 5-fold. Figure 7 (d) reflects a significant polarization of pro-inflammatory and anti-tumor phenotypes. Intratumoral PMN-MDSCs (CD45) + CD11b + Ly6G + Ly6C - The proportion decreased from 47.4% in the PBS group to 23.2% in group VI (compared to 33.3% in group III). Figure 7 (e). Intratumoral regulatory T cells (Tregs, CD45) + CD3 + CD25 + FoxP3 + The proportion decreased from 34.5% to 19.2% in group VI. Figure 7(f) indicates that combined nanotherapy significantly weakened the local immunosuppressive barrier. Notably, the proportion of intratumoral effector immune cells in group VI was significantly higher than that in the PBS group: NK cells increased from 5.89% to 18% (f). Figure 7 (g), CD8 + T cells increased from 8.64% to 44.6%. Figure 7 (h), CD4 + T cells increased from 24% to 51.7%. Figure 7 i).

[0141] Based on previous studies, siRNA targeting CD300ld can inhibit STAT3 phosphorylation and downstream S100A8 / 9 expression, thereby relieving the inhibition of T cells and NK cells by PMN-MDSCs and further amplifying the anti-tumor effect. Figure 7 (j). By binding to CH-CuNPs and activating via ultrasound, this system achieves a strong synergistic effect across multiple targets and pathways: it can powerfully activate various anti-tumor effector cells, selectively inhibit major immunosuppressive cell subsets, and effectively reshape the tumor microenvironment from an "immunely cold" to an "immunely hot" state. These findings provide a solid immunological basis for improving the response rate of tumor immunotherapy, maintaining durable anti-tumor effects, and reducing the risk of metastasis. Further in-depth investigation and validation of the core anti-tumor and immune activation molecular mechanisms will be conducted through transcriptome analysis.

[0142] Figure 5(a) Schematic diagram of 3D hydrogel suppositories placed in the vagina of mice; (b) Anatomical diagram of 3D hydrogel suppositories placed in the vagina of mice; (c, d) In vivo imaging showing tumor enrichment and in vivo distribution of m-THPP-labeled CH-CuNPs and CH-CuNPs@MM in the cervix of mice with orthotopic cervical tumors at different time points; (e) Schematic diagram of the establishment and treatment regimen of the mouse model of orthotopic cervical tumors; (f) Quantitative analysis of fluorescence intensity of cervical tumors in different groups of mice after tumor enrichment experiment; (g) Analysis of CD300ld mRNA expression level in spleen of mice with tumors in different treatment groups; (h) Analysis of CD300ld mRNA expression level in tumor tissues of different treatment groups; (i) Quantitative analysis of bioluminescence intensity in tumor areas of each group after treatment; (j) Survival curves of mice with orthotopic cervical tumors under different treatments; (k) In vivo bioluminescence imaging of mice on days 0, 6, and 12 of treatment and images of the appearance of reproductive organs in different treatment groups at the experimental endpoint. (I: PBS; II: 3D hydrogel suppositories loaded with CH-CuNPs@MM; III: 3D hydrogel suppositories loaded with LNPs-siCD300ld; IV: Solution-type CH-CuNPs@MM + LNPs-siCD300ld combined with ultrasound; V: 3D hydrogel suppositories loaded with CH-CuNPs@MM combined with ultrasound; VI: 3D hydrogel suppositories loaded with CH-CuNPs@MM + LNPs-siCD300ld combined with ultrasound).

[0143] Figure 6 In the image, (a) representative H&E staining images of tumor tissues from mice in different treatment groups; (b) representative TUNEL (red) immunofluorescence staining images of tumor tissues from different treatment groups, indicating apoptosis; (c) CD8+ staining images of tumor tissues from mice in different treatment groups. +(d) Representative images of T cells (red) and Ly6G (green) immunofluorescence staining; Western blot analysis of P-STAT3 and STAT3 protein levels in tumor samples from different groups. (I: PBS; II: 3D hydrogel suppositories loaded with CH-CuNPs@MM; III: 3D hydrogel suppositories loaded with LNPs-siCD300ld; IV: Solution-type CH-CuNPs@MM + LNPs-siCD300ld combined with ultrasound; V: 3D hydrogel suppositories loaded with CH-CuNPs@MM combined with ultrasound; VI: 3D hydrogel suppositories loaded with CH-CuNPs@MM + LNPs-siCD300ld combined with ultrasound); (e) Schematic diagram of the modeling and treatment protocol for peritoneal metastases from in situ cervical tumors; (f) Gross anatomical comparison of peritoneal effusion, peritoneal metastatic nodules (blue arrows), and primary cervical tumors after treatment in each group; (g) Quantitative comparison of peritoneal effusion volume in each treatment group; (h) Quantitative comparison of the number of peritoneal metastatic nodules in each group. (I: PBS; II: 3D hydrogel suppositories loaded with LNPs-siCD300ld; III: 3D hydrogel suppositories loaded with CH-CuNPs@MM combined with ultrasound; IV: 3D hydrogel suppositories loaded with CH-CuNPs@MM+LNPs-siCD300ld combined with ultrasound).

[0144] Figure 7 In the figures, (a) flow cytometry analysis and quantification of CD8+ T cells in the spleen; (b) flow cytometry analysis and quantification of PMN-MDSCs in the spleen; (c) flow cytometry analysis and quantification of mature dendritic cells (DCs) in tumor tissue; (d) flow cytometry analysis and quantification of M1 and M2 macrophages and M1 / M2 ratio in tumor tissue; (e) flow cytometry analysis and quantification of PMN-MDSCs in tumor tissue; (f) flow cytometry analysis and quantification of regulatory T cells (Tregs) in tumor tissue; (g) flow cytometry analysis and quantification of NK cells in tumor tissue; (h) flow cytometry analysis and quantification of CD8+ T cells in tumor tissue; (i) flow cytometry analysis and quantification of CD4+ T cells in tumor tissue; and (j) schematic diagram of the mechanism by which CD300ld regulates the tumor immune microenvironment. (I: PBS; II: 3D hydrogel suppositories loaded with CH-CuNPs@MM; III: 3D hydrogel suppositories loaded with LNPs-siCD300ld; IV: Solution-type CH-CuNPs@MM + LNPs-siCD300ld combined with ultrasound; V: 3D hydrogel suppositories loaded with CH-CuNPs@MM combined with ultrasound; VI: 3D hydrogel suppositories loaded with CH-CuNPs@MM + LNPs-siCD300ld combined with ultrasound).

[0145] Experimental Example 5

[0146] This experimental example demonstrates the analysis of RNA sequencing systems for the regulatory mechanisms of the tumor microenvironment.

[0147] Building upon the aforementioned material synthesis, ROS generation verification, cell function experiments, and animal experiments, a comprehensive transcriptome analysis was further conducted to systematically elucidate the molecular regulatory mechanisms of different treatment modalities on the cervical cancer tumor microenvironment. Mouse tumor tissues were divided into four groups: Group A was the PBS control group; Group B received 3D hydrogel suppositories loaded with LNPs-siCD300ld; Group C received 3D hydrogel suppositories loaded with CH-CuNPs@MM combined with ultrasound; and Group D received both CH-CuNPs@MM and LNPs-siCD300ld combined with ultrasound treatment. All groups underwent high-throughput RNA sequencing analysis.

[0148] Principal component analysis (PCA) Figure 8 (a) shows that the transcriptomic characteristics of group D were significantly different from those of other groups, highlighting the profound impact of dual-nanoparticle-loaded hydrogel suppositories combined with ultrasound on the tumor microenvironment (TME). Venn diagram analysis ( Figure 8 (b) revealed the group-specific and shared differentially expressed gene profiles, indicating that the combination therapy induced unique molecular markers. GO enrichment analysis ( Figure 8 Differential expression between group B (LNP-siCD300ld) and group A (PBS) showed that siCD300ld delivery significantly activated immune-related biological processes such as inflammatory responses and T cell activation. KEGG enrichment analysis ( Figure 8 Further analysis (i) revealed that differentially expressed genes in group B were mainly enriched in pathways closely related to immune activation and inflammation, such as the "cytokine-receptor interaction," the "C-type lectin receptor signaling pathway," and the "IL-17 signaling pathway." Activation of these pathways suggests that LNP-siCD300ld delivery can effectively enhance immune signaling, strengthen the recruitment and activation of immune effector cells, thereby comprehensively improving local anti-tumor immunity in the tumor.

[0149] In the combination therapy group (Group D), transcriptome analysis revealed extensive reprogramming at the molecular level, with 4239 genes upregulated and 5602 downregulated. Figure 8 (f). Functional enrichment analysis ( Figure 8 Further analysis (using the IL-17 signaling pathway) revealed that pro-tumor biological processes (such as angiogenesis, cell cycle, cell adhesion, extracellular matrix remodeling, and tumor cell survival-related pathways, including PI3K-Akt, NF-κB, and JAK / STAT) were inhibited, while immune-related and pro-apoptotic signaling pathways were significantly activated. Heatmap analysis of the IL-17 signaling pathway (…) Figure 8 c) and GSEA analysis ( Figure 8Further studies (j) confirmed that the combination therapy significantly upregulated inflammatory and anti-tumor effector genes while downregulating immunosuppressive genes such as S100A8 / 9 and CXCL2 / 3, shifting the tumor microenvironment from an immunosuppressive state to an immune-activated state. GSEA enrichment curves also showed significant negative enrichment of the estrogen signaling pathway and the Staphylococcus aureus infection pathway after combination therapy, indicating a strong downregulation of molecular networks related to tumor growth and immunosuppression. The inhibition of the estrogen signaling pathway (which is known to contribute to cervical cancer progression), combined with the blockade of infection-related and pro-inflammatory pathways, further supports the shift of the tumor microenvironment towards "low tumor promotion and high immune stimulation."

[0150] In terms of mechanism, such as Figure 8 As shown in Figure k, ultrasound-triggered CH-CuNPs can rapidly generate ROS, activate the p53 / PTEN axis, and simultaneously inhibit PI3K-Akt / mTOR and NF-κB signaling, directly inducing tumor apoptosis. Simultaneously, LNP-siCD300ld can silence CD300ld in PMN-MDSCs and, through ligand capture at the membrane of high-PD-1 macrophages, downregulate the PD-1 / PD-L1 axis, thereby relieving T-cell inhibition. These two mechanisms synergistically form a highly efficient paradigm of "targeted ROS attack + immune unlocking," significantly enhancing anti-tumor function and providing a solid molecular mechanistic basis for targeted combination therapy of cervical cancer.

[0151] Figure 8In the figure, (a) PCA plots show the distribution of groups A, B, C, and D on PC1 and PC2 (A: PBS; B: 3D hydrogel suppositories loaded with LNPs-siCD300ld; C: 3D hydrogel suppositories loaded with CH-CuNPs@MM combined with ultrasound; D: 3D hydrogel suppositories loaded with both CH-CuNPs@MM and LNPs-siCD300ld combined with ultrasound); (b) Venn plots of differentially expressed genes in tumors of each group; (c) Changes in the expression of IL-17 signaling pathway-related genes in tumors after treatment in each group (red: upregulated, green: downregulated); (d) Volcano plots of group B versus group A (B vs A), a total of 1122 upregulated genes and 627 downregulated genes were detected (|log2FC|>1, padj<0.05); (e) GO enrichment analysis of group B versus group A, which showed a significant downregulation of the immunosuppressive process (cyan); (f) Volcano plots of group D versus group A (D vs A). A) A total of 4239 genes were upregulated and 5602 genes were downregulated, reflecting the maximum transcriptome influence; (g) GO analysis of group D over group A: extracellular matrix (ECM) remodeling, adhesion / migration and myeloid differentiation were strongly inhibited, while ERK / Wnt negative feedback regulators were significantly upregulated; (h) Analysis of the top 10 KEGG pathways of group D over group A (D vs A): cell adhesion, PI3K-Akt, JAK-STAT, NF-κB, PD-L1 checkpoint and other pathways were widely downregulated; (i) KEGG enrichment results of group B over group A: cytokine-receptor and C-type lectin pathways were upregulated, and cell adhesion-related pathways were downregulated; (j) GSEA analysis of group B over group A: DNA damage checkpoint and telomere maintenance pathways were positively enriched, and mitochondrial gene expression was negatively enriched, suggesting stress-energy reprogramming; (k) Synergistic anti-tumor mechanism.

[0152] Experimental Example 6

[0153] This experimental example is an in vivo biosafety evaluation.

[0154] To comprehensively evaluate the in vivo biocompatibility of the 3D-printed hydrogel suppositories loaded with CH-CuNPs and LNPs-siCD300ld, we performed histological and hematological analyses on all treatment groups. HE staining of major organs such as the heart, liver, spleen, lungs, and kidneys showed that the tissue structures in all groups were intact, with no evidence of inflammatory cell infiltration or cell necrosis. Meanwhile, peripheral blood routine and serum biochemical tests showed that white blood cell, hemoglobin, and platelet levels, as well as liver and kidney function indicators, were all within the normal range. In summary, these results indicate that the composite hydrogel system has excellent in vivo biocompatibility and did not induce significant systemic toxicity under experimental conditions, providing strong support for its further development and clinical translation in the treatment of cervical cancer.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing an acoustic-dynamic nanocatalyst, characterized in that, The preparation method includes the following steps: (a) Compound 1 and Ni-4 were added to an organic solvent to react. After the reaction was completed, the solvent was removed and the mixture was purified to obtain Ni-5. The structural formula of compound 1 is shown in Formula 1, and the structural formula of Ni-4 is shown in Formula II. In formula I, R 1 It is n-undecyl; R 2 It is 2-hexyldecyl; in formula II, R 3 It is phenyl; (b) Under argon protection, LiAlH4 was added to Ni-5 tetrahydrofuran solution and stirred. Then, glacial acetic acid was added and extracted with dichloromethane. The organic phase was collected, dried, solvent removed and purified to obtain H-1. (c) Under argon protection, phosphorus oxychloride was added to a 1,2-dichloroethane solution containing H-1 and dimethylformamide and refluxed. After the reaction was completed, the mixture was cooled, and then a saturated sodium acetate solution was added and stirred for a period of time. Subsequently, the mixture was extracted with dichloromethane, the organic phase was collected, and the organic phase was dried, the solvent was removed, and the mixture was purified to obtain H-CHO. (d) Under argon protection, copper acetate was added to a tetrahydrofuran solution containing H-CHO and N,N-dimethylformamide and stirred. After the reaction was completed, the mixture was extracted, the organic phase was collected, and the organic phase was dried, the solvent was removed, and the mixture was purified to obtain Cu-CHO. (e) Under argon protection, Cu-CHO, 2-(5,6-difluoro-3-oxo-2,3-dihydro-1H-inden-1-ylidene) malononitrile and chloroform were mixed, and pyridine was added dropwise to carry out the reaction under heating. After the reaction was completed, the mixture was cooled, and then methanol was added, filtered, and purified to obtain ADA-type copper-centered organometallic molecule CH-Cu. (f) Under ultrasonic and water bath conditions, CH-Cu tetrahydrofuran solution and DSPE-mPEG 5000 tetrahydrofuran emulsion were mixed, and after continuous ultrasonic treatment for a period of time, deionized water was added, the mixture was allowed to stand, filtered through a membrane, ultrafiltered, concentrated and dried to obtain the sonic dynamic nanocatalyst.

2. The preparation method according to claim 1, characterized in that, In step (a), the reaction temperature is 100–120°C and the time is 20–28 h.

3. The preparation method according to claim 1, characterized in that, In step (b), the stirring temperature is room temperature and the time is 0.8 to 2 minutes.

4. The preparation method according to claim 1, characterized in that, In step (c), the reflux reaction temperature is 85–90°C and the time is 10–14 h; cooling to 0°C; stirring treatment temperature is room temperature and the time is 1.5–3 h.

5. The preparation method according to claim 1, characterized in that, In step (d), the stirring reaction temperature is 60–80°C and the time is 1–3 hours. In step (e), the heating reaction temperature is 60–80°C and the time is 10–14 h; In step (f), the ultrasonic treatment is continued for 15 to 25 minutes, and the settling time is 20 to 28 hours.

6. The acoustic-dynamic nanocatalyst prepared by any one of the preparation methods described in claims 1 to 5.

7. The use of the acoustic-dynamic nanocatalyst prepared by any one of claims 1 to 5 in combination with siRNA targeting CD300ld in the preparation of drugs for treating cervical cancer.

8. A method for preparing a GelMA-based hydrogel for treating cervical cancer, characterized in that, The preparation method includes the following steps: (1) The acoustic-dynamic nanocatalyst prepared by any of the preparation methods described in claims 1 to 5 is encapsulated in the membrane of M1 macrophages overexpressing PD-1 to obtain cell membrane-coated nanoparticles. (2) Cell membrane-coated nanoparticles and lipid nanoparticles loaded with siRNA targeting CD300ld were added to a mixture of GelMA-based hydrogel prepolymers to obtain a precursor solution. (3) The precursor solution was 3D printed to obtain GelMA-based hydrogel for the treatment of cervical cancer.

9. The preparation method according to claim 8, characterized in that, The cell membrane-coated nanoparticles were prepared by the following method: RAW264.7 macrophages were cultured to the logarithmic growth phase, and then IL-1β was added to a final concentration of 40-60 ng / mL. Palmitic acid DMSO solution was then added to a palmitic acid concentration of 20-30 μM and the cells were cultured for another 20-28 h. The cells were then collected and the cell membranes were extracted. The cell membrane and the acoustic-dynamic nanocatalyst prepared by any of the preparation methods described in claims 1 to 5 are mixed and repeatedly co-extruded to obtain the cell membrane-coated nanoparticles. The lipid nanoparticles carrying siRNA targeting CD300ld were prepared by the following method: DSPE-PEG, DOTAP, DSPE-PEG-SA, DSPC and cholesterol were co-dissolved in chloroform, and the solvent was removed by rotary evaporation to form a uniform lipid film and then hydrated to obtain a lipid vesicle suspension. The siRNA targeting CD300ld was added to a lipid vesicle suspension for incubation, repeated extrusion, and then ultrafiltration to obtain lipid nanoparticles loaded with the siRNA targeting CD300ld.

10. A GelMA-based hydrogel for treating cervical cancer prepared by the method of claim 8 or 9.

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