Preparation method and application of targeted nanoparticles for delivering gas and small molecule drugs based on ultrasonic piezoelectric catalysis

Abstract

The invention belongs to the field of biological medicine and ultrasonic medicine, and provides a barium titanate (BTO) piezoelectric material-based ultrasonic responsive targeting nano system (BTO at BAL), which is used for matrix microenvironment remodeling and immunopotentiation of compact tumors such as pancreatic cancer and the like. According to the nanoparticles, oleic acid modified BTO is taken as a core, ROS responsive prodrug molecules containing a small molecule PD-L1 inhibitor BMS1166 and a nitric oxide donor (Arg) 9 are loaded on the surface, and specific targeting on pancreatic cancer cells is realized by combining a targeting peptide LFC131. Under ultrasonic excitation, BTO generates reactive oxygen species (ROS), on one hand, (Arg) 9 is oxidized to release NO, tumor matrix is degraded, tumor hardness is reduced, drug delivery efficiency is improved, and meanwhile, ROS and NO synergistically enhance tumor immunogenicity; on the other hand, the ROS breaks a thioketal bond to release BMS1166, PD-L1 expression is down-regulated, and immunosuppression is reversed. According to the present invention, the significant tumor inhibition and immune activation effects are represented in the animal model, and the clinical transformation potential is provided.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an ultrasound-targeted nanoparticle that simultaneously delivers nitric oxide gas and the PD-L1 inhibitor BMS1166 deep into the body, its preparation method, and its application. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a common malignant tumor of the digestive tract, characterized by insidious onset, rapid progression, and low surgical resection rate. The tumor microenvironment of PDAC is characterized by abundant stroma, hypoxia, insufficient blood supply, and strong immunosuppression, posing significant challenges to its treatment. For example, the dense extracellular matrix (ECM) within the tumor hinders drug penetration, significantly reducing the efficacy of treatment. Nitric oxide (NO) is an important gaseous signaling molecule that exhibits multiple biological roles in tumor therapy. It can improve tumor tissue blood perfusion and permeability by degrading ECM and relaxing vascular smooth muscle, thereby alleviating the high-density and high-pressure state of the tumor microenvironment (TME). In dense stromal tumors such as PDAC, abundant collagen fibers and glycosaminoglycans form a strong physical barrier, severely hindering the penetration of therapeutic drugs and immune cells. Nitric oxide can selectively degrade collagen and other structural components in the ECM, reducing stroma density and tumor stroma pressure, thereby significantly improving the distribution and accumulation of drugs in tumor tissue. Furthermore, nitric oxide can react with superoxide (O2)... - The reaction produces highly reactive peroxynitrite (ONOO). - This further disrupts the tumor cell membrane and cytoskeleton structure, enhancing drug entry and cytotoxicity. On the other hand, LFC131 is a small peptide ligand that binds specifically to the CXCR4 receptor with high affinity. CXCR4 is highly expressed in various tumor cells and metastases, and participates in tumor cell migration, invasion, and immune escape. Utilizing the targeting effect of LFC131 on CXCR4 can achieve precise drug delivery at the tumor cell level, reducing uptake by non-target tissues and systemic toxicity. Combining a nitric oxide release system with the LFC131 targeting peptide not only improves drug diffusion and accumulation within tumors through nitric oxide-mediated matrix remodeling and improved blood flow, but also relies on CXCR4 targeting to precisely deliver drugs to tumor cells, achieving enhanced delivery efficiency at both the tissue and cellular levels. This synergistic strategy has groundbreaking significance and broad clinical application prospects in tumors with dense matrix and severe immunosuppression, such as PDAC.

[0003] The dense extracellular matrix of PDAC is a significant factor affecting treatment efficacy. Immunosuppressive cells in the tumor microenvironment promote the activation of pancreatic stellate cells by secreting cytokines and growth factors. Activated pancreatic stellate cells synthesize large amounts of extracellular matrix proteins within the tumor stroma, leading to matrix hardening and density. Excessive deposition of extracellular matrix components can form a physical barrier, limiting drug diffusion. Nitric oxide is a multifunctional endogenous gaseous messenger that plays a crucial role in vasodilation, angiogenesis, tumor formation, and progression. Simultaneously, nitric oxide and its derivatives can degrade extracellular matrix components such as collagen and fibronectin. To efficiently deliver high concentrations of nitric oxide, a series of nitric oxide donors have been developed, primarily including nitroglycerin, tetranitrate, and L-arginine. Furthermore, nitric oxide can react with superoxide anions to generate peroxynitrite anions (ONO). - Nitric oxide (NO) is a major reactive nitrogen species with a stronger peroxidation capacity than reactive oxygen species (ROS), leading to apoptosis in cancer cells. However, nitric oxide and ONOO... - Both have very short half-lives. Therefore, exploring and developing technologies that can regulate nitric oxide and ONOO... - The release of the drug or carrier system to maintain appropriate concentrations and durations of action in vivo is crucial. Nonamericanine reacts with superoxide anions and has the potential to generate nitric oxide.

[0004] During ultrasound irradiation, the piezoelectric catalytic effect of BTO combined with ultrasound can generate reactive oxygen species, leading to nucleic acid damage, protein oxidation, mitochondrial or plasma membrane disruption, and triggering immunogenic cell death. However, tumor cells with high PD-L1 expression can inhibit T cell activity by binding to PD-1, resulting in immune escape. To address this issue, there is an urgent need for regulators that inhibit self-protection to effectively enhance tumor-killing efficiency. PD-L1 (programmed death ligand 1) is an important immune checkpoint protein. Tumor cells can suppress T cell activity by overexpressing PD-L1, thereby evading the surveillance of the immune system. PD-L1 inhibitors exert their anti-cancer effects by blocking the PD-1 / PD-L1 signaling pathway and enhancing T cell activity. Summary of the Invention

[0005] The purpose of this invention is to provide an ultrasound-targeted nanoparticle for deep delivery of nitric oxide gas and the PD-L1 inhibitor BMS1166, its preparation method, and its application. The ultrasound-targeted nanoparticle for deep delivery of nitric oxide gas and the PD-L1 inhibitor BMS1166 provided by this invention enhances the accumulation of nitric oxide gas and BMS1166 in tumors and generates reactive oxygen species and reactive nitrogen species under ultrasound to achieve a therapeutic effect on tumors.

[0006] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0007] A nanoparticle for deep delivery of nitric oxide gas and the PD-L1 inhibitor BMS1166, wherein the nanoparticle comprises a piezoelectric core material BTO, a ROS-responsive prodrug molecule BMS1166-TK-(Arg)9, an LFC131 targeting peptide, and DSPE-PEG2k. The mass ratio of BTO:BMS1166-TK-(Arg)9:LFC131-PEG2k-DSPE:DSPE-PEG2k is 2:1:1:4.

[0008] In the above-mentioned targeted nanoparticles, the average particle size of the nanoparticles is 115 nm; the zeta potential of the nanoparticles is -15.6 ± 0.95 mV.

[0009] The aforementioned targeted nanoparticles can actively target tumor sites via the targeting peptide LFC131, while simultaneously increasing the accumulation of nitric oxide gas and the PD-L1 inhibitor BMS1166 at the tumor site. Under ultrasound irradiation, reactive oxygen species and nitric oxide are generated, thereby promoting tumor therapy. The ultrasound frequency range is 1-3 MHz, with a duty cycle of 10%-50%.

[0010] This invention also provides a method for preparing the above-mentioned targeted nanoparticles that simultaneously deliver nitric oxide gas and the PD-L1 inhibitor BMS1166, comprising the following steps:

[0011] Step 1: Dissolve BMS1166 and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in methanol to obtain solution A;

[0012] N-hydroxysuccinimide (NHS) was dissolved in methanol to obtain solution B, which was then added to solution A. The mixture was reacted under magnetic stirring for 10 minutes to obtain solution C.

[0013] Step 3: Add the above solution C to methanol solution D containing TK-NH2 to obtain solution E; then purify the product by column chromatography to remove unreacted compounds and other byproducts, and obtain BMS1166-TK.

[0014] Step 4: Dissolve the nitric oxide precursor in N,N-dimethylformamide to obtain solution F;

[0015] Step 5: Add solution F to EDC, NHS and BMS1166-TK, react under magnetic stirring for 24 h to obtain solution H, freeze dry to obtain the target prodrug molecule BMS1166-TK-(Arg)9.

[0016] Step 6: Dissolve LFC131 peptide in N,N-dimethylformamide and add EDC, NHS and DSPE-PEG2k-NH2. React under magnetic stirring for 24 h to obtain the target peptide DSPE-PEG2k-LFC131.

[0017] Step 7: Disperse the synthesized oleic acid-modified BTO in n-hexane to obtain solution G;

[0018] Step 8: Mix the products obtained in steps 5, 6, and 7 with DSPE-PEG2k and react under magnetic stirring for 24 hours. Then, inject the mixture into PBS solution or physiological saline under magnetic stirring to form nanoparticles. Then, dialyze the mixture in PBS solution or physiological saline to remove the organic solvent, thereby forming nanoparticles loaded with BMS1166 and nitric oxide precursor.

[0019] The present invention also provides the application of the targeted nanoparticles prepared according to the above technical solution in the diagnostic reagents or treatment of cancer, such as for the treatment of pancreatic cancer.

[0020] In this invention, piezoelectric nanomaterial BTO is used as the core, and the small molecule PD-L1 inhibitor BMS1166 and nitric oxide precursor (Arg)9 are linked by a thioketal bond to form an amphiphilic prodrug molecule. The self-assembly properties of Arg9 are then utilized to combine with BTO to prepare nanoparticles BTO@BAL. Nitric oxide, by degrading the extracellular matrix, can alleviate the high interstitial pressure state in dense stromal tumors such as pancreatic cancer, thereby promoting the penetration and accumulation of nanoparticles deep within the tumor. Simultaneously, the LFC131 peptide, targeting the CXCR4 receptor, is modified onto the surface of the nanoparticles, enabling specific recognition and accumulation of nanoparticles at the molecular level. Under ultrasound, BTO exhibits a piezoelectric catalytic effect, continuously generating reactive oxygen species (ROS). ROS directly cause oxidative damage to tumor cells and oxidize Arg9 to release nitric oxide. Nitric oxide can react with superoxide (O2-) to generate highly reactive peroxynitrite (ONOO-), further enhancing cytotoxicity; the PD-L1 inhibitor BMS1166 can block the PD-1 / PD-L1 immune checkpoint pathway, restore T cell activity, and enhance the immune system's ability to kill tumors. In this synergistic system, ultrasound-triggered ROS and nitric oxide not only directly kill tumor cells but also enhance tumor cell immunogenicity, further reshaping the tumor microenvironment and amplifying the immunotherapeutic effect.

[0021] The beneficial effects of this invention are as follows: This invention uses piezoelectric nanomaterial BTO as the core, and links the small molecule PD-L1 inhibitor BMS1166 with nitric oxide precursor (Arg)9 via a thioketal bond to form an amphiphilic prodrug molecule. Utilizing its self-assembly properties, it combines with BTO to prepare nanoparticles BTO@BAL. These nanoparticles possess piezoelectric catalytic properties, efficiently generating reactive oxygen species (ROS) under ultrasound, oxidizing the nitric oxide precursor to release nitric oxide. This degrades the dense tumor matrix, significantly improving the tumor microenvironment and promoting the nanoparticles' penetration into tumor tissue. Simultaneously, the combined action of targeting peptides increases the local accumulation of nanoparticles in the tumor, reducing non-specific accumulation and effectively increasing its bioavailability. Furthermore, ultrasound-triggered ROS and nitric oxide not only directly kill tumor cells but also enhance tumor cell immunogenicity, further reshaping the tumor microenvironment. The PD-L1 inhibitor BMS1166 can block the PD-1 / PD-L1 immune checkpoint pathway, restore T cell activity, enhance the immune system's ability to kill tumors, and further amplify the immunotherapeutic effect. This system achieves efficient delivery of nanoparticles and immune enhancement, significantly amplifying the anti-tumor effect of piezoelectric catalysis combined therapy. Attached Figure Description

[0022] Figure 1 This is a functional schematic diagram of the multifunctional nanoparticles in specific embodiment 1; Figure 2 This is the mass spectrum of the compound obtained by covalently linking BMS1166 and nitric oxide precursor in the multifunctional nanoparticles in Specific Example 2; Figure 3 The potential and hydration particle size of the multifunctional nanoparticles prepared in Specific Example 3; Figure 4 This is the in vitro electron spin resonance diagram of nitric oxide generated by the multifunctional nanoparticles in Specific Embodiment 4; Figure 5 These are flow cytometry images of cells being taken up by cancer cells in specific embodiment 5. Figure 6 This is a fluorescence microscope image of reactive oxygen species generated after the multifunctional nanoparticles are taken up by cancer cells in specific embodiment 6. Figure 7 This is a fluorescence microscope image of nitric oxide produced by cancer cells after the multifunctional nanoparticles are taken up in specific embodiment 7. Figure 8 This is a fluorescence microscope image of peroxynitrite produced by cancer cells after the multifunctional nanoparticles are taken up in Specific Embodiment 8; Figure 9 This is a fluorescence microscopy image of the downregulation of PD-L1 expression after the multifunctional nanoparticles were taken up by cancer cells in Specific Example 9; Figure 10These are flow cytometry images of multifunctional nanoparticles in embodiment 10, showing how cancer cells respond to ultrasound irradiation to kill tumor cells. Figure 11 This is a fluorescence microscopy image of immunogenic cell death mediated by the uptake of multifunctional nanoparticles by cancer cells in Specific Embodiment 11. Figure 12 This is a fluorescence microscope image of the multifunctional nanoparticles in Specific Example 12, showing how nitric oxide dissolves the dense tumor matrix after being taken up by cancer cells. Figure 13 This is a fluorescence imaging image of the enrichment of multifunctional nanoparticles in animal tumor tissue in specific embodiment 13; Figure 14 This is the tumor growth curve after treatment with multifunctional nanoparticles in specific embodiment 14. Detailed Implementation

[0023] The following detailed descriptions will help to understand the present invention, but do not limit the scope of the invention.

[0024] Example 1

[0025] BMS1166 and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were dissolved in methanol and added to a round-bottom flask, stirred at room temperature in the dark. N-hydroxysuccinimide (NHS) was then dissolved in methanol and added to the mixture, stirred at room temperature in the dark. The reacted mixture was then added dropwise to a pre-prepared methanol solution containing TK-NH2, and stirred at room temperature in the dark for 24 hours. The reaction mixture was then concentrated using a rotary evaporator and purified by column chromatography. After evaporation of the solvent, the product (BMS1166-TK) was obtained, and its structure was confirmed by mass spectrometry. (Arg)9 and EDC were dissolved in DMF and added to a round-bottom flask, stirred at room temperature in the dark. NHS was then dissolved in DMF and added to the mixture, stirred at room temperature in the dark. The purified BMS1166-TK was added to the mixture, and stirred at room temperature in the dark for 24 hours. The reaction mixture was concentrated using a rotary evaporator, then added dropwise to deionized water under vigorous stirring. Dialysis (MWCO 8-14 kDa, 2 hours) was then performed, followed by freeze-drying to obtain a white powder, namely the target prodrug molecule BMS1166-TK-(Arg)9. It was characterized using Fourier transform infrared spectroscopy and mass spectrometry. Following a similar method, the target peptide DSPE-PEG2k-LFC131 was prepared. The prepared barium titanate was then evaporated to remove n-hexane and dissolved in a solution containing DSPE-PEG2K. The mixture was then sonicated in a water bath to ensure thorough mixing. This mixture was then dissolved in a methanol mixture containing BMS1166-TK-(Arg)9 and LFC131-PEG2K-DSPE, and added dropwise to deionized water under vigorous stirring. After purification by dialysis (MWCO 8-14 kDa, 2 hours), barium titanate-loaded piezoelectric nanoparticles BTO@BAL (with attached...) were obtained. Figure 1 ).

[0026] Example 2

[0027] The compound obtained by covalently linking the PD-L1 inhibitor BMS1166 obtained in Example 1 with the nitric oxide precursor nonapolyarginine was purified, freeze-dried, and 1 mg of the compound was redissolved in 1 mL of methanol. The molecular weight of the compound was characterized by high-resolution mass spectrometry (see attached figure). Figure 2 The peak at a mass-to-charge ratio of 2222 indicates the presence of the compound formed by the covalent linkage of the PD-L1 inhibitor BMS1166 and the nitric oxide precursor nonamericarginine via an amide bond. This signifies the successful synthesis of a small molecule compound covalently linked to a PD-L1 inhibitor and a nitric oxide precursor.

[0028] Example 3

[0029] The multifunctional nanoparticles obtained in Example 1, internally loaded with barium titanate and covalently linked to the surface-linked targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericarginine prodrug molecule, were used to measure the hydration size and potential of the nanoparticles using a dynamic light scattering instrument (see attached). Figure 3 The results show that the average hydrated particle size of the multifunctional nanoparticles is 115 nm and the potential is -15.6 ± 0.95 mV, confirming the successful preparation of the nanoparticles.

[0030] Example 4

[0031] The ability of the multifunctional targeted nanoparticles obtained in Example 1 to generate nitric oxide was tested under ultrasonic irradiation, using 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) as a spin trapper and a laser power of 5 mW / cm². 2 The laser irradiation time was 1 minute, and the electron spin resonance spectroscopy was used for detection. The electron spin resonance diagram showed that the prepared targeted nanoparticles possessed a specific ultrastructure of nitric oxide (see attached image). Figure 4 The electron spin resonance spectrum (ESR) of the nanoparticles, which appears as a seven-line spectrum, shows no characteristic signal in the electron spin resonance diagram of the group without ultrasonic irradiation. This indicates that the prepared targeted nanoparticles generate nitric oxide in response to ultrasonic irradiation.

[0032] Example 5

[0033] To track the uptake of targeted nanoparticles by tumor cells, multifunctional nanoparticles labeled with CY5, internally loaded with barium titanate and covalently linked to the surface-linked targeting peptide LFC131 and the PD-L1 inhibitor BMS1166, and containing a nonamericarginine prodrug molecule, were cultured in a 37°C incubator. After cell adhesion, the nanoparticles and cells were co-incubated for 1 h, 2 h, 4 h, and 6 h. Residual nanoparticles were then washed away, and cells were digested with trypsin. Flow cytometry was used to track the fluorescence of CY5. Specific results are attached. Figure 5 As shown, cells exhibit time-dependent uptake of nanoparticles, with fluorescence from CY5 in the nanoparticles increasing over time.

[0034] Example 6

[0035] To evaluate the ability of the multifunctional nanoparticles to generate reactive oxygen species after being taken up by tumor cells, KPC cells were seeded in 24-well plates and incubated overnight to allow them to adhere. Then, different treatments were performed according to the following groups: (G1) saline; (G2) sonication alone; (G3) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule; (G4) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule, combined with sonication; (G5) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule; Nanoparticles containing FC131 and the PD-L1 inhibitor BMS1166 covalently linked to the nonacyclic lysine prodrug molecule were subjected to ultrasound treatment; (G6) nanoparticles containing barium titanate internally and the surface linked to the targeting peptide LFC131 and nonacyclic lysine were subjected to ultrasound treatment; (G7) nanoparticles containing barium titanate internally and the surface linked to the PD-L1 inhibitor BMS1166 covalently linked to the nonacyclic arginine prodrug molecule were subjected to ultrasound treatment; (G8) nanoparticles containing barium titanate internally and the surface linked to the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 covalently linked to the nonacyclic arginine prodrug molecule were subjected to ultrasound treatment (the concentration of BTO in each group was 100 μg / mL). Different drugs were diluted with serum-free medium and added to the corresponding culture dishes. Cells were incubated for 4 h, then the supernatant was discarded, the cells were washed with PBS, and incubated with the DCFH-DA probe at 37°C under light-shielded conditions for 20 min. Then, ultrasound irradiation (1.0 MHz, 1.5 W / cm²) was performed according to the grouping. 2 (50% duty cycle, 2 min), and the fluorescence images were observed using a fluorescence microscope (see attached). Figure 6 ).

[0036] Example 7

[0037] To evaluate the ability of the multifunctional nanoparticles to generate nitric oxide after being taken up by tumor cells, KPC cells were seeded in 24-well plates and incubated overnight to allow them to adhere. Then, different treatments were performed according to the following groups: (G1) saline; (G2) sonication alone; (G3) nanoparticles internally loaded with barium titanate and with a surface-coated targeting peptide LFC131 and PD-L1 inhibitor BMS1166 covalently linked to a nonamericine prodrug molecule; (G4) nanoparticles internally loaded with barium titanate and with a surface-coated targeting peptide LFC131 and PD-L1 inhibitor BMS1166 covalently linked to a nonamericine prodrug molecule combined with sonication; (G5) nanoparticles internally loaded with barium titanate and with a surface-coated targeting peptide... Nanoparticles covalently linked to LFC131 and the PD-L1 inhibitor BMS1166, along with a nonacyclic lysine prodrug, were subjected to ultrasound treatment. (G6) Nanoparticles internally loaded with barium titanate and surface-linked with the targeting peptides LFC131 and nonacyclic lysine were subjected to ultrasound treatment. (G7) Nanoparticles internally loaded with barium titanate and surface-linked with the PD-L1 inhibitor BMS1166, along with a nonacyclic arginine prodrug, were subjected to ultrasound treatment. (G8) Nanoparticles internally loaded with barium titanate and surface-linked with the targeting peptides LFC131 and the PD-L1 inhibitor BMS1166, along with a nonacyclic arginine prodrug, were subjected to ultrasound treatment (BTO concentration was 100 μg / mL in each group). Different drugs were diluted with serum-free medium and added to the corresponding culture dishes. Cells were incubated for 4 hours, then the supernatant was discarded, the cells were washed with PBS, and incubated with DAF-FM DA probes at 37°C under light-shielded conditions for 20 minutes. Then, ultrasound irradiation (1.0 MHz, 1.5 W / cm²) was performed according to the grouping. 2 (50% duty cycle, 2 min), and the fluorescence images were observed using a fluorescence microscope (see attached). Figure 7 ).

[0038] Example 8

[0039] To evaluate the ability of the multifunctional nanoparticles to generate peroxynitrite after being taken up by tumor cells, KPC cells were seeded in 24-well plates and incubated overnight to allow them to adhere. Then, different treatments were performed according to the following groups: (G1) saline; (G2) sonication alone; (G3) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule; (G4) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule, combined with sonication; (G5) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule; Nanoparticles containing LFC131 peptide and PD-L1 inhibitor BMS1166 covalently linked to a nonacyclic lysine prodrug molecule were subjected to ultrasound treatment; (G6) nanoparticles containing barium titanate internally and LFC131 and nonacyclic lysine targeting peptides were covalently linked to the surface, and (G7) nanoparticles containing barium titanate internally and BMS1166 covalently linked to a nonacyclic arginine prodrug molecule were covalently linked to the surface, and (G8) nanoparticles containing barium titanate internally and LFC131 and BMS1166 covalently linked to a nonacyclic arginine prodrug molecule were covalently linked to the surface, and (BTO concentration in each group was 100 μg / mL) were subjected to ultrasound treatment. Different drugs were diluted with serum-free medium and added to the corresponding culture dishes. Cells were incubated for 4 h, then the supernatant was discarded, the cells were washed with PBS, and incubated with a DHE probe at 37°C under light-shielded conditions for 20 min. Then, ultrasound irradiation (1.0 MHz, 1.5 W / cm²) was performed according to the grouping. 2 (50% duty cycle, 2 min), and the fluorescence images were observed using a fluorescence microscope (see attached). Figure 8 ).

[0040] Example 9

[0041] KPC cells were seeded in 12-well plates and incubated overnight until adherence. Then, they were treated according to the following groups: (G1) saline; (G2) sonication alone; (G3) nanoparticles internally loaded with barium titanate and covalently linked to the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule; (G4) nanoparticles internally loaded with barium titanate and covalently linked to the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule, combined with sonication; (G5) nanoparticles internally loaded with barium titanate and covalently linked to the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule. Nanoparticles covalently linked to nona-lysine prodrug molecule S1166 were subjected to ultrasound; (G6) nanoparticles internally loaded with barium titanate and surface-linked with targeting peptide LFC131 and nona-lysine were subjected to ultrasound; (G7) nanoparticles internally loaded with barium titanate and surface-linked with PD-L1 inhibitor BMS1166 and nona-arginine prodrug molecule were subjected to ultrasound; (G8) nanoparticles internally loaded with barium titanate and surface-linked with targeting peptide LFC131 and PD-L1 inhibitor BMS1166 and nona-arginine prodrug molecule were subjected to ultrasound (BTO concentration in each group was 100 μg / mL). Different drugs were diluted with serum-free medium and added to corresponding culture dishes, incubated with cells for 4 h, washed with serum-free medium to remove untaken nanoparticles, subjected to ultrasound irradiation, and then incubated for another 24 h (ultrasound parameters: 1.0 MHz, 1.5 W / cm²). 2 (50% duty cycle, 2 min), staining was performed using anti-PD-L1 antibody, and the fluorescence of each group was observed using a fluorescence microscope (see attached). Figure 9 This demonstrates that the targeted nanoparticles have a good ability to downregulate PD-L1 expression in cells.

[0042] Example 10

[0043] To evaluate the cell-killing ability of nanoparticles in response to ultrasound irradiation, KPC cells were seeded in 12-well plates and incubated overnight to allow them to adhere. Then, different treatments were administered according to the following groups: (G1) saline; (G2) ultrasound alone; (G3) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166, along with a nonamericine prodrug molecule; (G4) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166, combined with ultrasound; (G5) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166, along with ultrasound; 1. Nanoparticles covalently linked to the PD-L1 inhibitor BMS1166 and the nonaconjugated lysine prodrug molecule were subjected to ultrasound treatment; (G6) Nanoparticles internally loaded with barium titanate and surface-linked with the targeting peptide LFC131 and nonaconjugated lysine were subjected to ultrasound treatment; (G7) Nanoparticles internally loaded with barium titanate and surface-linked with the PD-L1 inhibitor BMS1166 and the nonaconjugated lysine prodrug molecule were subjected to ultrasound treatment; (G8) Nanoparticles internally loaded with barium titanate and surface-linked with the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 and the nonaconjugated lysine prodrug molecule were subjected to ultrasound treatment (BTO concentration in each group was 200 μg / mL). Different drugs were diluted with serum-free medium and added to the corresponding culture dishes, incubated with cells for 4 h, and then subjected to ultrasound irradiation (1.0 MHz, 1.5 W / cm²). 2 (50% duty cycle, 2 min), continue incubation for 16 h, then co-incubate with the cells using the Annexin V-APC / PI apoptosis detection kit for 20 min, and detect apoptosis by flow cytometry (see attached). Figure 10 ).

[0044] Example 11

[0045] Immunofluorescence staining was used to assess the immunogenic cell death effect induced by BTO@BAL nanoparticles. KPC cells were seeded in confocal microplates and incubated for 24 h to allow cell adhesion. Then, different treatments were performed according to the following groups: (G1) saline; (G2) sonication alone; (G3) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericarginine prodrug molecule; (G4) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericarginine prodrug molecule combined with sonication; (G5) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericarginine prodrug molecule combined with sonication; Nanoparticles containing lysine prodrug molecules were subjected to ultrasound treatment; (G6) nanoparticles internally loaded with barium titanate and surface-linked with the targeting peptide LFC131 and nonapolylysine were subjected to ultrasound treatment; (G7) nanoparticles internally loaded with barium titanate and surface-linked with the PD-L1 inhibitor BMS1166 and nonapolylysine prodrug molecules were subjected to ultrasound treatment; (G8) nanoparticles internally loaded with barium titanate and surface-linked with the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 and nonapolylysine prodrug molecules were subjected to ultrasound treatment (BTO concentration in each group was 100 μg / mL). Cells were then fixed with 4% paraformaldehyde (room temperature, 15 min), infiltrated with Triton X-100 (0.2%) for 5 min, and blocked with BSA (1%) at room temperature for 1 h. Subsequently, the treated cells were incubated overnight at 4°C with primary antibodies of Alexa Fluor@594 anti-HMGB1, Alexa Fluor@594 anti-CRT, and Alexa Fluor@594 anti-HSP70, respectively. KPC cell nuclei were stained with DPAI (room temperature, 10 min). Finally, KPC cells were subjected to immunofluorescence imaging using different bandpass filters (see attached image). Figure 11 The fluorescence channels are as follows: DAPI: λex: 405nm; λem: 410-490nm; Alexa Fluor@594: λex: 590nm; λem: 618nm.

[0046] Example 12

[0047] Immunofluorescence staining was used to evaluate the ability of BTO@BAL nanoparticles combined with ultrasound-generated nitric oxide to dissolve dense tumor matrix. Pathological sections were taken from mouse tumors treated with different drugs. The sections were incubated with FN, α-SMA, and Collagen-1 antibodies, and KPC cell nuclei were stained with DPAI, followed by immunofluorescence imaging (see attached image). Figure 12The different treatments are as follows: (G1) saline; (G2) sonication alone; (G3) nanoparticles internally loaded with barium titanate, with the surface linked to the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 covalently linked to the nonamericarin prodrug molecule; (G4) nanoparticles internally loaded with barium titanate, with the surface linked to the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 covalently linked to the nonamericarin prodrug molecule combined with sonication; (G5) nanoparticles internally loaded with barium titanate, with the surface linked to the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 covalently linked to the nonamericarin prodrug molecule. Nanoparticles containing drug molecules were subjected to combined ultrasound; (G6) nanoparticles containing barium titanate internally and surface-linked targeting peptide LFC131 and nonapolylysine were subjected to combined ultrasound; (G7) nanoparticles containing barium titanate internally and surface-linked PD-L1 inhibitor BMS1166 and nonapolylysine prodrug molecules were subjected to combined ultrasound; (G8) nanoparticles containing barium titanate internally and surface-linked targeting peptide LFC131 and PD-L1 inhibitor BMS1166 and nonapolylysine prodrug molecules were subjected to combined ultrasound (the concentration of BTO in each group was 100 μg / mL).

[0048] Example 13

[0049] To assess the in vivo distribution of nanoparticles, in vivo near-infrared fluorescence imaging was performed. First, a mouse subcutaneous KPC tumor model was established. Nanoparticles containing CY5-labeled barium titanate internally loaded with the target peptide LFC131 and the PD-L1 inhibitor BMS1166 covalently linked to a nonamericargine prodrug molecule, and nanoparticles containing CY5-labeled untargeted peptide internally loaded with barium titanate and covalently linked to the PD-L1 inhibitor BMS1166 to a nonamericargine prodrug molecule, were injected into mice via the tail vein. Near-infrared fluorescence imaging was performed on the mice at 3h, 9h, 12h, 24h, and 48h (see attached image). Figure 13 The nanoparticles with the targeting peptide linked to the target peptide exhibited stronger fluorescence at the tumor site compared to nanoparticles without the target peptide linked to them, indicating that the linkage of the target peptide improved the tumor targeting of the nanoparticles. This strongly demonstrates that the prepared targeting nanoparticles can effectively increase the enrichment amount at the tumor site.

[0050] Example 14

[0051] In vivo therapeutic combination experiments investigated whether the prepared BTO@BAL nanoparticles could effectively inhibit tumor growth. Mice with KPC subcutaneous tumors were randomly divided into 8 groups and given different treatment regimens, including (G1) saline; (G2) ultrasound alone; (G3) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule; (G4) nanoparticles internally loaded with the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 covalently linked to a nonamericine prodrug molecule combined with ultrasound; and (G5) nanoparticles internally loaded with barium titanate and covalently linked to the surface of the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 with a nonamericine prodrug molecule combined with ultrasound. Nanoparticles covalently linked to the BMS1166 nonacyclic lysine prodrug molecule and combined with ultrasound; (G6) Nanoparticles internally loaded with barium titanate and surface-linked with the targeting peptide LFC131 and nonacyclic lysine and combined with ultrasound; (G7) Nanoparticles internally loaded with barium titanate and surface-linked with the PD-L1 inhibitor BMS1166 covalently linked to the nonacyclic arginine prodrug molecule and combined with ultrasound; (G8) Nanoparticles internally loaded with barium titanate and surface-linked with the targeting peptide LFC131 and the PD-L1 inhibitor BMS1166 covalently linked to the nonacyclic arginine prodrug molecule and combined with ultrasound (Dosage: 10 mg / kg BaTiO3, once every 3 days, for a total of 2 times; ultrasound therapy was performed 24 hours after injection, with a frequency of 1.0 MHz and a power of 1.5 W / cm²). 2 (50% duty cycle, duration 5 minutes), with 200 μL administered via tail vein injection in all groups. Tumor volume and body weight changes were recorded daily after treatment in each group of mice (tumor volume = 1 / 2 × length × width²). The BTO@BAL+US group showed a significant inhibitory effect on tumor growth, with tumor growth only one-tenth that of the Control group, indicating that ultrasonic piezoelectric catalysis combined with NO gas therapy effectively enhanced the efficacy of immunotherapy. Results are attached. Figure 14 As shown.

Claims

1. Targeted nanoparticles that simultaneously deliver nitric oxide gas and the PD-L1 inhibitor BMS1166, wherein, The nanoparticles comprise a piezoelectric core BTO, a ROS-responsive prodrug molecule BMS1166-TK-(Arg)9, and an LFC131 targeting peptide and a DSPE-PEG2kBTO:prodrug molecule BMS1166-TK-(Arg)9 in a mass ratio of 2:

1.

2. The ultrasound-targeted nanoparticles as described in claim 1, characterized in that, The nitric oxide precursor is nonamericarginine; The piezoelectric core is a tetragonal phase oleic acid-modified BTO; the targeting peptide is LFC131 covalently linked with DSPE-PEG2k; and the PD-L1 inhibitor is the small molecule drug BMS1166.

3. The ultrasound-targeted nanoparticles as described in claim 2, characterized in that, The prodrug molecule contains the nitric oxide precursor nonamericarginine, whose structural formula is as follows:

4. The ultrasound-targeted nanoparticles as described in claim 2, characterized in that, The PD-L1 inhibitor BMS1166 has the following structural formula:

5. The ultrasonically targeted nanoparticles as described in claim 2, characterized in that, The prodrug molecule BMS1166-TK-(Arg)9 has the following structural formula:

6. The ultrasound-targeted nanoparticles as described in claim 2, characterized in that, The targeting peptide is DSPE-PEG2k-LFC131, covalently linked to LFC131 and DSPE-PEG2k, and its structural formula is as follows:

7. The ultrasound-targeted nanoparticles as described in claim 1, characterized in that, The average particle size of the nanoparticles is approximately 115 nm.

8. The ultrasound-targeted nanoparticles as described in claim 1, characterized in that, The zeta potential of the nanoparticles is -15.6 ± 0.95 mV.

9. A method for preparing ultrasound-targeted nanoparticles that simultaneously deliver nitric oxide gas and PD-L1 inhibitor BMS1166 as described in any one of claims 1 to 8, comprising the following steps: 1) Dissolve BMS1166 and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in methanol to obtain solution A; 2) Dissolve N-hydroxysuccinimide (NHS) in methanol to obtain solution B, and add it to solution A to obtain solution C; 3) Add the above solution C to methanol solution D containing TK-NH2 to obtain solution E; The product was then purified by column chromatography to remove unreacted compounds and other byproducts, yielding BMS1166-TK. 4) Dissolve the nitric oxide precursor in N,N-dimethylformamide to obtain solution F; 5) Add solution F to EDC, NHS and BMS1166-TK, react under magnetic stirring for 24 h to obtain solution H, freeze dry to obtain the target prodrug molecule BMS1166-TK-(Arg)9. 6) Dissolve LFC131 peptide in N,N-dimethylformamide and add EDC, NHS and DSPE-PEG2k-NH2. React under magnetic stirring for 24 h to obtain the target peptide DSPE-PEG2k-LFC131. 7) The synthesized oleic acid-modified BTO was dispersed in n-hexane to obtain solution G; 8) The products obtained in steps 5 and 6) are mixed with BTO and DSPE-PEG2k dispersed in n-hexane and reacted under magnetic stirring for 24 h. Then, the mixture is injected into PBS solution or physiological saline under magnetic stirring to form nanoparticles. The nanoparticles are then dialyzed in PBS solution or physiological saline to remove the organic solvent and form nanoparticles loaded with BMS1166 and nitric oxide precursor.

10. The preparation method according to claim 6, characterized in that, The BTO formed in step 7), the prodrug molecule formed in step 5), the targeted peptide formed in step 6), and DSPE-PEG2k are mixed in a mass ratio of 2:1:1:

4.

11. The use of the targeted nanoparticles that simultaneously deliver nitric oxide gas and the PD-L1 inhibitor BMS1166 as described in any one of claims 1 to 10 in a medicament for diagnosing and / or treating cancer, wherein the cancer is pancreatic cancer.

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