A pH-ultrasound dual-responsive albumin nanodrug and a preparation method thereof

Abstract

The application relates to the technical field of drug synthesis, in particular to a pH-ultrasound double-response albumin nano-drug and a preparation method thereof. The specific technical scheme is as follows: a preparation method of a pH-ultrasound double-response albumin nano-drug, steps are as follows: (1) synthesizing SrO2NPs: in a PVP solution, SrCl2, NaOH and H2O are sequentially added 2， After stirring, centrifugation is carried out, SrO2NPs precipitate is obtained, and dry SrO2NPs powder is obtained after drying; (2) activating Ce6: Ce6 is dissolved in DMSO, EDC and NHS are added, and reaction is carried out at normal temperature in the dark; (3) SrO2NPs are dissolved in ultrapure water, after ultrasonic treatment, HSA aqueous solution, Ce6 DMSO solution and Ti DMSO solution are added, after stirring, centrifugation and washing, the pH-ultrasound double-response albumin nano-drug is obtained. The application successfully synthesizes a multifunctional novel nano-drug HSA-SrO2-Ce6-TGFbeta inhibitor (HSCTi NPs) which has targeting property, can relieve the hypoxic microenvironment in tumors and can jointly deliver the ultrasound-responsive cytotoxic drug Ce6 and the TGFbeta inhibitor, so as to realize precise and controllable liver cancer sonodynamic- immune combined treatment.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis technology, specifically to a pH-ultrasound dual-response albumin nanomedicine and its preparation method. Background Technology

[0002] Liver cancer is the sixth leading cause of cancer incidence and the third leading cause of cancer death worldwide, with over 800,000 new cases annually and a mortality-to-incidence ratio as high as 0.88. Liver cancer seriously threatens the health and lives of people worldwide. The low cure rate of liver cancer may be attributed to a variety of factors, such as the lack of obvious early clinical symptoms, the lack of precise and effective treatment methods, and the presence of an immunosuppressive microenvironment. Studies have found that liver cancer often suffers from insufficient immunogenicity, as dendritic cells (DCs) cannot effectively recognize and present cancer cell antigens, thus failing to efficiently recruit and activate CD8+. + T cells, CD4 + Effector T cells, such as T cells, are also present. Simultaneously, the tumor often infiltrates with a large number of immunosuppressive cells, including regulatory T cells (Tregs) and tumor-associated macrophages (TAMs, primarily M2 type), giving liver cancer an overall immunologically "cold tumor" state. This immunosuppressive microenvironment not only severely restricts the effectiveness of immunotherapy but also prevents cytotoxic drugs from reaching their full therapeutic potential. Therefore, there is an urgent need to develop therapeutic strategies that can target and kill tumor cells effectively, overcoming the immunosuppressive microenvironment.

[0003] Immunotherapy is widely used in cancer treatment due to its high specificity, broad applicability, ability to eliminate residual cancer cells, and prevention of tumor recurrence. However, immunotherapy alone often faces many challenges, such as the fact that the immune system of most cancer patients cannot effectively recognize cancer cells, resulting in limited efficacy. Furthermore, tumors often evade immune system attacks through immune escape mechanisms, further limiting the effectiveness of immunotherapy. To address these issues, Watabe et al. developed a personalized immunotherapy based on smart nanovesicles to overcome breast cancer resistance to immunotherapy. By anchoring bioactive interleukin-2 (IL2) and enriching T-cell co-stimulatory factors on the surface of dendritic cell-derived small exosomes, an immune response was promoted. These nanovesicles can activate IL2 receptors in lymphocytes, perform antigen presentation, significantly enhance the immune response, and effectively prevent the metastasis and recurrence of breast cancer. Liu et al. designed a multifunctional colloidal microreactor that encapsulates catalase in colloidal vesicles assembled from calcium carbonate nanoparticles to neutralize the acidic environment of tumors and alleviate tumor hypoxia. When applied within tumors, this microreactor not only activates a robust anti-tumor immune response and significantly enhances the therapeutic effect of anti-PD-1 antibodies, but also strengthens CAR-T cell tumor infiltration and effector cytokine secretion, thereby improving its therapeutic efficacy against tumors. Therefore, combining nanotechnology with immunotherapy to construct multifunctional nanomedicines may offer an innovative solution for overcoming immune resistance and improving therapeutic efficacy.

[0004] In recent years, sonodynamic therapy (SDT) has received widespread attention in cancer treatment due to its non-invasive nature, excellent tissue penetration (>10cm), and low toxicity. The principle of sonodynamic therapy is to utilize ultrasound (US) of appropriate frequency and intensity, along with an ultrasound-responsive sonosensitive agent, to generate a large amount of reactive oxygen species (ROS, mainly singlet oxygen) from oxygen (O2). 1O2 (oxygen-2) has a killing effect on tumor cells. Therefore, in practical applications, the efficacy of sonodynamic therapy is affected by the harsh tumor microenvironment (such as hypoxia, high infiltration of immunosuppressive cells, etc.) and cannot achieve the expected results. Recent studies have found that multimodal therapies (such as sonodynamic therapy combined with immunotherapy) have shown great application potential in anti-tumor treatment. For example, Wang et al. constructed a multifunctional biomimetic nanodrug, MPIRx, using IR780 as a sonosensitive agent, adding a CD47 inhibitor, and coating it with osteosarcoma (OS) cell membranes to achieve sonodynamic therapy combined with immunotherapy for osteosarcoma lung metastases. Experimental results showed that the ultrasound (US) activated MPIRx nanodrug significantly inhibited tumor development and lung metastases, reduced CD47 expression, and promoted the polarization of tumor-associated macrophages (TAMs) to the M1 type (anti-tumor type). Furthermore, numerous studies have reported that SDT can induce immunogenic cell death (ICD) by translocating to calreticulin (CRT), secreting human high-mobility histone 1 (HMGB-1), and releasing adenosine triphosphate (ATP) in dying cells. This promotes the maturation of dendritic cells (DCs), ultimately activating cytotoxic T lymphocytes (CTLs) and triggering anti-tumor immune responses. Zhao et al. developed biomimetic nanoparticles coated with a hybrid membrane formed from cancer cell membranes and macrophage membranes. These nanoparticles were loaded with the semiconductor polymer PFODBT, atovaquinone (ATO), and TMP195 to achieve a novel strategy combining sonodynamic therapy and immunotherapy for tumor treatment. In vivo experiments demonstrated that the synergistic effect of enhanced SDT-mediated ICD and TAM polarity reprogramming significantly inhibited the proliferation of primary and distant tumors in a 4T1 subcutaneous tumor model and effectively reduced lung and liver metastases. Therefore, sonodynamic therapy combined with immunotherapy for targeted treatment of liver cancer may be an effective therapeutic strategy.

[0005] Immunosuppression and insufficient drug targeting are among the major challenges in the treatment of liver cancer. Sonodynamic therapy, as an emerging and spatiotemporally controllable effective method for cancer treatment, has shown great clinical potential. Therefore, how to achieve targeted synergistic use of sonodynamic therapy and immunotherapy in the treatment of liver cancer is of great significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a pH-ultrasound dual-responsive albumin nanomedicine and its preparation method. A novel multifunctional nanomedicine, HSA-SrO2-Ce6-TGFβ inhibitor (HSCTiNPs), was successfully synthesized. This nanomedicine possesses targeting capabilities, can alleviate the hypoxic microenvironment within tumors, and can deliver ultrasound-responsive cytotoxic drugs Ce6 and TGFβ inhibitors in combination, aiming to achieve precise and controllable sonodynamic-immunotherapy for liver cancer.

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

[0008] This invention discloses 1. a method for preparing pH-ultrasound dual-response albumin nanomedicine, the steps of which are as follows:

[0009] (1) Synthesis of SrO2 NPs: SrCl2, NaOH and H2O were added sequentially to the PVP solution. 2， After stirring and centrifugation, SrO2 NPs precipitate was obtained, and after drying, dry SrO2 NPs powder was obtained.

[0010] (2) Activation of Ce6: Dissolve Ce6 in DMSO, add EDC and NHS, and react at room temperature in the dark;

[0011] (3) Dissolve SrO2 NPs in ultrapure water, sonicate, add HSA aqueous solution, Ce6DMSO solution and TiDMSO solution, stir, centrifuge and wash to obtain pH-ultrasound dual-response albumin nanomedicine.

[0012] Preferably, in step (1), the concentration of the PVP solution is 20-60 g / L, the concentration of SrCl2 is 0.5-3 mol / L, the concentration of NaOH is 2-10 mol / L, and the concentration of H2O2 is 20-30 wt%.

[0013] Preferably, in step (1), the stirring time is 20-35 hours and the centrifugation is performed at 7000-10000 rpm for 10-30 minutes.

[0014] Preferably, in step (2), the mass ratio of Ce6, EDC and NHS is 1:1.5 to 3:1.5 to 3.

[0015] Preferably, in step (2), the reaction is carried out in the dark for 2 to 5 hours.

[0016] Preferably, in step (3), the amount of SrO2 NPs is 0.01-0.7 mg / mL, the concentration of HSA aqueous solution is 2-5 mg / mL, the concentration of Ce6 DMSO solution is 0.5-3 mg / mL, and the concentration of Ti DMSO solution is 0.1-1.0 mg / mL.

[0017] Preferably, in step (3), the volume ratio of the HSA aqueous solution, Ce6 DMSO solution and Ti DMSO solution is 10-30:2-4:1-3.

[0018] Preferably, in step (3), the ultrasonic treatment time is 10-30 min, the stirring time is 20-35 h, and the centrifugation speed is 10000-15000 rpm.

[0019] Preferably, the prepared pH-ultrasound dual-response albumin nanomedicine is stored in a PBS solution at pH 7.4.

[0020] Correspondingly, the pH-ultrasound dual-response albumin nanomedicine prepared by the above preparation method.

[0021] The present invention has the following beneficial effects:

[0022] This invention successfully constructed a novel multifunctional nanomedicine, HSCTi NPs, using a one-pot method. It possesses a spherical nanostructure with a stable particle size of approximately 140 nm. Furthermore, it exhibits excellent tumor targeting and pH-ultrasound dual responsiveness, capable of alleviating tumor cell hypoxia and efficiently killing tumor cells, thus reshaping the tumor immunosuppressive microenvironment. Simultaneously, in vitro and in vivo experiments confirmed the antitumor effect of HSCTi NPs combined with ultrasound-mediated sonodynamic immunotherapy. This nanomedicine also demonstrates good biocompatibility, providing a new approach for mobilizing a systemic immune response and combining it with cytotoxic drugs to efficiently kill tumor cells in the treatment of liver cancer. Attached Figure Description

[0023] Figure 1 Characterization of HSCTi NPs: (A) HSCTi NPs synthesis steps; (B) TEM of HSC NPs; (C) TEM of HSCTi NPs; (D) Particle size of HS NPs, HSC NPs, and HSCTi NPs; (E) Potential of HS NPs, HSC NPs, and HSCTi NPs; (FH) HAADF (High Angle Ring Dark Field Imaging) and EDS of HSCTi NPs; (I) UV-Vis spectrum; (J) Infrared spectrum; (K) XRD of SrO2 NPs and HSCTi NPs; (L) XPS full spectrum of SrO2 NPs and HSCTi NPs; (MN) High-resolution XPS spectra of Sr 3d and O1s in HSCTi NPs.

[0024] Figure 2The study aimed to evaluate the efficacy of in vitro antitumor therapy. The results included: (A) UV spectral changes of Ce6+DPBF solution and (B) HSCTi NPs+DPBF solution after ultrasound irradiation; (C) Cell viability analysis (n=3) of tumor cells (Hepa1-6 cells) treated with Ce6, HSC NPs, and HSCTi NPs for 24 h with and without ultrasound; (D) Cell viability analysis (n=3) of normal hepatocytes (MIHA cells) treated with HSCTi NPs for 24 h without ultrasound; (EF) TNF-α and IFN-γ secretion in tumor cells treated with Ce6, HSC NPs, and HSCTi NPs with and without ultrasound; and (G) Apoptosis of Hepa1-6 cells detected by Annexin-VFITC / PI double staining and flow cytometry in the following groups: ①PBS group, ②US group, ③Ce6 group, ④HSC NPs group, ⑤HSSCTi NPs group, ⑥Ce6+US group, and ⑦HSC NPs+US group, ⑧HSCTi NPs+US group;

[0025] Figure 3 For in vitro antitumor efficacy evaluation; (A) HSCTi NPs and (B) CLSM images of Hepa1-6 cells at different time points after Ce6 treatment; scale: 10 μm; (C) Quantitative analysis of cell uptake by flow cytometry; (D) Images of Hepa1-6 cells incubated with H2DCFDA probe after different drugs and with or without sonication treatment (scale: 100 μm) and (F) semi-quantitative fluorescence analysis (n=3); (G) Quantitative analysis by flow cytometry; (E) Live / dead cell staining images of Hepa1-6 cells after different drugs and with or without sonication treatment (scale: 200 μm) and (H) semi-quantitative analysis.

[0026] Figure 4 The study aimed to evaluate the efficacy of in vitro antitumor therapy. The results included: (A) scratch assay (scale: 50 μm); (B) quantitative semi-analysis of migration ability after different drug treatments (n=3); (C) quantitative analysis of ATP in Hepa1-6 cells after different drug treatments and with or without sonication (n=3); (D) quantitative analysis of HMBG1 ELISA in Hepa1-6 cells after different drug treatments and with or without sonication (n=3); (E) hemolysis assay (n=3); (F) immunofluorescence images of CD11c in tumor tissues after different treatments (scale: 50 μm); and (G) immunofluorescence images of CD86 in tumor tissues after different treatments (scale: 50 μm).

[0027] Figure 5For in vivo antitumor efficacy evaluation; (A) Schematic diagram of tumor treatment; (B) Real-time fluorescence imaging of C57 mice after intravenous injection of HSCTi NPs; (C) Tumor tissue photograph on day 15 (scale: 1cm); (D, G) Tumor volume changes after different treatments, n=6; (E) Tumor weight obtained after euthanizing mice 15 days after treatment, n=6; (F) H&E staining images of the heart, liver, spleen, lungs and kidneys of mice after different treatments; (H) Changes in body weight of mice during treatment, n=6; (I) Changes in body temperature at the tumor site of mice during treatment, n=6.

[0028] Figure 6 Complete blood count and serum biochemical analysis were performed on mice euthanized 15 days after treatment (n=3).

[0029] Figure 7 Transcriptomic analysis (n=3) was performed, including (A) observation of upregulation and downregulation of differentially expressed genes in the HSCTi+US group using the volcano plot method; (B) KEGG pathway enrichment analysis; (C, D) heatmap of differentially expressed immune-related genes positively regulating innate immune response and T cell activation after HSCTi NPs treatment with or without ultrasound application, with C57 tumor-bearing mice that did not receive any treatment as blank controls;

[0030] Figure 8 The sonodynamic-immunotherapy mechanism mediated by HSCTi NPs. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0033] This invention successfully synthesized a novel multifunctional nanomedicine, HSA-SrO2-Ce6-TGFβ inhibitor (HSCTi NPs), which possesses targeting capabilities, alleviates the hypoxic microenvironment within tumors, and can deliver in combination with the ultrasound-responsive cytotoxic drug Ce6 and a TGFβ inhibitor. This aims to achieve precise and controllable sonodynamic-immunotherapy for liver cancer. In vitro experiments showed that HSCTi NPs exhibit dual pH and ultrasound responsiveness, generating large amounts of reactive oxygen species (ROS) to kill tumor cells, and the dead tumor cells successfully induced tumor immunogenic death (ICD). Importantly, in vivo studies confirmed that HSCTi NPs effectively inhibited tumor growth and significantly activated the anti-tumor immune response. Therefore, this invention provides new insights for the synergistic treatment of liver cancer with sonodynamic therapy and immunotherapy, as well as for other multimodal targeted cancer therapies.

[0034] Albumin is the most abundant plasma protein and a reservoir of various compounds in the physiological environment. Its unique advantages (such as good biocompatibility, long half-life (19 days), low cost, good stability, and ease of preparation) have made it a popular choice for multifunctional drug nanocarriers. Furthermore, using albumin as a nanocarrier for tumor therapy drugs can enable autonomous targeting of lesions: firstly, tumor tissue itself has a high uptake of albumin; secondly, albumin has a high binding affinity for cysteine-rich acidic secretory proteins (SPARC, a metastasis-associated extracellular matrix glycoprotein overexpressed in cancer cells).

[0035] Dihydroporphyrin e6 (Ce6) is the most commonly used porphyrin-based small-molecule organic somatosensitizer. Its advantages include good biocompatibility and easy metabolism in the liver or kidneys of live mice. Ce6 has been shown to be a common somatosensitizer (such as acridine orange (AO), curcumin (CUR), methylene blue (MB), indocyanine green (ICG), protoporphyrin IX (PpIX), etc.). 1 The sonodynamic agent with the highest O2 production rate. However, the hypoxic microenvironment of tumor tissue greatly limits the efficiency of sonodynamic therapy.

[0036] SrO2 can generate a large amount of O2 in an acidic environment, improving the hypoxic microenvironment of tumors. Furthermore, O2 can serve as a raw material for sonodynamic therapy, enhancing the efficiency of sonodynamic therapy (SDT). TGFβ expression is typically increased in tumor cells. TGFβ can induce phenotypic changes in tumor cells through epithelial-mesenchymal transition (EMT), thereby increasing their migration and invasive properties. In addition, TGFβ can reduce the activity of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) while increasing the number of regulatory T lymphocytes, thus enhancing immune tolerance. Therefore, inhibiting TGFβ signaling is considered a prerequisite and a major pathway to improve the efficacy of current and future immunotherapies.

[0037] This invention discloses the synthesis process of pH-ultrasound dual-response albumin nanomedicine as follows:

[0038] (1) Synthesis of SrO2 NPs: In a solution containing 20–60 g / L PVP, 0.5–3 mol / L SrCl2, 2–10 mol / L NaOH, and 20–30 wt% H2O2 were added sequentially. The mixture was stirred vigorously for 20–35 h, then transferred to a 50 mL centrifuge tube and centrifuged at 7000–10000 rpm for 10–30 min. The supernatant was decanted to obtain SrO2 NPs precipitate. Finally, the sample was dried overnight in a 70 °C oven to obtain dried SrO2 NPs powder.

[0039] (2) Activation of Ce6: Ce6 is dissolved in DMSO, EDC and NHS are added, and the mixture is reacted at 25°C in the dark for 2-5 hours to activate the carboxyl group. The mass ratio of Ce6, EDC and NHS is 1:1.5-3:1.5-3.

[0040] (3) Dissolve SrO2 NPs in ultrapure water (DIW) and sonicate for 10–30 min. Add HSA aqueous solution, Ce6 DMSO solution, and Ti DMSO solution to the solution. Stir for 20–35 h, then centrifuge at 10,000–15,000 rpm, wash three times with pure water, and finally store in PBS solution at pH 7.4. The amount of SrO2 NPs used is 0.01–0.7 mg / mL, the concentration of HSA aqueous solution is 2–5 mg / mL, the concentration of Ce6 DMSO solution is 0.5–3 mg / mL, and the concentration of Ti DMSO solution is 0.1–1.0 mg / mL. The volume ratio of HSA aqueous solution, Ce6 DMSO solution, and Ti DMSO solution is 10–30:2–4:1–3. Ce6 DMSO solution refers to DMSO solution containing Ce6, and similarly, Ti DMSO solution also refers to DMSO solution containing Ce6.

[0041] The present invention will be further described below with reference to specific embodiments.

[0042] The reagents and solvents used in the examples are: polyvinylpyrrolidone (PVP), strontium chloride (SrCl2), sodium hydroxide (NaOH), hydrogen peroxide (H2O2), Ce6 (dihydroporphyrin e6), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dimethyl sulfoxide (DMSO), human serum albumin (HSA), TGFβ inhibitor (SB505124, Ti), and phosphate buffered saline (PBS).

[0043] Example 1: Synthesis of pH-ultrasound dual-response albumin nanomedicines (HSCTi NPs)

[0044] The synthesis process is as follows:

[0045] (1) Synthesis of SrO2 NPs: 1 mol / L SrCl2, 5 mol / L NaOH, and 30 wt% H2O2 were added sequentially to a solution containing 40 g / L PVP. The mixture was stirred vigorously for 24 h, then transferred to a 50 mL centrifuge tube and centrifuged at 8000 rpm for 20 min. The supernatant was decanted to obtain SrO2 NPs precipitate. Finally, the sample was dried overnight in a 70 °C oven to obtain dried SrO2 NPs powder.

[0046] (2) Activation of Ce6: Dissolve Ce6 (3 mg) in 3 mL DMSO, add EDC (6 mg) and NHS (6 mg), and react at 25 °C in the dark for 3 h to activate the carboxyl group.

[0047] (3) Dissolve 2 mg SrO2 NPs in 20 mL of ultrapure water (DIW) and sonicate for 15 min. Add 20 mL of 3 mg / mL HSA aqueous solution, 3 mL of 1 mg / mL Ce6 DMSO solution, and 2 mL of 0.5 mg / mL Ti DMSO solution to the solution. Stir for 24 h, then centrifuge at 12000 rpm, wash three times with pure water, and finally store in PBS solution at pH 7.4.

[0048] HS NPs (HSA-SrO2 nanoparticles) consist only of HSA and SrO2, and their synthesis method is as follows:

[0049] 1) Synthesis of SrO2 NPs: In a solution containing 40 g / L PVP, 1 mol / L SrCl2, 5 mol / L NaOH, and 30 wt% H2O2 were added sequentially. The mixture was vigorously stirred for 24 h, then transferred to a 50 mL centrifuge tube and centrifuged at 8000 rpm for 20 min. The supernatant was decanted to obtain SrO2 NPs precipitate. Finally, the sample was dried overnight in a 70 °C oven to obtain dried SrO2 NPs powder.

[0050] 2) Dissolve 2 mg SrO2 NPs in 20 mL of ultrapure water (DIW) and sonicate for 15 min. Add 20 mL of 3 mg / mL HSA aqueous solution, stir for 24 h, then centrifuge at 12000 rpm, wash three times with pure water, and finally store in PBS solution at pH 7.4.

[0051] HSC NPs (HSA-SrO2-Ce6 nanoparticles) consist only of HSA, SrO2, and Ce6. The synthesis method is as follows:

[0052] 1) Synthesis of SrO2 NPs: In a solution containing 40 g / L PVP, 1 mol / L SrCl2, 5 mol / L NaOH, and 30 wt% H2O2 were added sequentially. The mixture was vigorously stirred for 24 h, then transferred to a 50 mL centrifuge tube and centrifuged at 8000 rpm for 20 min. The supernatant was decanted to obtain SrO2 NPs precipitate. Finally, the sample was dried overnight in a 70 °C oven to obtain dried SrO2 NPs powder.

[0053] 2) Dissolve 2 mg SrO2 NPs in 20 mL of ultrapure water (DIW) and sonicate for 15 min. Add 20 mL of 3 mg / mL HSA aqueous solution and 3 mL of 1 mg / mL Ce6 DMSO solution to the solution, stir for 24 h, then centrifuge at 12000 rpm, wash three times with pure water, and finally store in PBS solution at pH 7.4.

[0054] like Figure 1 As shown, the shape of the synthesized HSCTi NPs was characterized using transmission electron microscopy (TEM). Figure 1 (B) and (C) indicate that HSC NPs and HSCTi NPs are uniformly dispersed spherical.

[0055] Dynamic light scattering (DLS) measurements revealed that the average hydrodynamic sizes of HS NPs (HSA-SrO2 nanoparticles), HSC NPs, and HSCTi NPs were concentrated at 110.8 nm, 142.2 nm, and 144.5 nm, respectively. Figure 1 (D)).

[0056] Zeta potential testing showed that HS NPs, HSC NPs, and HSCTi NPs all had negative potentials, with potentials of -17mV, -13.6mV, and -14.3mV, respectively. Figure 1 (E)).

[0057] Energy-dispersive X-ray spectroscopy (EDS) analysis of HSCTi NPs showed that Sr and O elements were uniformly distributed within them. Figure 1 (FH)).

[0058] The phase structures of SrO2 NPs and HSCTi NPs were analyzed using X-ray diffraction (XRD) patterns. Figure 1(K)), and the XRD patterns of both are very similar to those of the SrO2 standard database card number (ICDD 98-000-0417). Furthermore, X-ray photoelectron spectroscopy (XPS) studies were conducted to gain a deeper understanding of the chemical and electronic structures of SrO2 NPs and HSCTi NPs. Figure 1 As shown in (L), O, N, C, and Sr signals can be clearly detected. To further analyze the changes in functional groups, high-resolution spectral analysis was performed on O1s and Sr 3d. Figure 1 (MN)). The O1s orbital state of HSCTi NPs shows a strong peak with a binding energy (BE) of 530.96 eV. The XPS spectrum of Sr 3d shows binding energies of 133.16 eV and 134.95 eV, representing the Sr 3d5 / 2 and Sr3d3 / 2 orbital states, respectively.

[0059] Ultraviolet absorption spectrum ( Figure 1 (I) shows that HSCTi NPs exhibited characteristic absorption peaks for each drug component (HSA: 278 nm; Ti: 320 nm; Ce6: 660 nm), indicating successful loading of SrO2, Ce6, and Ti. The characteristic absorption peak of Ti was not clearly visible, possibly because the wavelengths of Ti's characteristic absorption peak are similar to those of HSA, and the fluctuations in HSA's characteristic absorption peak masked the trend of Ti's characteristic peak. However, compared to HSC NPs, a significant increase in the UV absorbance of HSCTi NPs at 320 nm was observed, indicating successful Ti loading. Simultaneously, the composition of HSCTi NPs was observed using infrared spectroscopy. Figure 1 As shown in (J), HSCTi NPs contained the characteristic infrared peaks of all components, further verifying the successful preparation of the nanomedicine. Subsequently, the drug loading was quantitatively analyzed using ICP-MS (inductively coupled plasma mass spectrometry) and UV-Vis spectroscopy. The drug loading rates of SrO2 in HSCTi NPs were 7.32%, Ce6 was 49.51%, and Ti was 20.01%.

[0060] The prepared multifunctional nanomedicines were characterized using a variety of methods.

[0061] The experimental procedure is as follows:

[0062] 1,1,3-Diphenylisobenzofuran (DPBF) test

[0063] DPBF was used as an indicator to evaluate ROS generation of HSCTi NPs in vitro. Specifically, Ce6 and HSCTi NPs (Ce6 concentration of 10 μM) were mixed with DPBF (10 μM, DMSO) in phosphate buffer at pH 6.5 at a volume ratio of 3:1, followed by induction at 1.0 W / cm².2 Irradiation was performed at different intensities for 0, 1, 3, 5, 7, and 10 minutes. The ultraviolet spectra were recorded using a microplate reader.

[0064] 2. Drug release test

[0065] To investigate the pH-sonic responsive release of nanomaterials, 3 mL of HSCTi NPs were placed in a dialysis bag (MWCO: 30 kDa) and immersed in 30 mL of phosphate-buffered saline solution with different pH values ​​(pH = 7.4, 6.5). The solution in the dialysis bag was either sonicated or not. The test solution was placed in a 37°C constant-temperature shaker (150 rpm) to simulate a living environment. Then, at predetermined time points, 100 μL of dialysate was extracted from each sample, and an equal volume of fresh buffer was added. The Ce6 release was quantitatively determined using a microplate reader. Three replicate samples were prepared for each group.

[0066] 3. Cell proliferation-toxicity assay (Cell Counting Kit-8, CCK-8)

[0067] Hepa1-6 cells (mouse-derived hepatocellular carcinoma cells) and MIHA cells (human-derived normal hepatocytes) were seeded in 96-well plates. Cells were treated with different concentrations of Ce6, HSC NPs (HSA-SrO2-Ce6 nanomaterials), and HSCTi NPs, respectively. Some groups were subjected to sonication (3 min, 1 W / cm²). 2 The cells were incubated for 24 hours, then cultured in cell culture medium containing Cell Counting Kit-8 (CCK-8) for 4 hours, and then the cell viability was detected by CCK-8 assay.

[0068] 4. Cell uptake assay

[0069] Hepa1-6 cells were seeded in culture dishes and cultured for 24 h. Cells were then treated with Ce6, HSC NPs, and HSCTi NPs (Ce6 concentration 2 μg / mL) for 2, 4, 6, and 8 h, respectively. Cells were fixed with 4% paraformaldehyde, and intracellular fluorescence was observed using a laser scanning confocal microscope (CLSM). Furthermore, cellular uptake was quantitatively analyzed by flow cytometry.

[0070] 5. Apoptosis assay

[0071] Hepa1-6 cells were seeded in 12-well plates and treated with PBS, Ce6, HSC NPs, and HSCTiNPs (Ce6 concentration 5 μg / mL) for 6 h, respectively. Some groups were sonicated (3 min, 1 W / cm²). 2Incubate for another 3 hours. Then, collect the cells and resuspend them in PBS. Stain the cells with Annexin V-FITC and PI in the dark for 10 minutes. After washing three times with PBS, analyze the cells by flow cytometry.

[0072] 6. Assessment of intracellular ROS levels

[0073] Hepa1-6 cells were seeded in 12-well plates and treated with PBS, Ce6, HSC NPs, and HSCTiNPs (Ce6 concentration 2 μg / mL) for 6 h, respectively. Some groups were sonicated (3 min, 1 W / cm²). 2 The cells were then incubated for another 3 hours. Afterward, they were stained with H2DCFDA (5 μm), and fluorescence images were captured using an automated cell imaging system (EVOS) to analyze intracellular ROS levels. Furthermore, the amount of ROS produced by the cells was quantified by flow cytometry.

[0074] 7. Live / Dead Cell Staining

[0075] Hepa1-6 cells were seeded in 12-well plates and treated with PBS, Ce6, HSC NPs, and HSCTiNPs (Ce6 concentration 10 μg / mL) for 8 h, respectively. Some groups were sonicated (3 min, 1 W / cm²). 2 Then incubate for another 3 hours. Next, stain with Calcein-AM / PI kit and capture fluorescence images using an automated cell imaging system (EVOS) to observe cell survival and death.

[0076] 8. Scratch test

[0077] Hepa1-6 cells were seeded in 6-well plates. When the cell density reached 70%, they were cultured in serum-free medium for 12 hours. A scratch was made with a pipette tip, and PBS, Ce6, HSC NPs, and HSCTiNPs (Ce6 concentration of 5 μg / mL) were added. After 6 hours, 10 μg / L TGF-β was added. Cell migration was evaluated by taking pictures after 24 hours.

[0078] 9. Measure the levels of HMGB1, TNF-α, and IFN-γ using an ELISA kit.

[0079] Hepa1-6 cells were seeded in 24-well plates. Cells were treated with PBS, Ce6, HSC NPs, and HSCTiNPs (Ce6 concentration 5 μg / ml) for 8 h, respectively. Some groups underwent sonication (3 min, 1 W / cm²). 2Incubate overnight. Collect cell-free supernatant (1 mL) and centrifuge (3000 rpm, 20 min) to remove cell debris. Add the harvested supernatant (100 μL) to a 96-well plate and add HRP-labeled antibody (100 μL), incubate at 37°C for 1 hour. Then, discard the liquid, add 100 μL of washing buffer to each well (washing buffer is prepared by diluting concentrated washing buffer and distilled water at a ratio of 1:20; washing buffer: 0.05% Tween 20), let stand for 20 seconds, discard the washing buffer, pat dry on absorbent paper, repeat 5 times. Then add 100 μL of substrate mixture to each well (substrate mixture is prepared by a volume ratio of substrate solution A:substrate solution B = 1:1; substrate solution A: 0.01% hydrogen peroxide, substrate solution B: 0.1% TMB), incubate for 15 min. Finally, add 50 μL of stop solution and measure the absorbance at 450 nm using a microplate reader.

[0080] 10. Construction of a subcutaneous tumor model in C57 mice

[0081] Six-week-old female C57 mice were subcutaneously injected with Hepa1-6 cells (1.5 × 10⁻⁶) into the right axilla. 6 (each tumor), when the tumor grows to 100mm 3 The subjects were randomly divided into 4 groups (n=6) and treated as follows: PBS group (injected with PBS buffer), US group (sonicated for 1 min at 1 W / cm²). 2 HSCTi group (injected with HSCTi nanomedicine at a Ce6 concentration of 5 mg / kg), HSCTi+US group (injected with HSCTi nanomedicine at a Ce6 concentration of 5 mg / kg and subjected to ultrasound, 1 min, 1 W / cm²) 2 Treatment was administered every two days. The tumor size was measured using calipers, and its volume was calculated: V = length × width. 2 / 2, and recorded mouse weight. Mice were sacrificed 15 days after treatment, and fresh whole blood and serum were collected for complete blood count and blood biochemistry analysis. Tumor tissue was collected, photographed, weighed, and sectioned for H&E staining, Ki67 staining, and TUNEL staining. Major tissues (heart, liver, spleen, lung, and kidney) were collected for H&E staining. The mechanism of HSCTi NPs-mediated sonodynamic-immunotherapy is as follows: Figure 8 As shown.

[0082] The characterization results are as follows:

[0083] 1. Evaluation of in vitro antitumor efficacy

[0084] First, the ability of HSCTi NPs to generate reactive oxygen species (ROS) was evaluated at the solution level. The DPBF probe exhibits a characteristic UV absorption peak around 410 nm and can react with ROS, thus being irreversibly oxidized, causing the characteristic absorption peak to decrease. For example... Figure 2As shown in (AB), under ultrasound (US) irradiation, the absorbance of DPBF at 410 nm in both the Ce6 and HSCTi NPs groups decreased significantly in a time-dependent manner, indicating that US induced ROS generation. Furthermore, compared to the Ce6+US group, the decrease in DPBF absorbance was more pronounced in the HSCTi NPs+US group. This may be attributed to the fact that O2 generated by SrO2 under acidic conditions can promote the oxygen-consuming SDT process, thereby generating more ROS.

[0085] To confirm the biocompatibility and antitumor effects of nanomedicines in biomedical applications, the cytotoxicity of Ce6, HSC NPs, and HSCTi NPs was investigated using the CCK-8 assay. Figure 2 As shown in (C), at the same Ce6 concentration as HSCTi NPs, Ce6 showed almost no inhibitory effect on tumor cell proliferation regardless of whether ultrasound was applied, likely due to limited internalization. In contrast, HSCTi NPs exhibited significantly enhanced cytotoxicity after ultrasound application, indicating the great potential of this multifunctional nanomedicine in sonodynamic tumor therapy. Furthermore, without ultrasound application, HSCTi NPs showed very low toxicity to normal hepatocytes, suggesting superiority of HSCTi NPs in selective tumor therapy using local ultrasound. Figure 2 (D)).

[0086] Furthermore, flow cytometry was used to assess apoptosis levels with and without the application of different drugs and ultrasound. Consistent with the results of the CCK-8 assay, the HSCTi NPs+US group induced the highest number of apoptosis. Figure 2 (G)).

[0087] Furthermore, the antitumor activity of HSCTi NPs was evaluated using a live / dead cell staining assay. For example... Figure 3 As shown in (E), Hepa1-6 cells treated with Ce6 alone exhibited bright green fluorescence regardless of whether ultrasound was applied or not, indicating no significant cell damage. However, the cell density after Ce6+US treatment was sparser than that in the Ce6 group, suggesting that even Ce6 SDT induces some cell death, which may be due to cell detachment during incubation and treatment, resulting in the presence of cells without red fluorescence. Notably, the weakest green fluorescence and the strongest red fluorescence were observed in cells treated with HSCTi NPs and ultrasound, confirming potent antitumor efficacy. In vivo experiments showed that on day 11 of treatment, the tumor site temperature in the HSCTi+US group mice was increased, and this difference was statistically significant compared to other groups. Figure 5(I)). A possible reason is that under the dual influence of tumor cell immunogenic death (ICD) and TGFβ inhibitors, significantly increased cytotoxic T cells (CTLs) secrete large amounts of TNF-α (tumor necrosis factor-α) and IFN-γ (interferon-γ). These cytokines can not only further enhance the immune response but may also trigger inflammatory heat effects. Based on this, we measured the amounts of TNF-α and IFN-γ in the cell supernatant, such as... Figure 2 As shown in (EF), tumor cells treated with HSCTi+US secreted TNF-α and IFN-γ at higher levels than those in other groups.

[0088] Subsequently, we evaluated the cellular uptake capacity of HSCTi NPs using confocal laser scanning microscopy (CLSM) and flow cytometry. Figure 3 As shown in (AC), Hepa1-6 cells exhibited enhanced red fluorescence with increasing incubation time, confirming the time-dependent cellular uptake of Ce6 and HSCTi NPs. Importantly, the red fluorescence observed in cells treated with HSCTi NPs was significantly stronger than that observed in cells treated with Ce6, indicating that nanotechnology improves intracellular drug delivery efficiency. This is attributed to the improved stability of Ce6 after assembly into nanomedicines. Furthermore, particle size also influences the cellular uptake behavior of nanomedicines.

[0089] Before evaluating the SDT-induced ICD response, we also used the H2DCFDA probe as a sensor to assess the sonodynamic performance of HSCTi NPs in generating ROS within cells. It is well known that H2DCFDA can be oxidized by ROS and emits green fluorescence. Figure 3 As shown in (D), Hepa1-6 cells exhibited negligible fluorescence when treated with Ce6 alone, indicating minimal ROS production. However, the use of nanomedicines significantly increased green fluorescence, and cells incubated with HSC NPs and HSCTi NPs after sonication showed bright fluorescence, suggesting elevated intracellular ROS levels during SDT. Notably, HSC NPs and HSCTi NPs generated more ROS in Hepa1-6 cells compared to Ce6, likely due to improved cellular uptake following nanomedicine formation.

[0090] In addition, we performed semi-quantitative fluorescence analysis and quantitative flow cytometry analysis of ROS. For example... Figure 3As shown in (FH), under ultrasound application, the intracellular DCF fluorescence intensity induced by HSCTi NPs was more than three times that induced by Ce6. Furthermore, it was significantly higher than the fluorescence intensity induced by HSC NPs, which is consistent with the conclusions drawn from cytotoxicity assays. This may be related to the successful induction of ICD response by SDT and the enhanced cell-killing ability assisted by TGF inhibitors. The excellent acoustic-dynamic properties of HSCTi NPs demonstrate the advantages of this multifunctional nanomedicine in highly efficient tumor therapy.

[0091] In addition, we also conducted a scratch test ( Figure 4 (A) and its semi-quantitative analysis Figure 4 (B) The migration ability of Hepa1-6 cells was assessed. It is well known that cancer cells are generally metastatic, such as... Figure 4 As shown in (A), Hepa1-6 cells migrated immediately after 24 hours of culture without any treatment. TGF-β can accelerate cell migration by inducing phenotypic changes in tumor cells through EMT. Conversely, TGF-β blockade induced by Ti and the Ti-loaded nanodrug HSCTi helped to inhibit cell migration. Notably, HSCTi NPs reduced migration by approximately 40% compared to cells treated with PBS and TGFβ. This result suggests that HSCTi NPs have great potential in inhibiting tumor metastasis.

[0092] When local SDT is performed on a tumor, tumor cells exhibit varying degrees of damage and release large amounts of tumor-associated antigens, triggering anti-tumor immune responses. To elucidate the ICD induced by SDT, we examined damage-associated molecular pattern (DAMP) signaling, including ATP, HMGB1, and CRT. ATP and HMGB1 can be recognized by dendritic cells, thereby enhancing dendritic cell (DC) recruitment and activation. Figure 4 As shown in (CD), the expression levels of ATP and HMGB1 in cells treated with the HSCTi+US group were significantly increased, as detected by the ATP kit and the HMGB1 Elisa kit.

[0093] Immunofluorescence analysis of CD11c and CD86 ( Figure 4 (FG) further confirmed this, indicating that the recruitment and activation levels of dendritic cells in tumor tissue were higher after HSCTi+US treatment. In summary, HSCTi+US can induce cell damage and the release of damage-associated molecular patterns (DAMPs), further stimulating dendritic cell maturation. Meanwhile, to ensure the safety of in vivo studies, we performed a hemolysis test before conducting animal experiments. Figure 4As shown in (E), when the Ce6 concentration is 3.125–100 μg / mL, the hemolysis rate of erythrocytes after incubation with HSCTi NPs for 4 h is less than 5.0%, indicating that the hemolytic effect of the nanoparticles is negligible.

[0094] 2. Evaluation of in vivo antitumor efficacy

[0095] C57 tumor-bearing mice were injected with PBS or treatment drugs via the tail vein on day 1, and injected every two days. Ultrasound irradiation was performed 24 hours after each injection. Figure 5 (A)). Next, to optimize the timing of ultrasound therapy for cancer, the accumulation of nanoparticles in tumor tissue was investigated, and the biodistribution of HSCTi NPs was detected using fluorescence imaging. Figure 5 As shown in (B), intravenously injected HSCTi NPs showed accumulation at the tumor site as early as 1 hour after injection. This may be due to multiple factors, including the inherent properties of albumin and its EPR effect after being combined into nanomedicines. The highest fluorescence intensity of HSCTi NPs in mouse tumors was observed at 24 hours after injection, and accumulation continued even after 48 hours. Frozen sections of mouse tumor tissue 48 hours after drug injection clearly showed the accumulation of HSCTi NPs at the tumor site and their red fluorescence.

[0096] To evaluate the antitumor effect of HSCTi NPs-mediated sonodynamic-immunotherapy, tumor volume was measured every two days using calipers from the start of treatment. Compared with the PBS group, US group, and HSCTi group, the HSCTi+US group significantly inhibited tumor growth and demonstrated a more significant antitumor effect. Figure 5 (C) demonstrates the promising potential of HSCTi NPs-mediated sonodynamic-immunotherapy in tumor treatment. From tumor volume ( Figure 5 (D, G)) and tumor weight ( Figure 5 (E) also reached the same conclusion. The change in mouse body weight during treatment was negligible, indicating low systemic toxicity of the multifunctional nanomedicine. Figure 5 (H)).

[0097] Furthermore, immunohistochemical staining of tumor tissues using H&E, Ki67, and TUNEL revealed that the HSCTi+US group exhibited more severe tumor tissue damage and lower tumor cell proliferation compared to other groups. These results confirm the excellent antitumor activity of HSCTi NPs. To assess the in vivo biosafety of HSCTi NPs, H&E staining was performed on the major organs of mice under different treatments. Figure 5 As shown in (F), no obvious pathological abnormalities were found in these organs, indicating low toxicity. Blood routine examination and serum biochemical indicators were also measured in mice. Figure 6 The results showed that all indicators were normal after HSCTi NP injection, and no significant damage was observed in vital organs such as the liver and kidneys. In conclusion, HSCTi NP-mediated sonodynamic immunotherapy demonstrated superior tumor-killing effects with few adverse reactions.

[0098] 3. Transcriptomics analysis

[0099] TGF-β plays a crucial role in maintaining the immunosuppressive tumor microenvironment (TME) to protect tumor cells from the host immune response. To further explore the immune status of the TME, transcriptome analysis was performed on tumor tissues treated with HSCTi NPs with or without ultrasound. Untreated tumor tissue served as a blank control. A total of 14,540 tumor tissue genes were analyzed, and 1,454 differentially expressed genes (fold change >1.5 and p <0.05) were identified. Volcano plots showed that after HSCTi+US treatment, 1,276 genes were upregulated and 178 genes were downregulated. Figure 7 (A) Furthermore, differentially expressed immune-related genes associated with "positive regulation of innate immune response" and "positive regulation of T cell activation" were screened out. Figure 7 (CD) was used to assess innate and cellular immunity in tumor tissues. Compared with PBS, HSCTi NPs did not significantly upregulate these immune-related genes, indicating a poorer immune response. Conversely, tumors treated with HSCTi NPs after ultrasound irradiation showed effective immune activation, confirming SDT-amplified immune activation. Further KEGG pathway enrichment analysis was performed on differentially expressed immune-related genes (CD). Figure 7 (B) indicates that the activated immune response largely depends on "cytokine-cytokine receptor interactions," "chemokine signaling pathways," and "natural killer cell-mediated cytotoxicity." Furthermore, we categorized differentially expressed upregulated genes associated with these pathways into "biological processes," "molecular functions," and "cellular components" using gene ontology (GO) annotation analysis. Clearly, the upregulated genes play crucial roles in cellular processes, cellular responses to organic matter, cell communication regulation, immune response regulation, and MHC protein complex binding, suggesting their important roles in cell signal transduction and immune function. Overall, HSCTi NPs combined with ultrasound can modulate the immune microenvironment, which is highly beneficial for enhancing the anti-tumor effects of immunotherapy.

[0100] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a pH-ultrasound dual-response albumin nanomedicine, characterized in that: The steps are as follows: (1) Synthesis of SrO2 NPs: SrCl2, NaOH and H2O were added sequentially to the PVP solution. 2， After stirring and centrifugation, SrO2 NPs precipitate was obtained, and after drying, dry SrO2 NPs powder was obtained. (2) Activation of Ce6: Dissolve Ce6 in DMSO, add EDC and NHS, and react at room temperature in the dark; (3) SrO2 NPs were dissolved in ultrapure water, and after ultrasonic treatment, HSA aqueous solution, Ce6 DMSO solution and Ti DMSO solution were added. After stirring, centrifugation and washing were performed to obtain pH-ultrasound dual-response albumin nanomedicine. The Ti in the Ti DMSO solution is a TGFβ inhibitor, and the TGFβ inhibitor is SB505124.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of the PVP solution is 20-60 g / L, the concentration of SrCl2 is 0.5-3 mol / L, the concentration of NaOH is 2-10 mol / L, and the concentration of H2O2 is 20-30 wt%.

3. The preparation method according to claim 1, characterized in that: In step (1), the stirring time is 20-35 hours, and the centrifugation is performed at 7000-10000 rpm for 10-30 minutes.

4. The preparation method according to claim 1, characterized in that: In step (2), the mass ratio of Ce6, EDC and NHS is 1:1.5 to 3:1.5 to 3.

5. The preparation method according to claim 1, characterized in that: In step (2), the reaction is carried out in the dark for 2 to 5 hours.

6. The preparation method according to claim 1, characterized in that: In step (3), the amount of SrO2NPs used is 0.01-0.7 mg / mL, the concentration of HSA aqueous solution is 2-5 mg / mL, the concentration of Ce6 DMSO solution is 0.5-3 mg / mL, and the concentration of Ti DMSO solution is 0.1-1.0 mg / mL.

7. The preparation method according to claim 1, characterized in that: In step (3), the volume ratio of the HSA aqueous solution, Ce6 DMSO solution and Ti DMSO solution is 10-30:2-4:1-3.

8. The preparation method according to claim 1, characterized in that: In step (3), the ultrasonic treatment time is 10-30 min, the stirring time is 20-35 h, and the centrifugation speed is 10000-15000 rpm.

9. The preparation method according to claim 1, characterized in that: The prepared pH-ultrasound dual-response albumin nanomedicine was stored in PBS solution at pH 7.

4.

10. The pH-ultrasound dual-response albumin nanomedicine prepared by the preparation method according to any one of claims 1 to 9.

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