A physiological pressure-driven piezocatalytic therapeutic nanodrug delivery system with pH-responsive drug release function and a preparation method and application thereof

Abstract

The application belongs to the field of medicine, and particularly relates to a physiological pressure driven piezoelectric catalytic treatment nano drug delivery system with pH response drug release function and a preparation method and application thereof. The preparation method comprises preparation of metal nanoparticles, preparation of yolk-shell structure nanoparticles, loading of drugs and preparation of the drug delivery system. The application can utilize physiological pressure fluctuation to drive piezoelectric catalysis, combine metabolic intervention and pH response drug release, achieve efficient treatment of body cavity diseases such as malignant pleural effusion, and has good targeting, biological safety and anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a physiological pressure-driven piezoelectric catalytic therapeutic nanodrug delivery system with pH-responsive drug release function, its preparation method, and its application. Background Technology

[0002] Malignant pleural effusion (MPE) is a common and challenging serious complication of advanced malignant tumors, especially lung and breast cancer. It is often accompanied by chest tightness, shortness of breath, chest pain, and recurrent effusion, significantly impacting patients' quality of life and usually indicating disease progression and poor prognosis. Its formation is mainly related to pleural metastasis, increased vascular permeability induced by tumor-associated factors, and obstructed lymphatic drainage, ultimately leading to the continuous accumulation of protein-rich fluid in the pleural cavity. This effusion is not merely fluid retention; it constitutes a "fluid tumor microenvironment" involving tumor cells, immune cells, cytokines, and metabolites, which can promote malignant progression and create an immunosuppressive niche.

[0003] Current treatments for malignant pleural effusion mainly include thoracentesis, pleurodesis, intrapleural chemotherapy, biotherapy, and, in recent years, pressure-triggered piezoelectric catalytic therapy. While these techniques can alleviate symptoms or inhibit lesion progression to some extent, they still have the following limitations:

[0004] 1. Current clinical treatments are mostly palliative, with limited ability to continuously control tumor burden and effusion progression: thoracentesis is mainly used to temporarily relieve compression symptoms, but effusion is prone to recurrence; pleurodesis can reduce the recurrence rate to some extent, but it is often accompanied by significant irritation and is not suitable for all patients; intrapleural chemotherapy and biological therapy are limited by factors such as insufficient drug penetration, tumor heterogeneity, and systemic toxicity, resulting in limited stability of treatment effects. Overall, current clinical protocols do not adequately address the pro-tumor pleural microenvironment formed by malignant pleural effusion.

[0005] 2. Current piezoelectric catalytic therapy still has limited sustained catalytic capacity under low-intensity physiological pressure conditions in the pleural cavity: the mechanical stimulation in the pleural cavity mainly originates from intrapleural pressure fluctuations caused by respiratory movements, which are characterized by low intensity and obvious rhythmicity. For general piezoelectric materials, under such low-intensity physiological conditions, the electromechanical conversion efficiency, charge separation efficiency, and reactive oxygen generation capacity may still be insufficient, thus affecting the sustainability of treatment.

[0006] 3. Existing ozone-dependent piezoelectric catalytic schemes rely on exogenous oxidizing substrates, resulting in limited system stability and controllability: Existing intrapleural pressure-triggered piezoelectric catalytic systems enhance reactive oxygen species (ROS) generation by introducing ozone as an exogenous oxidizing substrate. However, the effectiveness of this strategy depends to some extent on the distribution, stability, and local oxygen availability of ozone. Due to the reactive nature of ozone, it is easily depleted during preparation, storage, and delivery, and its local release process is difficult to control precisely, which may affect treatment repeatability and system stability.

[0007] 4. Existing solutions do not fully utilize the synergistic effect between drug delivery, metabolic regulation and piezoelectric catalysis: In existing technologies, local treatment systems often focus on single physical stimulus response or single drug action, and have not yet adequately considered the synergistic relationship between low-pressure mechanical response, local stable retention, microenvironment response release and metabolic substrate enrichment and piezoelectric catalytic amplification. Therefore, there is still room for improvement in continuous treatment and comprehensive regulation of the tumor microenvironment. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a method for preparing a physiological pressure-driven piezoelectric catalytic therapeutic nanodrug delivery system with pH-responsive drug release function, the preparation method comprising the following steps:

[0009] (I) Preparation of metal nanoparticles

[0010] The aqueous solution of the metal source was heated to reflux, and then sodium citrate solution was added to react. After the reaction was completed, polyvinylpyrrolidone (PVP) solution was added, and after mixing evenly, metal nanoparticles were obtained by centrifugation.

[0011] (II) Preparation of egg yolk-shell structured nanoparticles

[0012] (II-1) After dispersing the metal nanoparticles, add alkaline solution and silicon source, stir and react to obtain gold-silica nanoparticles;

[0013] (II-2) The gold-silica nanoparticles are dispersed in a mixed solvent, and then a titanium source is added to react and obtain gold-silica-titanium dioxide nanoparticles;

[0014] (II-3) After dispersing the gold-silica-titanium dioxide nanoparticles, a barium source is added to react and obtain egg yolk-shell structured nanoparticles;

[0015] (III) Drug load

[0016] After dispersing the egg yolk-shell structured nanoparticles, a solution of metabolic regulating drugs was added for incubation. After completion, the nanoparticles were centrifuged and washed to obtain drug-loaded nanoparticles.

[0017] (IV) Preparation of drug delivery system

[0018] (IV-1) CHEMS, DOPE, DSPE-PEG2000 and DSPE-PEG2000-Folate were mixed and dissolved in a solvent, and the resulting solution was distilled under reduced pressure to obtain a lipid film.

[0019] (IV-2) After dispersing the drug-loaded nanoparticles, they are mixed with the lipid film and hydrated to obtain the drug delivery system.

[0020] Preferably, in step (I), the tetrachloroauric acid (HAuCl4) aqueous solution is heated to reflux, then sodium citrate solution is added and reacted for 30-40 minutes. After the reaction is complete, polyvinylpyrrolidone solution is added, and the mixture is mixed evenly and centrifuged to obtain metal nanoparticles (AuNPs). This invention uses gold (Au) nanoparticles as the metal core. Any metal or conductive core capable of forming a metal-piezoelectric interface with piezoelectric materials, promoting charge separation, or enhancing local field effects can be used. Examples include: Ag, Pt, Pd, Cu, Ru, Ir, Rh, etc.; metal alloy nanoparticles; doped conductive oxide particles; carbon-based conductive nanocores, etc.

[0021] And / or, the concentration of the tetrachloroauric acid aqueous solution is 1-2 mM;

[0022] And / or, the concentration of the sodium citrate solution is 1-2 wt%;

[0023] And / or, the concentration of the polyvinylpyrrolidone solution is 10-20 mg / mL.

[0024] Preferably, in step (II-1), the metal nanoparticles are dispersed in isopropanol, and ammonia and tetraethyl orthosilicate (TEOS) are added sequentially under stirring conditions. After stirring and reacting, gold-silica (Au@SiO2) nanoparticles are obtained.

[0025] And / or, the concentration of the ammonia solution is 28-35 wt%.

[0026] Preferably, in step (II-2), the gold-silica nanoparticles are dispersed in a mixed solvent of ethanol and acetonitrile, and then titanium isopropoxide (TTIP) is added and reacted at 5-10°C for 6-8 hours to obtain gold-silica-titanium dioxide (Au@SiO2@TiO2) nanoparticles.

[0027] And / or, in the mixed solvent of ethanol and acetonitrile, the volume ratio of ethanol to acetonitrile is 3:1.

[0028] Preferably, in step (II-3), the gold-silica-titanium dioxide nanoparticles are dispersed in ethanol, and barium hydroxide solution is added and reacted at 160-180°C for 6-8 hours. During this process, the TiO2 shell is converted in situ to BaTiO3, while the SiO2 component is gradually etched, ultimately yielding Au@h-BaTiO3 (AhBT) nanoparticles, i.e., yolk-shell structured nanoparticles.

[0029] Preferably, in step (III), the yolk-shell structured nanoparticles are dispersed in methanol, and a solution of metabolic regulatory drugs is added for incubation for 12-16 hours. After incubation, the nanoparticles are collected by centrifugation at 10000-15000 r / min, washed 2-3 times with PBS solution, and then lyophilized to obtain drug-loaded (AhBT∩AA5) nanoparticles. It should be emphasized that the mitochondrial complex II inhibitor Atpenin A5 (AA5) is preferably used in this embodiment of the invention. Any metabolic regulatory drug that can reduce tumor cell oxygen consumption, improve local oxygen availability, and / or promote endogenous H2O2 accumulation can be used as a substitute. Examples include: other complex II inhibitors, drugs that induce mitochondrial electron leakage, drugs that inhibit oxidative phosphorylation, drugs that inhibit respiratory chain complexes I, III, or IV, metabolic inhibitors that intervene in tumor oxygen consumption metabolism, siRNA, miRNA, or nucleic acid metabolic regulatory molecules, etc.

[0030] Preferably, in step (IV-1), CHEMS, DOPE, DSPE-PEG2000 and DSPE-PEG2000-Folate are mixed in a molar ratio of 4:6:0.45:0.05 and dissolved in a mixed solvent of chloroform and methanol. The resulting solution is then distilled under reduced pressure at 40-50°C for 1-2 hours to obtain a lipid film (Lipo).

[0031] And / or, in the chloroform and methanol mixed solvent, the volume ratio of chloroform to methanol is 9:1.

[0032] Preferably, in step (IV-2), the drug-loaded nanoparticles are dispersed in a PBS solution and hydrated with the lipid film at 40-45°C for 1-2 hours to obtain the drug delivery system (AhBT∩AA5@Lipo nanoparticles).

[0033] Based on the same technical concept, another aspect of the present invention is to provide a physiological pressure-driven piezoelectric catalytic therapeutic nanodrug delivery system with pH-responsive drug release function obtained by the preparation method described above.

[0034] Based on the same technical concept, another aspect of the present invention provides the application of a physiological pressure-driven piezoelectric catalytic therapeutic nanomedicine delivery system with pH-responsive drug release function in the preparation of drugs for treating body cavity diseases with endogenous periodic physiological pressure fluctuations. Related diseases include, but are not limited to: malignant pleural effusion, malignant ascites, pericardial effusion-related tumors, peritoneal metastases, gastrointestinal motility-related tumors, and cavity tumors such as those in the bladder and uterus that present with a periodic mechanical stimulation environment.

[0035] The beneficial effects of this invention are as follows:

[0036] 1. It can better utilize the physiological pressure fluctuations of the pleural cavity itself as activation conditions: This invention uses the intrapleural pressure fluctuations caused by respiration as the source of endogenous mechanical stimulation, and achieves local activation by constructing a piezoelectric catalytic nanosystem. It does not rely on external ultrasound, light irradiation or other equipment, so it is more suitable for the special environment of the pleural cavity where there are continuous physiological pressure changes.

[0037] 2. Good piezoelectric response and catalytic basis under low-intensity physiological pressure conditions: The present invention adopts a gold core-hollow BaTiO3 shell structure. With the combined effect of the metal-piezoelectric interface and the hollow structure, the electromechanical conversion efficiency, charge separation ability and local reaction activity are improved. Therefore, compared with ordinary piezoelectric materials, it is more conducive to maintaining the piezoelectric catalytic process under low-intensity pressure fluctuations in the pleural cavity.

[0038] 3. Reduced dependence on exogenous highly reactive oxidizing substrates: Compared to schemes that rely on ozone loading to enhance ROS generation, this invention reduces tumor cell oxygen consumption and promotes endogenous H2O2 accumulation by loading the mitochondrial complex II inhibitor AA5, thereby improving the availability of catalytic substrates. This strategy mainly relies on tumor metabolic regulation and endogenous substrate enrichment, rather than simply on exogenous oxidizing components, thus making it more reasonable in terms of system stability and duration of action.

[0039] 4. It takes into account the needs of drug delivery, drug release and local retention: The present invention adopts a lipid encapsulation system, which enables the nanoplatform to have good colloidal stability and local retention ability in the pleural cavity. At the same time, it uses the CHEMS / DOPE system to realize pH-responsive drug release in an acidic microenvironment. Therefore, compared with systems that only emphasize catalytic function, it is more conducive to improving the utilization efficiency of drugs in the lesion area.

[0040] 5. Synergistic effect of piezoelectric catalysis and metabolic intervention: Existing technologies mostly focus on single physical catalysis or single drug action, while this invention integrates piezoelectric catalysis, metabolic regulation and local drug delivery into the same nanosystem, so that mechanical stimulation-triggered ROS generation and AA5-mediated oxygen metabolism reprogramming work together, thereby enhancing local oxidative stress and improving anti-tumor effect.

[0041] 6. While inhibiting tumor burden and effusion progression, it also regulates the tumor microenvironment: This invention not only kills tumor cells locally but also induces immunogenic cell death (ICD) by enhancing oxidative stress. During ICD, the endoplasmic reticulum chaperone protein calreticulin (CRT) translocates to the cell surface, acting as an "eat-me" signal to promote dendritic cell recruitment and activation. Meanwhile, high-mobility group box 1 (HMGB1) is released from the nucleus into the extracellular space, promoting the activation of antigen-presenting cells. It can also promote CD8... + T-cell infiltration and improvement of tumor immunosuppression. Therefore, compared with palliative decompression alone or cytotoxic therapy alone, this invention is more comprehensive in terms of local microenvironment regulation.

[0042] 7. Good local therapeutic suitability and biosafety basis: This invention adopts a local intrapleural administration method. The nanosystem has good retention and tumor enrichment characteristics in the pleural cavity, which can reduce systemic exposure. At the same time, animal experiments show that it has no obvious toxicity in major organs, so it has a certain basis for local application and further transformation. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a synthesis pathway diagram for AhBT∩AA5@Lipo nanoparticles.

[0045] Figure 2 These are TEM images of BT, hBT, and AhBT.

[0046] Figure 3 The amplitude-voltage butterfly curve was obtained under a ±10V bias condition.

[0047] Figure 4 These are the electrochemical impedance spectroscopy (EIS) and photoluminescence (PL) spectra of BT, hBT, and AhBT.

[0048] Figure 5 This is a graph showing drug release data for AhBT@Lipo.

[0049] Figure 6 This is an in vitro cellular uptake fluorescence image of AhBT@Lipo.

[0050] Figure 7This is a graph showing the measurement data of intracellular hydrogen peroxide levels and oxygen consumption rate (OCR).

[0051] Figure 8 This is a graph showing data from a CCK-8 cytotoxicity assay.

[0052] Figure 9 This is a graph showing the results of intracellular ROS analysis.

[0053] Figure 10 This is a graph showing the evaluation results of HMGB1 release.

[0054] Figure 11 This is an in vivo distribution map of AhBT@Lipo.

[0055] Figure 12 This is a flowchart of the in vivo treatment plan.

[0056] Figure 13 It is an in vivo imaging image of the therapeutic effect in vivo.

[0057] Figure 14 It is an internal therapeutic effect diagram of an anatomical tumor.

[0058] Figure 15 This is a diagram illustrating the body's therapeutic effects in inhibiting ascites formation.

[0059] Figure 16 It is an in vivo ICD image.

[0060] Figure 17 This is a diagram showing the promotion of immune infiltration in the body.

[0061] Figure 18 It's a survival curve. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0063] Example 1

[0064] This embodiment provides a method for preparing a physiological pressure-driven piezoelectric catalytic nanomedicine delivery system with pH-responsive drug release function. The preparation method includes the following steps:

[0065] (I) Preparation of AuNPs

[0066] An aqueous solution of HAuCl4 (1 mM, 80 mL) was heated to reflux, followed by the rapid addition of sodium citrate solution (1%, 5 mL). The reaction was maintained for 30 min until the solution turned wine-red. PVP solution (10 mg / mL, 5 mL) was then added, and the mixture was cooled to room temperature. The resulting AuNPs were collected by centrifugation and redispersed in ethanol.

[0067] (II) Preparation of AhBT nanoparticles

[0068] AuNPs were dispersed in 18 mL of isopropanol. Ammonia (28 wt%) and TEOS were added sequentially under magnetic stirring. The mixture was stirred at room temperature for 16 h to form Au@SiO2 nanoparticles. The product was collected by centrifugation, washed with deionized water, and redispersed in a mixed solvent of ethanol / acetonitrile (3:1, v / v). Then, 1.8 mL of titanium isopropoxide (TTIP) was added, and the reaction was carried out at 5 °C for 6 h to obtain Au@SiO2@TiO2 nanoparticles. The obtained particles were dispersed in ethanol and reacted with Ba(OH)2 solution (0.05 M) under hydrothermal conditions at 160 °C for 6 h. During this process, the TiO2 shell was converted in situ to BaTiO3, while the SiO2 component was gradually etched, ultimately yielding Au@h-BaTiO3 (AhBT) nanoparticles.

[0069] (III) Drug loading on AhBT nanoparticles

[0070] 10 mg AhBT nanoparticles were dispersed in 2 mL of methanol and incubated with 200 μL of AA5 methanol solution (10 mg / mL) under light-protected conditions for 12 h. After incubation, the nanoparticles were collected by centrifugation (10000 r / min, 15 min), washed three times with PBS, and lyophilized to obtain drug-loaded (AhBT∩AA5) nanoparticles.

[0071] (IV) Preparation of drug delivery system

[0072] CHEMS, DOPE, DSPE-PEG2000, and DSPE-PEG2000-Folate were mixed in a molar ratio of 4:6:0.45:0.05 and dissolved in 10 mL of chloroform / methanol (9:1, v / v). The solution was transferred to a 100 mL round-bottom flask and evaporated under reduced pressure at 40 °C for 1 h to form a homogeneous lipid film. AhBT∩AA5 nanoparticles were dispersed in 10 mL of PBS by sonication and stirring to form a stable suspension, which was then added to the flask. Hydration was carried out at 40 °C for 1 h to obtain AhBT∩AA5@Lipo nanoparticles.

[0073] Comparative Example 1

[0074] The difference between this comparative example and Example 1 is that, in step (II), AuNPs are not introduced, and hollow BaTiO3 (hBT) nanoparticles are obtained using the same process.

[0075] Comparative Example 2

[0076] The difference between this comparative example and Example 1 is that in step (II), AuNPs are not introduced, TEOS is not used, and BaTiO3 (BT) nanoparticles are obtained using the same process.

[0077] Comparative Example 3

[0078] The difference between this comparative example and Example 1 is that in step (III), AA5 is replaced with Nile Red, and the same process is used to obtain AhBT∩NR@Lipo nanoparticles.

[0079] Comparative Example 4

[0080] The difference between this comparative example and Example 1 is that step (III) is omitted, and the AhBT@Lipo nanoparticles are obtained using the same process.

[0081] Characterization detection example

[0082] (I) Material property characterization

[0083] The synthetic route of the AhBT∩AA5@Lipo hollow piezoelectric nanoplatform is as follows: Figure 1 As shown. By Figure 1 As can be seen, gold nanoparticles (AuNPs, approximately 20 nm) were used as the core. First, a SiO2 shell was deposited on the AuNP surface using a sol-gel method. Then, an amorphous TiO2 layer was coated onto the SiO2 shell using TTIP as a precursor, resulting in Au@SiO2@TiO2 nanoparticles. Next, a hydrothermal reaction with Ba(OH)2 was performed to transform the TiO2 layer in situ into a BaTiO3 shell. Simultaneously, under the weakly alkaline conditions provided by Ba(OH)2, the SiO2 component was gradually etched, ultimately forming Au@h-BaTiO3 yolk-shell structured nanoparticles (AhBT). Notably, Ba(OH)2 played a dual role, acting as both a BaTiO3 precursor and a controllable etching medium, allowing for the gradual removal of SiO2 while maintaining structural integrity. Subsequently, AA5 was loaded into the mesoporous AhBT framework (AhBT∩AA5). Finally, AhBT∩AA5 was co-assembled with CHEMS, DOPE, DSPE-PEG2000 and DSPE-PEG2000-folate using a thin-film hydration method to obtain the tumor-targeting, pH-responsive nanoplatform AhBT∩AA5@Lipo.

[0084] Figure 2It is a high-resolution TEM image, by Figure 2 It can be seen that the AhBT nanoparticles exhibit a clear yolk-shell structure morphology, with the Au core confined in the internal cavity and the BaTiO3 shell thickness being approximately 10 nm.

[0085] Figure 3 This is an amplitude-voltage butterfly curve obtained under a ±10V bias condition. Figure 3 It can be seen that all samples exhibit characteristic ferroelectric switching behavior, but the response amplitudes differ significantly. The maximum amplitude of AhBT is 1087.6 pm, significantly higher than that of BT (104 pm) and hBT (157 pm), reflecting a significantly enhanced electromechanical deformation. The piezoelectric coefficients (d) of BT, hBT, and AhBT were calculated. 33 The values ​​are approximately 102.56, 206.18, and 688.61 pm / V, respectively. AhBT's d 33 The value is 6.7 times higher than that of BT and 3.4 times higher than that of hBT.

[0086] Electrochemical impedance spectroscopy (EIS) and photoluminescence (PL) spectroscopy further confirmed the above viewpoints. Nyquist plot ( Figure 4 A) shows that AhBT has the smallest semicircle radius, corresponding to a lower interfacial charge transport resistance and higher electron mobility. Meanwhile, compared to BT and hBT, AhBT exhibits a significantly reduced PL intensity ( Figure 4 (B) indicates that electron-hole recombination is effectively suppressed. These results collectively demonstrate that AhBT possesses superior charge separation efficiency and interfacial charge transport capability.

[0087] The drug loading and release behavior of AA5 were quantitatively analyzed using LC-MS / MS. The drug loading (LC) was calculated using the following formula: LC = (Total drug - Drug in supernatant) / Total drug × 100%. The cumulative release of AA5 from AhBT∩AA5@Lipo was assessed using dialysis. AhBT∩AA5@Lipo nanoparticles were placed in a dialysis bag (molecular weight cutoff: 1000 Da) and immersed in 40 mL of PBS at different pH values ​​(pH 7.4, pH 6.5, and pH 5.5). Samples were taken from the release medium at preset time points for LC-MS / MS analysis, and an equal volume of fresh buffer was added.

[0088] AA5 was monitored using the characteristic ion pair m / z 366.2 / 276.3, with a retention time of 4.5 min. The calibration curve showed excellent linearity across the test concentration range, with a maximum drug loading of 12.76% in AhBT@Lipo. AA5 release from AhBT@Lipo exhibited a significant pH dependence, with cumulative release rates of 13.23%, 48.17%, and 81.94% at pH 7.4, 6.5, and 5.5, respectively (e.g., ...). Figure 5 (As shown). This pH-responsive behavior is primarily regulated by intramolecular interactions within the CHEMS / DOPE lipid bilayer. Under acidic conditions, protonation of CHEMS reduces headgroup charge density, thereby destabilizing the DOPE-stabilized layered bilayer structure and promoting its transition to a non-bilayer phase (such as the hexagonal HII phase). This structural rearrangement enhances membrane permeability and promotes AA5 diffusion.

[0089] (ii) In vitro experiments

[0090] Cell uptake: LLC and 4T1 cells were added at a rate of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 μg / mL in 24-well glass plates. After overnight culture, AhBT∩NR@Lipo nanoparticles were added to each well to a final concentration of 50 μg / mL, and the plates were incubated for 24 h. Cells were then washed with PBS to remove excess nanoparticles, fixed with cold methanol, and briefly stained with WGA488 to visualize the cell membrane. After rinsing, the nuclei were counterstained with a mounting medium containing DAPI. All procedures were performed in the dark. Cell endocytosis was observed using a confocal laser scanning microscope.

[0091] Experimental results are as follows Figure 6 As shown: Confocal laser scanning microscopy revealed that both LLC and 4T1 cells could effectively take up AhBT∩NR@Lipo, with strong red fluorescence mainly localized in the cytoplasmic region.

[0092] Measurement of intracellular hydrogen peroxide levels and oxygen consumption rate (OCR): 4T1 and LLC cells were cultured at 2 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured overnight. Afterward, cells were treated with 1 μM Atpenin A5 (AA5) or a vector for 2 hours. Following treatment, cells were collected and lysed, and hydrogen peroxide was quantitatively detected using a hydrogen peroxide detection kit (S0038, Beyotime, China) according to the manufacturer's instructions. The detection principle is that H2O2 decomposes Fe […]. 2+ Oxidized to Fe 3+ The latter forms a purple complex with xylene orange under acidic conditions. Cell lysate and serially diluted H2O2 standards were incubated with the chromogenic working solution at 25°C for 15 min. The absorbance was measured at 560 nm using a microplate reader, and the H2O2 concentration was calculated based on the standard curve.

[0093] Cell OCR was measured using a Seahorse XF24 Analyzer (Agilent Technologies). Cells were loaded at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 mcg / well in XF microplates and analyzed after AA5 treatment for 24 h. OCR was performed using the XFCell Mito Stress Test Kit (#103015, Agilent) according to the manufacturer's instructions. Before testing, the medium was replaced with Seahorse assay medium (#103575, Agilent) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. Cells were then incubated at 37°C in a CO2-free incubator for 60 min to equilibrate. During the assay, oligomycin (ATP synthase inhibitor), FCCP (mitochondrial uncoupling agent), and rotenone / antimycin A (complex I / III inhibitor) were injected sequentially to interfere with mitochondrial respiration, while OCR was continuously recorded. After the experiment, cell counts were performed for data normalization.

[0094] The experimental results show that AA5 treatment resulted in an increase in intracellular H2O2 levels (e.g., Figure 7 As shown in Figure A, accumulated H2O2, a key redox substrate, can be effectively utilized in mechanically triggered piezoelectric catalytic reactions. AA5 also regulates cellular oxygen metabolism. Oxygen consumption rate (OCR) measurements showed that AA5 significantly inhibited mitochondrial respiration in LLC and 4T1 cells, manifested as a significant reduction in both basal and maximal respiratory capacity (e.g., as shown in Figure A). Figure 7 (As shown in B). The reduction in oxygen utilization increases the relative availability of molecular oxygen within the cell, thereby maintaining its role as an electron acceptor in redox reactions. Therefore, AA5 treatment may be associated with intracellular H2O2 accumulation while maintaining relatively stable O2 availability, which may help create a metabolically favorable oxidation microenvironment, thus supporting the amplification of piezoelectrically catalyzed ROS.

[0095] CCK-8 Cytotoxicity Assay: Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. LLC and 4T1 cells were seeded at a density of 1000 cells per well in 96-well plates. After 24 h of incubation, different concentrations of the specified treatments were administered. After 72 h of treatment, 10 μL of CCK-8 reagent was added to each well and incubated for 2 h. The absorbance was then measured at 450 nm using a multi-mode microplate reader.

[0096] Experimental results: AA5 alone moderately reduced cell viability, while AhBT∩AA5@Lipo (AhBT∩AA5@Lipo+IPP) under IPP-mimicked mechanical stimulation induced significant cytotoxicity in all tested tumor cell lines (e.g., Figure 8 (As shown).

[0097] Intracellular ROS analysis: Intracellular ROS levels were detected using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). LLC, 4T1, and H1299 cells were seeded on 24-well plates and cultured overnight. Subsequently, cells were treated according to the same protocol as in the cytotoxicity assay. After 4 h of incubation, cells were washed with PBS and incubated with DCFH-DA (5 μM) at 37 °C for 30 min. Cells were washed three times with PBS to remove excess probe. Cells were then fixed with 4% paraformaldehyde for 30 min and mounted with mounting medium containing DAPI. ROS-related fluorescence signals were observed using a confocal laser scanning microscope (excitation wavelength: 488 nm).

[0098] Experimental results: Intracellular ROS levels significantly increased after combined treatment. Fluorescence imaging showed that the ROS signal was significantly enhanced in the AhBT∩AA5@Lipo+IPP group (e.g., ...). Figure 9 (As shown). This enhancement is attributed to the synergistic effect between piezoelectric charge generation and AA5-induced redox substrate enrichment.

[0099] Assessment of HMGB1 release: Intracellular HMGB1 localization was analyzed using immunofluorescence staining. LLC and 4T1 cells were seeded on 24-well plates and cultured overnight. After specified treatment, cells were fixed with cold methanol and incubated overnight at 4°C with anti-HMGB1 primary antibody (Abcam, #ab79823, RRID: AB_1603373, 1:250) in the dark. After washing, cells were incubated with Alexa Fluor 594 conjugated with secondary antibody (Invitrogen, #A-21207, RRID: AB_141637, 1:2000) at room temperature for 1 h. Fluorescence images were acquired using confocal laser scanning microscopy.

[0100] Experimental Results: Further analysis showed that this treatment induced characteristic manifestations of immunogenic cell death (ICD). Immunofluorescence imaging revealed significantly reduced HMGB1 signaling in the mechanical stimulation group, while the AhBT∩AA5@Lipo+IPP group was almost completely exhausted (e.g., Figure 10 (As shown). The results indicate that AA5-enhanced piezoelectric catalytic therapy not only exerts direct cytotoxic effects on tumor cells but also enhances tumor immunogenicity.

[0101] (III) In vivo experiments

[0102] Establishment and in vivo distribution of the MPE model: An MPE model was established in 6-8 week old female C57BL / 6 and BALB / c mice. Hair was shaved from the anterior chest to expose the skin, and the injection site on the right chest wall was disinfected with alcohol. A 26-G catheter was carefully inserted into the right pleural cavity along the midaxillary line through the 10th or 11th intercostal space. After confirming successful catheter placement, the stylet was removed, and 50 μL of LLC-Luc or 4T1-Luc cell suspension (1.2 × 10⁻⁶) was slowly injected into the pleural cavity. 5 (Cells). After injection, the catheter was removed, and the area was gently pressed to prevent leakage. For in vivo distribution analysis, DiR-labeled AhBT@Lipo nanoparticles (AhBT∩DiR@Lipo, 0.5 mg / kg) were administered intrapleurally on day 12 post-tumor inoculation. Whole-body fluorescence imaging was performed at preset time points (1, 3, 6, 12, 24, 48, and 72 h) using the IVIS Spectrum imaging system. To further assess organ-level distribution, ex vivo imaging was performed on heart, liver, spleen, lung, kidney, brain, stomach, intestine, tumor tissue, and pleural effusion.

[0103] Experimental results: IVIS imaging showed that after intrapleural administration, the fluorescence signal was mainly confined to the pleural cavity and lasted for at least 72 hours (e.g., Figure 11 As shown in Figure A). Further ex vivo imaging revealed that AhBT@Lipo significantly accumulated in pleural effusion and tumor tissue, with signal intensity significantly higher than in other major organs (such as...). Figure 11 As shown in B), this highlights the excellent intrathoracic retention and tumor targeting properties of the nanoplatform.

[0104] In vivo treatment regimen: To evaluate the efficacy of piezoelectric catalytic therapy in vivo, mice with pleural metastases and MPE were randomly assigned to different treatment groups, ensuring comparable baseline body weight and tumor burden across groups. Mice were anesthetized with isoflurane and received intrapleural injections every three days. Treatment agents included AA5 (0.5 mg / kg), AhBT@Lipo (10 mg / mL), or AhBT∩AA5@Lipo (10 mg / mL), while the control group received an equal volume of saline.

[0105] After treatment commencement, tumor progression and pleural effusion were monitored every two days via IVIS imaging and ultrasound. Throughout the study, animal health status, including weight and survival, was carefully recorded. At the experimental endpoint (day 22), mice were euthanized by CO2 asphyxiation. Pleural effusion was gently aspirated using a sterile syringe and its volume was measured. Tumor tissue and major organs were collected and fixed in 4% paraformaldehyde for subsequent histological and immunological analysis. Treatment regimens were as follows. Figure 12 As shown.

[0106] Experimental Results: IVIS imaging showed rapid tumor growth in the saline and AA5 groups, as evidenced by a gradual increase in fluorescence intensity. In contrast, AhBT@Lipo treatment significantly delayed tumor progression. Notably, throughout the treatment period, the AhBT∩AA5@Lipo group consistently exhibited the lowest tumor-related fluorescence signal, indicating its superior tumor-suppressive effect (e.g., Figure 13 (As shown in A and B). Anatomical observation and pleural effusion measurements further confirmed the imaging results. Extensive pleural tumor nodules were observed in the saline and AA5 groups, while AhBT@Lipo treatment reduced the nodule burden. The AhBT∩AA5@Lipo group showed the fewest number of tumor nodules (e.g., ...). Figure 14 (As shown in A and B). Furthermore, AhBT∩AA5@Lipo treatment significantly reduced the volume of malignant effusions (e.g., ...). Figure 15 (As shown in A and B), indicating that it has a dual inhibitory effect on tumor growth and fluid accumulation progression.

[0107] Immunohistochemical (IHC) and immunofluorescence (IF) staining: To assess ICD and CD8+ T cell infiltration, tumor tissue was analyzed using either immunohistochemistry (IHC) or immunofluorescence (IF). Tumor samples were fixed in formalin for 24 h, dehydrated, embedded in paraffin, and cut into 4 μm thick sections. The sections were placed on glass slides, dewaxed, and then stained.

[0108] For IHC, tissue sections were first subjected to antigen retrieval, followed by incubation with endogenous peroxidase blocking solution for 15 min. After blocking with 5% BSA / PBS at room temperature for 1 h, the sections were incubated overnight at 4 °C with primary antibodies against HMGB1 (Abcam, #ab79823, RRID: AB_1603373, 1:500) and CRT (Abcam, #ab92516, RRID: AB_10805991, 1:500). After washing with PBS, HRP-conjugated secondary antibody was added, and DAB substrate was used for staining. Subsequently, the sections were counterstained with hematoxylin, dehydrated, and mounted.

[0109] For IF staining, sections were processed using a similar procedure to IHC. Slides were incubated overnight at 4°C with anti-CD8 primary antibody (Abcam, #ab217344, RRID: AB_2892040, 1:500), followed by incubation at room temperature for 1 hour with Alexa Fluor 594-conjugated secondary antibody under light-protected conditions. Before mounting, cell nuclei were counterstained with Hoechst (1 μg / mL), and the slides were mounted using anti-fluorescence quenching mounting medium.

[0110] Experimental results: AhBT@Lipo treatment alone promoted CRT membrane exposure and HMGB1 release, while these effects were significantly enhanced in the AhBT∩AA5@Lipo group (e.g., Figure 16 (As shown in A and B). Compared with the control group, AhBT∩AA5@Lipo combination therapy significantly enhanced CD8+ T cell infiltration (e.g., ...). Figure 17 As shown in A and B), this treatment promotes anti-tumor immune activation in vivo. Kaplan-Meier survival analysis further demonstrated that AhBT∩AA5@Lipo treatment significantly prolonged survival (e.g., as shown in A and B). Figure 18 (As shown in A and B).

[0111] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a physiological pressure-driven piezoelectric catalytic therapeutic nanodrug delivery system with pH-responsive drug release function, characterized in that, The preparation method includes the following steps: (I) Preparation of metal nanoparticles The aqueous solution of the metal source was heated to reflux, and then sodium citrate solution was added to react. After the reaction was completed, polyvinylpyrrolidone solution was added, and after mixing evenly, metal nanoparticles were obtained by centrifugation. (II) Preparation of egg yolk-shell structured nanoparticles (II-1) After dispersing the metal nanoparticles, add alkaline solution and silicon source, stir and react to obtain gold-silica nanoparticles; (II-2) The gold-silica nanoparticles are dispersed in a mixed solvent, and then a titanium source is added to react and obtain gold-silica-titanium dioxide nanoparticles; (II-3) The gold-silica-titanium dioxide nanoparticles were dispersed in ethanol, and barium hydroxide solution was added. The mixture was then subjected to a hydrothermal reaction at 160-180℃ for 6-8 hours to obtain egg yolk-shell structured nanoparticles. (III) Drug load After dispersing the yolk-shell structured nanoparticles, a solution of a metabolic regulatory drug was added and incubated. After incubation, the nanoparticles were centrifuged and washed to obtain drug-loaded nanoparticles. The metabolic regulatory drug was Atpenin A5, a mitochondrial complex II inhibitor. (IV) Preparation of drug delivery system (IV-1) CHEMS, DOPE, DSPE-PEG2000 and DSPE-PEG2000-Folate were mixed in a molar ratio of 4:6:0.45:0.05 and dissolved in a mixed solvent of chloroform and methanol. The resulting solution was then distilled under reduced pressure at 40-50°C for 1-2 hours to obtain a lipid film. The volume ratio of chloroform to methanol in the mixed solvent was 9:

1. (IV-2) After dispersing the drug-loaded nanoparticles, they are mixed with the lipid film and hydrated to obtain the drug delivery system.

2. The preparation method of the physiological pressure-driven piezoelectric catalytic therapeutic nanomedicine delivery system with pH-responsive drug release function according to claim 1, characterized in that, In step (I), the tetrachloroauric acid aqueous solution is heated to reflux, and then sodium citrate solution is added to react for 30-40 minutes. After the reaction is completed, polyvinylpyrrolidone solution is added, and after mixing evenly, metal nanoparticles are obtained by centrifugation. And / or, the concentration of the tetrachloroauric acid aqueous solution is 1-2 mM; And / or, the concentration of the sodium citrate solution is 1-2 wt%; And / or, the concentration of the polyvinylpyrrolidone solution is 10-20 mg / mL.

3. The preparation method of the physiological pressure-driven piezoelectric catalytic therapeutic nanomedicine delivery system with pH-responsive drug release function according to claim 1, characterized in that, In step (II-1), the metal nanoparticles are dispersed in isopropanol, and ammonia and tetraethyl orthosilicate are added sequentially under stirring. After stirring and reacting, gold-silica nanoparticles are obtained. And / or, the concentration of the ammonia solution is 28-35 wt%.

4. The preparation method of the physiological pressure-driven piezoelectric catalytic therapeutic nanomedicine delivery system with pH-responsive drug release function according to claim 1, characterized in that, In step (II-2), the gold-silica nanoparticles are dispersed in a mixed solvent of ethanol and acetonitrile, and then titanium isopropoxide is added and reacted at 5-10°C for 6-8 hours to obtain gold-silica-titanium dioxide nanoparticles. And / or, in the mixed solvent of ethanol and acetonitrile, the volume ratio of ethanol to acetonitrile is 3:

1.

5. The preparation method of the physiological pressure-driven piezoelectric catalytic therapeutic nanodrug delivery system with pH-responsive drug release function according to claim 1, characterized in that, In step (III), the yolk-shell structured nanoparticles are dispersed in methanol, and a solution of metabolic regulating drugs is added for incubation for 12-16 hours. After completion, the nanoparticles are collected by centrifugation at 10000-15000 r / min, then washed 2-3 times with PBS solution, and finally freeze-dried to obtain drug-loaded nanoparticles.

6. The method for preparing the physiological pressure-driven piezoelectric catalytic therapeutic nanodrug delivery system with pH-responsive drug release function according to claim 1, characterized in that, In step (IV-2), the drug-loaded nanoparticles are dispersed in a PBS solution and hydrated with the lipid film at 40-45°C for 1-2 hours to obtain the drug delivery system.

7. A physiological pressure-driven piezoelectric catalytic therapeutic nanomedicine delivery system with pH-responsive drug release function, obtained by the preparation method according to any one of claims 1-6.

8. The application of the physiological pressure-driven piezoelectric catalytic therapeutic nanomedicine delivery system with pH-responsive drug release function as described in claim 7 in the preparation of a drug for treating malignant pleural effusion.

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